LMP1对人鼻咽癌细胞系CNE1癌基因微小RNA表达谱的影响
|
摘要
背景与目的
鼻咽癌是中国南方和东南亚地区最常见的恶性肿瘤之一,具有明显的区域分布特征、与EB病毒的密切关系。LMP1是EBVⅡ型潜伏感染的鼻咽癌细胞表达的一个病毒癌基因,他能促进上皮细胞转化,能通过NF-κB、p38、JNK等信号通路激活下游基因的转录,影响细胞的生长与增殖、凋亡以及侵蚀和转移能力。同时,它在EBVⅢ型潜伏感染的淋巴瘤细胞中能调节miRNAs的表达,并通过miRNAs的靶基因调控作用保持病毒的潜伏感染状态、影响肿瘤的生物学行为等等。
miRNAs一种新型的、内源性的、非蛋白编码小RNA分子,长约22个核苷酸,发挥转录后基因表达调控作用。miRNAs通过miRISC抑制靶mRNA的翻译或促使其降解,从而调节细胞的发育、分化、增殖、凋亡、代谢等生命活动。其中部分miRNAs具有癌基因和肿瘤抑制基因的功能,称为癌基因miRNAs (oncomiRs),对肿瘤的生物学行为起重要作用。
通过miRNA芯片进行高通量筛选,找出差异表达的miRNAs分子,特别是oncomiRs分子,能全面了解肿瘤发生和发展的分子机制、更为精确地对肿瘤的进行分型和预后。找出差异表达：miRNA分子后,可以通过多个靶基因预测软件对靶基因进行预测,对于了解其生物学功能具有重要意义。最近有学者通过对鼻咽癌组织与癌旁正常组织的差异表达miRNA的靶基因参与的信号通路进行计算机预测,来了解差异表达miRNA的生物学功能,并发现它们能通过靶基因干预多条与肿瘤生物学行为相关的信号通路,对肿瘤的生长与增殖、凋亡、转移及血管生成产生重要作用。
miRNA的功能研究及生物信息学预测结果可以通过转染miRNA mimics、antagomiRs或miRNA抑制剂,正向或反向诱导miRNA的功能,来研究或验证细胞中该miRNA分子的功能。该转染的有效性可以通过靶基因的表达水平的改变获得证实。同时可以通过检测相关靶基因的表达来探讨其功能发生的具体机制。
本实验旨在探讨LMP1对鼻咽癌细胞miRNA表达的调控作用,并探讨其对鼻咽癌生物学行为的影响。通过比较鼻咽癌细胞系CNE1与其EB病毒的潜伏膜蛋白1(latent membrane protein 1, LMP1)稳定转染细胞系CNE1-LMP1的癌基因微小RNA (oncogenic microRNAs, oncomiRs)表达谱的差异,探讨LMP1对EBVⅡ型潜伏感染的鼻咽癌细胞系CNE1oncomiRs表达的影响。并通过生物信息学分析,从中找出对细胞生物学功能影响较关键的分子；通过antagomiRs阻断后探讨其对鼻咽癌细胞生物学行为的影响,并在鼻咽癌组织中检测其表达及与LMP1的相关性及与临床病理特征的关系,探讨其在鼻咽癌组织中表达的意义。
方法
1.采用包含有132个oncomiRs分子的膜基microRNA芯片,检测鼻咽癌细胞系CNE1及其EBV LMP1的稳定转染细胞系oncomiRs的表达谱及差异表达谱。采用实时定量PCR检测验证表达差异较大的(达2倍)miRNAs分子,并对两者结果进行相关分析,验证芯片检测结果的可靠性。
2.采用miRNA分子功能预测在线工具DIANA-mirPath,分析差异表达miRNA参与的已知信号通路——京都基因及基因组百科全书(KEGG)信号通路,来阐明它们参与的生物学功能。
3.通过hsa-miR-19b knockdown探针阻断鼻咽癌细胞CNE1-LMP1中的hsa-miR-19b,然后分析其对细胞周期、增殖、凋亡和细胞迁移、侵袭的影响；及其对靶基因蛋白SOCS1及STAT3 (Signal transducer and activator of transcription 3)信号通路的影响,以初步探讨鼻咽癌细胞中hsa-miR-19b的功能和作用机制。
4.选择芯片筛选及qRT-PCR验证获得的表达差异较为明显的hsa-miR-19b分子,采用实时定量PCR检测46例鼻咽癌组织中差异表达最大的miRNA分子hsa-miR-19b表达,并采用免疫组化技术原位检测46例鼻咽癌组织中LMP1表达,并探讨其与LMP1表达的相关性。并探讨两者表达与临床病理特征之间的关系,探讨其表达作为诊断分子标记的意义。
结果
1.CNE1的oncomiRs表达谱
在芯片包含的132个oncomiRs中,CNE1中检出oncomiRs分子21个；其中高表达的(表达量与内参RUN48比大于1)有4个,低表达(表达量与内参RUN48比小于1)的有17个。
2. CNE1-LMP1的oncomiRs表达谱
在芯片包含的132个oncomiRs中,NE1-LMP1中检出30个；其中高表达的(表达量与内参RUN48比大于1)有7个,低表达(表达量与内参RUN48比小于1)的有23个。
3.CNE1和CNE1-LMP1的oncomiRs差异表达谱
通过比较较鼻咽癌细胞系CNE1与其LMP1稳定转染细胞系CNE1-LMP1的肿瘤相关miRNAs (oncomiRs)表达谱的差异,其中CNE1检出oncomiRs分子21个,而CNE1-LMP1中检出30个,显示LMP1能调节miRNAs的表达。与CNE1比较,CNE1-LMP1中miRNAs分子表达总量增加,有9个在CNE1-LMP1中特异性表达。分析共同表达的21个miRNA分子发现,CNE1-LMP1中有8个轻度降低,13个表达升高,其中表达升高达两倍以上的miRNA分子有7个:hsa-miR-19b、hsa-miR-17-3p、hsa-miR-22、hsa-miR-149>、hsa-miR-150、hsa-miR-188和hsa-miR-205。9个在CNE1-LMP1中特异性表达的miRNA中,hsa-miR-122a呈高水平表达,其表达量相对值大于1。综上所述,CNE1-LMP1中miRNA表达的总体水平升高。
4. qRT-PCR验证两细胞系中差异表达较大的miRNA分子
通过荧光定量RT-RCR检测,对芯片检测发现的7个(hsa-miR-19b、hsa-miR-17-3p、hsa-miR-22、hsa-miR-149、hsa-miR-150、hsa-miR-188和hsa-miR-205)表达差异较大的miRNA进行验证,芯片检测发现结果差异与qRT-RCR检测发现的结果差异相关(r=0.970,P=0.000)。两种方法检测结果一致。
5. DIANA-mirPath对差异表达miRNA参与的信号通路及功能预测
通过软件预测到与细胞信号通路相关的靶基因数目总共有95个；在差异表达的8个miRNA分子中,miR-17有预测参与信号通路靶基因44个,miR-19b有53个,其余6个共计4个。由此可见miR-17和miR-19b对信号通路的贡献占了绝大部分。两者参与了多条信号通路,与肿瘤密切相关的有环境信息的信号转导,细胞的运动性、生长与死亡及细胞通讯,多种肿瘤相关通路等。同时前期发现miR-19b的改变倍数及表达量显著高于miR-17,因此将miR-19b挑出进行后续功能研究。
6.鼻咽癌细胞CNE1-LMP1中hsa-miR-19b功能初探
hsa-miR-19b阻断后,细胞增殖下降(5pmol组抑制率为20.45%,11pmol组为44.50%)；细胞周期阻滞于G1,hsa-miR-19b阻断后G1期细胞百分比明显增加,而细胞凋亡明显增加；hsa-miR-19b阻断后细胞的迁移、侵袭能力下降。同时,其靶基因SOCS1蛋白表达明显升高,使STAT3磷酸化水平下降。
7.鼻咽癌组织中LMP1与hsa-miR-19b的表达及与临床病理特征的关系
NPC组织中LMP1蛋白阳性表达率为60.95%(28/46), hsa-miR-19b的表达量为68.27±69.00；LMP1蛋白表达及hsa-miR-19b表达均与肿瘤分期和淋巴结转移相关(P<0.05)。同时LMP1蛋白表达与hsa-miR-19b表达相关(r=0.390,P<0.05)。
结论
1.鼻咽癌细胞系CNE1与其LMP1稳定转染细胞系CNE1-LMP1的oncomiRs表达谱存在差异。
2.LMP1能调节鼻咽癌细胞系oncomiRs的表达,使miRNAs的总体表达水平升高,其可能机制为通过其转录激活功能促进miRNAs的编码基因表达miRNAs。
3.LMP1可能调节鼻咽癌细胞系oncomiRs的表达,可以成为其发挥病毒癌基因作用的另一重要的通路。
4.在8个差异表达较大的miRNA分子中,hsa-miR-17和hsa-miR-19b的靶基因对细胞信号通路的贡献最大,其参与的信号转导、细胞的运动、生长与死亡、细胞通讯的信号通路、肿瘤相关信号通路与肿瘤的发生发展密切相关。
5. hsa-miR-19b能促进鼻咽癌细胞生长与增殖、抑制细胞凋亡；其作用的可能途径有：通过抑制其靶基因SOCS1而抑制STAT3信号通路。hsa-miR-19b还能促进细胞迁移和侵袭。
6.鼻咽部低分化鳞状细胞癌组织中存在LMP1和hsa-miR-19b的表达。
7.鼻咽部低分化鳞状细胞癌组织中,LMP1可能参与了hsa-miR-19b的表达调控。
8.LMP1和hsa-miR-19b可以作为鼻咽部低分化鳞状细胞癌的分期及转移的诊断分子标记。
Background and objective:
The incidence of nasopharyngeal carcinoma (NPC) is most prevalent in Southeast Asia, particularly in Southern China. NPC is characterized by regional distribution and EBV-associated. LMP1 is a viral oncogene expressed in nasopharyngeal carcinoma cells, which exhibite latency type II of EBV infection. LMP1 can promote epithelial cells transformation through hijacking cell signaling pathway such as NF-κB, p38/MAPK and JNK signaling to activate the transcription of downstream genes. The activated protein expression profile are widely involed in cell growth, proliferation, apoptosis, invasion and metastasis. Meanwhile, LMP1 can regulate the expression of miRNAs in lymphoma that exhibte latency typeⅢof EBV infection.This regulation can keep the latent infection of EBV and change the biological characters of tumor cells.
miRNAs is a novel class of small(-22 nucleotide) non-protein-coding RNA molecular, which function as post-transcriptional gene regulator. miRNAs recruit other factors to assemble a complex named with miRISC(miRNA induced silencing complex), which suppress translation or promote degration of target mRNAs. miRNAs regulate diverse biological process such as development, differetiation, proliferation, apoptosis and metabolism. Differential expression profile study by highthrough screening through miRNA microarray to find differentially expressed miRNA, especially oncomiRs, is a good way to elucidate the globle molecular mechanism of development and progression of tumor, and also a good index in typing and prognosis of cancer. The targets of differentially expressed miRNA can be predicted by various predicting software to comprehend their biological function in cells. Recently, one group found the differentially expressed miRNAs between nasopharyngeal carcinoma tissue and adjacent normal tissue. Then the cell signal pathway mediated by their targets were predicted in silicon to elucidate their biological effects. Various signal pathway, which includes several pathway associated with tumoric biological characters, were interferred by their targets and were involved in cell growth, proliferation, apoptosis, metastasis and angiogenesis. Function of miRNA can be studied in model cells by transfecting with miRNA mimics and antagomiRs (miRNA inhibitors) to induced the function of miRNA positively or inversely. The results from bioinformatics can also be verified by transfecting assay. The efficacy of transfection can be validation by targets detection. Simultaneously, the detailed mechanism of miRNA functioning can be indicated by targets assay.
In order to explore the regulating effection of LMP1 on oncomiRs expression in CNE1 cell line and ultimate effection on biological characters in nasopharyngeal carcinoma. We investigate the differential oncomiRs expression profile between nasopharyngeal carcinoma cell line CNE1 and it's steady EBV-LMP1-transfected cell line CNE1-LMP1. Among the differentially expressed oncomiRs, the miRNAs play a key role in cellular biological function were picked out by bioinformatical assay. AntagomiRs were used to block the selected miRNAs to investigate it's role in biological characters of nasopharyngeal cells. It's expression in nasophayngeal carcinoma tissue were detected and the association with expression of LMP1 were assessed. Both of them were evaluated their association with clinopathological characters of nasopharyngeal carcinoma to explore the significance of their expression in nasopharyngeal carcinoma tissue.
Then detect the most differentially expressed miRNA in tissues of nasopharyngeal carcinoma, evaluate the association between it's expression value and LMP1 expression and their expression with clincopathological characters. And transfection knockdown probe into model cell to block the miRNA to elucidate its prelimilary function.
Methods:
1. A membrane-based microRNA array that targets 132 of the most well studied oncomiRs were used to detect the expression profile of CNE1 and CNE1-LMP1. And then real time qRT-PCR assay verified the expression data of the most differentiatedly expressed(changed over 2 folds) miRNAs. The data of miRNA array and qRT-PCR were analysised by correlation analysis to verify the reliability of miRNA array assay.
2. The KEGG (kyoto encyclopedia of genes and genomes) pathway employed by the differentially expressed oncomiRs to execute their biological function were predicted by DIANA-mirPath, a online functional prediction tool of miRNA.
3. Cell cycle, proliferation, apoptosis, cell migration and invasion in CNE1-LMP1 cells were evaluated after knockdown of hsa-miR-19b. STAT3 signaling pathway and SOCS1 (a target gene of hsa-miR-19b, also suppressor of STAT3 signaling) were also detected to elucidate the preliminary function and fundamental mechanism of hsa-miR-19b.
4.46 cases of nasopharyngeal carcinoma were enrolled in the study before radiotherapy. LMP1 protein detected by immunohistochemisty and hsa-miR-19b by real time qRT-PCR. The correlation between these two index were assessed. hsa-miR-19b detected by real time qRT-PCR. The correlation between LMP1 and hsa-miR-19b expression were assessed. Meanwhile, evaluate the correlation between LMP1 or hsa-miR-19b and clinicopathological characters.
Results:
1. The oncomiRs expression profile of CNE1 cells shows as below. Among the restricted 132 miRNAs,21 were detectable in CNE1. There is 4 miRNAs with higher expression levels(expression levels higher than internal reference RUN48); And 17 with lower expression levels(expression levels lower than internal reference RUN48).
2. The oncomiRs expression profile of CNE1-LMP1 cells shows as below. Among the restricted 132 miRNAs,30 were detectable in CNE1. There is 7 miRNAs with higher expression levels(expression levels higher than internal reference RUN48). And 23 with lower expression levels(expression levels lower than internal reference RUN48).
3. The differential expression profile between CNE1 and CNE1-LMP1. Among the restricted 132 miRNAs,30 were detectable in CNE1-LMP1 and 21 in CNE1. There is 21 shared miRNAs, among which 7 miRNAs'expression level (hsa-miR-19b, hsa-miR-17-3p, hsa-miR-22, hsa-miR-149, hsa-miR-150, hsa-miR-188 and hsa-miR-205) elevated over two folds. Among the 9 specifically expressed miRNAs in CNE1-LMP1, hsa-miR-122a has a highest expression level surpass the internal control sample. Altogether, CNE1-LMP1 showed higher total miRNAs levels.
4. qRT-PCR verify the expression detected by miRNA array. The expression differentiation of 7 most differentiatedly expressed miRNAs confirmed by the real qRT-PCR. The results show that they are closely correlated(r=0.970, P=0.000).
5. DIANA-miPath predict the pathway and function of differentially expressed miRNAs
The software predicts that the total targets involved in cell signal pathway is 95. Among 8 differentially expressed miRNAs, miR-17 has 44 targets, miR-19b has 53 and the other 6 miRNAs has only 4. From this, we included that miR-17 and miR-19b contribute to most of the biological function through signal pathway. The two major miRNAs involved in various pathway. Among them, the signal pathway which closely associated with tumor includes enviromental signal transduction, cell motibility, cell growth and death, cell communication and tumor associated pathway. Taking the previous expression data together(miR-19b shared the higher expression level and changed times), we pick out miR-19b for further functional assay.
6. The preliminary function of hsa-miR-19b in CNE1-LMP1
After knockdown the hsa-miR-19b, cell proliferation decreased (5pmol group cell proliferation inhibit rate is 20.45%, llpmol group is 44.50%); the percentage of cells in G1 stage increased, cell cycle is blocked in G1 stage; And the apoptosis increased. Meanwhile, the expression of it's target protein, SOCS1 increased; lead to lower phosphorylation level of STAT3.
7. The in situ expression of LMP1 and hsa-miR-19b and their correlation with clinopathological characters
The positive expression rate of LMP1 protein is 60.95%(28/46). The relative expression value of hsa-miR-19b is 68.27±69.00. Both LMP1 and hsa-miR-19b were associated with stage and lymphatic metastasis(P<0.05).The expression of LMP1 were associated with the expression of hsa-miR-19b (r=0.390, P<0.05).
Conclusions:
1. oncomiRs are differentially expressed between CNE1 cells and CNE1-LMP1 cells.
2. LMP1 can regulate the expression of oncomiRs in nasopharyngeal carcinoma cell line and elevate the total miRNAs level through it's transcription-activating function.
3. Regulating the expression of miRNAs would be another important pathway employed by LMP1 to function as a viral oncogene.
4. Among 8 differentially expressed miRNAs, hsa-miR-17 and has-miR-17 contribute most to the cell signal pathway, among which signal transduction, cell motibility, cell growth and death, cell communication and tumor associated pathway were closely associated with the development and progression of tumor.
5. Hsa-miR-19b can promote cell cycle and proliferation, inhibit apoptosis. The possible mechanism employed in these process may be constructively activate the STAT3 signaling. In which hsa-miR-19b inhibit the translation of it's target gene SOCS1, and ultimately abolished the blockage of the phosphorylation of STAT3. Hsa-miR-19b can also promote cell migration and ivasion in vitro.
6. LMP1 and hsa-miR-19b showed expression in low differentiated squamous cell carcinoma of nsaopharynx epithelia.
7. LMP1 would be involved in expression regulation of hsa-miR-19b in low differentiated squamous cell carcinoma of nsaopharynx epithelia.
8. LMP1 and hsa-miR-19b can functionate as a diagnosis molecular marker in low differentiated squamous cell carcinoma of nsaopharynx epithelia.
鼻咽癌是中国南方和东南亚地区最常见的恶性肿瘤之一,具有明显的区域分布特征、与EB病毒的密切关系。LMP1是EBVⅡ型潜伏感染的鼻咽癌细胞表达的一个病毒癌基因,他能促进上皮细胞转化,能通过NF-κB、p38、JNK等信号通路激活下游基因的转录,影响细胞的生长与增殖、凋亡以及侵蚀和转移能力。同时,它在EBVⅢ型潜伏感染的淋巴瘤细胞中能调节miRNAs的表达,并通过miRNAs的靶基因调控作用保持病毒的潜伏感染状态、影响肿瘤的生物学行为等等。
miRNAs一种新型的、内源性的、非蛋白编码小RNA分子,长约22个核苷酸,发挥转录后基因表达调控作用。miRNAs通过miRISC抑制靶mRNA的翻译或促使其降解,从而调节细胞的发育、分化、增殖、凋亡、代谢等生命活动。其中部分miRNAs具有癌基因和肿瘤抑制基因的功能,称为癌基因miRNAs (oncomiRs),对肿瘤的生物学行为起重要作用。
通过miRNA芯片进行高通量筛选,找出差异表达的miRNAs分子,特别是oncomiRs分子,能全面了解肿瘤发生和发展的分子机制、更为精确地对肿瘤的进行分型和预后。找出差异表达：miRNA分子后,可以通过多个靶基因预测软件对靶基因进行预测,对于了解其生物学功能具有重要意义。最近有学者通过对鼻咽癌组织与癌旁正常组织的差异表达miRNA的靶基因参与的信号通路进行计算机预测,来了解差异表达miRNA的生物学功能,并发现它们能通过靶基因干预多条与肿瘤生物学行为相关的信号通路,对肿瘤的生长与增殖、凋亡、转移及血管生成产生重要作用。
miRNA的功能研究及生物信息学预测结果可以通过转染miRNA mimics、antagomiRs或miRNA抑制剂,正向或反向诱导miRNA的功能,来研究或验证细胞中该miRNA分子的功能。该转染的有效性可以通过靶基因的表达水平的改变获得证实。同时可以通过检测相关靶基因的表达来探讨其功能发生的具体机制。
本实验旨在探讨LMP1对鼻咽癌细胞miRNA表达的调控作用,并探讨其对鼻咽癌生物学行为的影响。通过比较鼻咽癌细胞系CNE1与其EB病毒的潜伏膜蛋白1(latent membrane protein 1, LMP1)稳定转染细胞系CNE1-LMP1的癌基因微小RNA (oncogenic microRNAs, oncomiRs)表达谱的差异,探讨LMP1对EBVⅡ型潜伏感染的鼻咽癌细胞系CNE1oncomiRs表达的影响。并通过生物信息学分析,从中找出对细胞生物学功能影响较关键的分子；通过antagomiRs阻断后探讨其对鼻咽癌细胞生物学行为的影响,并在鼻咽癌组织中检测其表达及与LMP1的相关性及与临床病理特征的关系,探讨其在鼻咽癌组织中表达的意义。
方法
1.采用包含有132个oncomiRs分子的膜基microRNA芯片,检测鼻咽癌细胞系CNE1及其EBV LMP1的稳定转染细胞系oncomiRs的表达谱及差异表达谱。采用实时定量PCR检测验证表达差异较大的(达2倍)miRNAs分子,并对两者结果进行相关分析,验证芯片检测结果的可靠性。
2.采用miRNA分子功能预测在线工具DIANA-mirPath,分析差异表达miRNA参与的已知信号通路——京都基因及基因组百科全书(KEGG)信号通路,来阐明它们参与的生物学功能。
3.通过hsa-miR-19b knockdown探针阻断鼻咽癌细胞CNE1-LMP1中的hsa-miR-19b,然后分析其对细胞周期、增殖、凋亡和细胞迁移、侵袭的影响；及其对靶基因蛋白SOCS1及STAT3 (Signal transducer and activator of transcription 3)信号通路的影响,以初步探讨鼻咽癌细胞中hsa-miR-19b的功能和作用机制。
4.选择芯片筛选及qRT-PCR验证获得的表达差异较为明显的hsa-miR-19b分子,采用实时定量PCR检测46例鼻咽癌组织中差异表达最大的miRNA分子hsa-miR-19b表达,并采用免疫组化技术原位检测46例鼻咽癌组织中LMP1表达,并探讨其与LMP1表达的相关性。并探讨两者表达与临床病理特征之间的关系,探讨其表达作为诊断分子标记的意义。
结果
1.CNE1的oncomiRs表达谱
在芯片包含的132个oncomiRs中,CNE1中检出oncomiRs分子21个；其中高表达的(表达量与内参RUN48比大于1)有4个,低表达(表达量与内参RUN48比小于1)的有17个。
2. CNE1-LMP1的oncomiRs表达谱
在芯片包含的132个oncomiRs中,NE1-LMP1中检出30个；其中高表达的(表达量与内参RUN48比大于1)有7个,低表达(表达量与内参RUN48比小于1)的有23个。
3.CNE1和CNE1-LMP1的oncomiRs差异表达谱
通过比较较鼻咽癌细胞系CNE1与其LMP1稳定转染细胞系CNE1-LMP1的肿瘤相关miRNAs (oncomiRs)表达谱的差异,其中CNE1检出oncomiRs分子21个,而CNE1-LMP1中检出30个,显示LMP1能调节miRNAs的表达。与CNE1比较,CNE1-LMP1中miRNAs分子表达总量增加,有9个在CNE1-LMP1中特异性表达。分析共同表达的21个miRNA分子发现,CNE1-LMP1中有8个轻度降低,13个表达升高,其中表达升高达两倍以上的miRNA分子有7个:hsa-miR-19b、hsa-miR-17-3p、hsa-miR-22、hsa-miR-149>、hsa-miR-150、hsa-miR-188和hsa-miR-205。9个在CNE1-LMP1中特异性表达的miRNA中,hsa-miR-122a呈高水平表达,其表达量相对值大于1。综上所述,CNE1-LMP1中miRNA表达的总体水平升高。
4. qRT-PCR验证两细胞系中差异表达较大的miRNA分子
通过荧光定量RT-RCR检测,对芯片检测发现的7个(hsa-miR-19b、hsa-miR-17-3p、hsa-miR-22、hsa-miR-149、hsa-miR-150、hsa-miR-188和hsa-miR-205)表达差异较大的miRNA进行验证,芯片检测发现结果差异与qRT-RCR检测发现的结果差异相关(r=0.970,P=0.000)。两种方法检测结果一致。
5. DIANA-mirPath对差异表达miRNA参与的信号通路及功能预测
通过软件预测到与细胞信号通路相关的靶基因数目总共有95个；在差异表达的8个miRNA分子中,miR-17有预测参与信号通路靶基因44个,miR-19b有53个,其余6个共计4个。由此可见miR-17和miR-19b对信号通路的贡献占了绝大部分。两者参与了多条信号通路,与肿瘤密切相关的有环境信息的信号转导,细胞的运动性、生长与死亡及细胞通讯,多种肿瘤相关通路等。同时前期发现miR-19b的改变倍数及表达量显著高于miR-17,因此将miR-19b挑出进行后续功能研究。
6.鼻咽癌细胞CNE1-LMP1中hsa-miR-19b功能初探
hsa-miR-19b阻断后,细胞增殖下降(5pmol组抑制率为20.45%,11pmol组为44.50%)；细胞周期阻滞于G1,hsa-miR-19b阻断后G1期细胞百分比明显增加,而细胞凋亡明显增加；hsa-miR-19b阻断后细胞的迁移、侵袭能力下降。同时,其靶基因SOCS1蛋白表达明显升高,使STAT3磷酸化水平下降。
7.鼻咽癌组织中LMP1与hsa-miR-19b的表达及与临床病理特征的关系
NPC组织中LMP1蛋白阳性表达率为60.95%(28/46), hsa-miR-19b的表达量为68.27±69.00；LMP1蛋白表达及hsa-miR-19b表达均与肿瘤分期和淋巴结转移相关(P<0.05)。同时LMP1蛋白表达与hsa-miR-19b表达相关(r=0.390,P<0.05)。
结论
1.鼻咽癌细胞系CNE1与其LMP1稳定转染细胞系CNE1-LMP1的oncomiRs表达谱存在差异。
2.LMP1能调节鼻咽癌细胞系oncomiRs的表达,使miRNAs的总体表达水平升高,其可能机制为通过其转录激活功能促进miRNAs的编码基因表达miRNAs。
3.LMP1可能调节鼻咽癌细胞系oncomiRs的表达,可以成为其发挥病毒癌基因作用的另一重要的通路。
4.在8个差异表达较大的miRNA分子中,hsa-miR-17和hsa-miR-19b的靶基因对细胞信号通路的贡献最大,其参与的信号转导、细胞的运动、生长与死亡、细胞通讯的信号通路、肿瘤相关信号通路与肿瘤的发生发展密切相关。
5. hsa-miR-19b能促进鼻咽癌细胞生长与增殖、抑制细胞凋亡；其作用的可能途径有：通过抑制其靶基因SOCS1而抑制STAT3信号通路。hsa-miR-19b还能促进细胞迁移和侵袭。
6.鼻咽部低分化鳞状细胞癌组织中存在LMP1和hsa-miR-19b的表达。
7.鼻咽部低分化鳞状细胞癌组织中,LMP1可能参与了hsa-miR-19b的表达调控。
8.LMP1和hsa-miR-19b可以作为鼻咽部低分化鳞状细胞癌的分期及转移的诊断分子标记。
Background and objective:
The incidence of nasopharyngeal carcinoma (NPC) is most prevalent in Southeast Asia, particularly in Southern China. NPC is characterized by regional distribution and EBV-associated. LMP1 is a viral oncogene expressed in nasopharyngeal carcinoma cells, which exhibite latency type II of EBV infection. LMP1 can promote epithelial cells transformation through hijacking cell signaling pathway such as NF-κB, p38/MAPK and JNK signaling to activate the transcription of downstream genes. The activated protein expression profile are widely involed in cell growth, proliferation, apoptosis, invasion and metastasis. Meanwhile, LMP1 can regulate the expression of miRNAs in lymphoma that exhibte latency typeⅢof EBV infection.This regulation can keep the latent infection of EBV and change the biological characters of tumor cells.
miRNAs is a novel class of small(-22 nucleotide) non-protein-coding RNA molecular, which function as post-transcriptional gene regulator. miRNAs recruit other factors to assemble a complex named with miRISC(miRNA induced silencing complex), which suppress translation or promote degration of target mRNAs. miRNAs regulate diverse biological process such as development, differetiation, proliferation, apoptosis and metabolism. Differential expression profile study by highthrough screening through miRNA microarray to find differentially expressed miRNA, especially oncomiRs, is a good way to elucidate the globle molecular mechanism of development and progression of tumor, and also a good index in typing and prognosis of cancer. The targets of differentially expressed miRNA can be predicted by various predicting software to comprehend their biological function in cells. Recently, one group found the differentially expressed miRNAs between nasopharyngeal carcinoma tissue and adjacent normal tissue. Then the cell signal pathway mediated by their targets were predicted in silicon to elucidate their biological effects. Various signal pathway, which includes several pathway associated with tumoric biological characters, were interferred by their targets and were involved in cell growth, proliferation, apoptosis, metastasis and angiogenesis. Function of miRNA can be studied in model cells by transfecting with miRNA mimics and antagomiRs (miRNA inhibitors) to induced the function of miRNA positively or inversely. The results from bioinformatics can also be verified by transfecting assay. The efficacy of transfection can be validation by targets detection. Simultaneously, the detailed mechanism of miRNA functioning can be indicated by targets assay.
In order to explore the regulating effection of LMP1 on oncomiRs expression in CNE1 cell line and ultimate effection on biological characters in nasopharyngeal carcinoma. We investigate the differential oncomiRs expression profile between nasopharyngeal carcinoma cell line CNE1 and it's steady EBV-LMP1-transfected cell line CNE1-LMP1. Among the differentially expressed oncomiRs, the miRNAs play a key role in cellular biological function were picked out by bioinformatical assay. AntagomiRs were used to block the selected miRNAs to investigate it's role in biological characters of nasopharyngeal cells. It's expression in nasophayngeal carcinoma tissue were detected and the association with expression of LMP1 were assessed. Both of them were evaluated their association with clinopathological characters of nasopharyngeal carcinoma to explore the significance of their expression in nasopharyngeal carcinoma tissue.
Then detect the most differentially expressed miRNA in tissues of nasopharyngeal carcinoma, evaluate the association between it's expression value and LMP1 expression and their expression with clincopathological characters. And transfection knockdown probe into model cell to block the miRNA to elucidate its prelimilary function.
Methods:
1. A membrane-based microRNA array that targets 132 of the most well studied oncomiRs were used to detect the expression profile of CNE1 and CNE1-LMP1. And then real time qRT-PCR assay verified the expression data of the most differentiatedly expressed(changed over 2 folds) miRNAs. The data of miRNA array and qRT-PCR were analysised by correlation analysis to verify the reliability of miRNA array assay.
2. The KEGG (kyoto encyclopedia of genes and genomes) pathway employed by the differentially expressed oncomiRs to execute their biological function were predicted by DIANA-mirPath, a online functional prediction tool of miRNA.
3. Cell cycle, proliferation, apoptosis, cell migration and invasion in CNE1-LMP1 cells were evaluated after knockdown of hsa-miR-19b. STAT3 signaling pathway and SOCS1 (a target gene of hsa-miR-19b, also suppressor of STAT3 signaling) were also detected to elucidate the preliminary function and fundamental mechanism of hsa-miR-19b.
4.46 cases of nasopharyngeal carcinoma were enrolled in the study before radiotherapy. LMP1 protein detected by immunohistochemisty and hsa-miR-19b by real time qRT-PCR. The correlation between these two index were assessed. hsa-miR-19b detected by real time qRT-PCR. The correlation between LMP1 and hsa-miR-19b expression were assessed. Meanwhile, evaluate the correlation between LMP1 or hsa-miR-19b and clinicopathological characters.
Results:
1. The oncomiRs expression profile of CNE1 cells shows as below. Among the restricted 132 miRNAs,21 were detectable in CNE1. There is 4 miRNAs with higher expression levels(expression levels higher than internal reference RUN48); And 17 with lower expression levels(expression levels lower than internal reference RUN48).
2. The oncomiRs expression profile of CNE1-LMP1 cells shows as below. Among the restricted 132 miRNAs,30 were detectable in CNE1. There is 7 miRNAs with higher expression levels(expression levels higher than internal reference RUN48). And 23 with lower expression levels(expression levels lower than internal reference RUN48).
3. The differential expression profile between CNE1 and CNE1-LMP1. Among the restricted 132 miRNAs,30 were detectable in CNE1-LMP1 and 21 in CNE1. There is 21 shared miRNAs, among which 7 miRNAs'expression level (hsa-miR-19b, hsa-miR-17-3p, hsa-miR-22, hsa-miR-149, hsa-miR-150, hsa-miR-188 and hsa-miR-205) elevated over two folds. Among the 9 specifically expressed miRNAs in CNE1-LMP1, hsa-miR-122a has a highest expression level surpass the internal control sample. Altogether, CNE1-LMP1 showed higher total miRNAs levels.
4. qRT-PCR verify the expression detected by miRNA array. The expression differentiation of 7 most differentiatedly expressed miRNAs confirmed by the real qRT-PCR. The results show that they are closely correlated(r=0.970, P=0.000).
5. DIANA-miPath predict the pathway and function of differentially expressed miRNAs
The software predicts that the total targets involved in cell signal pathway is 95. Among 8 differentially expressed miRNAs, miR-17 has 44 targets, miR-19b has 53 and the other 6 miRNAs has only 4. From this, we included that miR-17 and miR-19b contribute to most of the biological function through signal pathway. The two major miRNAs involved in various pathway. Among them, the signal pathway which closely associated with tumor includes enviromental signal transduction, cell motibility, cell growth and death, cell communication and tumor associated pathway. Taking the previous expression data together(miR-19b shared the higher expression level and changed times), we pick out miR-19b for further functional assay.
6. The preliminary function of hsa-miR-19b in CNE1-LMP1
After knockdown the hsa-miR-19b, cell proliferation decreased (5pmol group cell proliferation inhibit rate is 20.45%, llpmol group is 44.50%); the percentage of cells in G1 stage increased, cell cycle is blocked in G1 stage; And the apoptosis increased. Meanwhile, the expression of it's target protein, SOCS1 increased; lead to lower phosphorylation level of STAT3.
7. The in situ expression of LMP1 and hsa-miR-19b and their correlation with clinopathological characters
The positive expression rate of LMP1 protein is 60.95%(28/46). The relative expression value of hsa-miR-19b is 68.27±69.00. Both LMP1 and hsa-miR-19b were associated with stage and lymphatic metastasis(P<0.05).The expression of LMP1 were associated with the expression of hsa-miR-19b (r=0.390, P<0.05).
Conclusions:
1. oncomiRs are differentially expressed between CNE1 cells and CNE1-LMP1 cells.
2. LMP1 can regulate the expression of oncomiRs in nasopharyngeal carcinoma cell line and elevate the total miRNAs level through it's transcription-activating function.
3. Regulating the expression of miRNAs would be another important pathway employed by LMP1 to function as a viral oncogene.
4. Among 8 differentially expressed miRNAs, hsa-miR-17 and has-miR-17 contribute most to the cell signal pathway, among which signal transduction, cell motibility, cell growth and death, cell communication and tumor associated pathway were closely associated with the development and progression of tumor.
5. Hsa-miR-19b can promote cell cycle and proliferation, inhibit apoptosis. The possible mechanism employed in these process may be constructively activate the STAT3 signaling. In which hsa-miR-19b inhibit the translation of it's target gene SOCS1, and ultimately abolished the blockage of the phosphorylation of STAT3. Hsa-miR-19b can also promote cell migration and ivasion in vitro.
6. LMP1 and hsa-miR-19b showed expression in low differentiated squamous cell carcinoma of nsaopharynx epithelia.
7. LMP1 would be involved in expression regulation of hsa-miR-19b in low differentiated squamous cell carcinoma of nsaopharynx epithelia.
8. LMP1 and hsa-miR-19b can functionate as a diagnosis molecular marker in low differentiated squamous cell carcinoma of nsaopharynx epithelia.
引文
[1]Bartel DP. MicroRNAs:genomics, biogenesis, mechanism, and function[J]. Cell,2004,116(2):281-297.
[2]Llave C, Xie Z, Kasschau KD, et al. Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA[J]. Science,2002, 297(5589):2053-2056.
[3]Aurora EK, Frank JS. Oncomirs-microRNAs with a role in cancer[J]. Nature Reviews Cancer,2006,6(4):259-269
[4]Cimmino A, Calin GA, Fabbri M, et al. miR-15 and miR-16 induce apoptosis by targeting BCL2[J]. Proc Natl Acad Sci USA,2005, 102(39):13944-13949.
[5]O'Donnell KA, Wentzel EA, Zeller KI, et al. c-Myc-regulated microRNAs modulate E2F1 expression[J]. Nature,2005,435(7043), 839-843.
[6]Si ML, Zhu S, Wu H, et al. miR-21-mediated tumor growth[J]. Oncogene, 2007,26(19):2799-2803.
[7]Liu CG, Calin GA, Meloon B, et al. An oligonucleotide microchip for genome-wide microRNA profiling in human and mouse tissues[J]. Proc Natl Acad Sci USA,2004,101(26):9740-9744.
[8]Calin GA, Dumitru CD, Shimizu M, et al. Frequent deletions and down-regulation of micro-RNA genes miR-15 and miR-16 at 13q14 in chronic lymphocytic leukemia[J]. Proc Natl Acad Sci USA,2002, 99(24):15524-15529.
[9]Raver-Shapira N, Marciano E, Meiri E, et al. Transcriptional activation of miR-34a contributes to p53-mediated apoptosis[J]. Mol Cell,2007, 26(5):731-743.
[10]Knipe DM, Howley PM, Griffin DE, et al. Fields virology[M]. Philadelphia PA:Lippincott Williams & Wilkins.2007,5th ed, vol 2: 2603-2654.
[11]Knipe DM, Howley PM, Griffin DE, et al. Fields virology[M]. Philadelphia PA:Lippincott Williams & Wilkins.2007,5th ed, vol 2: 2655-2700.
[12]Young LS, Murray PG. Epstein-Barr virus and oncogenesis:from latent genes to tumours[J]. Oncogene,2003,22(33):5108-5121.
[13]Kulwichit W, Edwards RH, Davenport EM, et al. Expression of the Epstein-Barr virus latent membrane protein 1 induces B cell lymphoma in transgenic mice[J]. Proc Natl Acad Sci U S A,1998,95(20):11963-11968.
[14]Gires O, Zimber-Strobl U, Gonnella R, et al. Latent membrane protein 1 of Epstein-Barr virus mimics a constitutively active receptor molecule[J]. EMBO J,1997,16(20):6131-6140.
[15]Hatzivassiliou E, Miller WE, Raab-Traub N, et al. A fusion of the EBV latent membrane protein-1 (LMP1) transmembrane domains to the CD40 cytoplasmic domain is similar to LMP1 in constitutive activation of epidermal growth factor receptor expression, nuclear factor-kappa B, and stress-activated protein kinase[J]. J Immunol,1998,160(3):1116-1121.
[16]Liebowitz D, Wang D, Kieff E. Orientation and patching of the latent infection membrane protein encoded by Epstein-Barr virus[J]. J Virol, 1986,58(1):233-237.
[17]Izumi KM, Kaye KM, Kieff ED. The Epstein-Barr virus LMP1 amino acid sequence that engages tumor necrosis factor receptor associated factors is critical for primary B lymphocyte growth transformation[J]. Proc Natl Acad Sci U S A,1997,94(4):1447-1452.
[18]Izumi KM, Kieff ED. The Epstein-Barr virus oncogene product latent membrane protein 1 engages the tumor necrosis factor receptor-associated death domain protein to mediate B lymphocyte growth transformation and activate NF-kappaB[J]. Proc Natl Acad Sci U S A,1997,94(23): 12592-12597.
[19]Kaye KM, Izumi KM, Mosialos G, et al. The Epstein-Barr virus LMP1 cytoplasmic carboxy terminus is essential for B-lymphocyte transformation; fibroblast cocultivation complements a critical function within the terminal 155 residues. J Virol,1995,69(2):675-683.
[20]Brodeur SR, Cheng G, Baltimore D, et al. Localization of the major NF-kappaB-activating site and the sole TRAF3 binding site of LMP-1 defines two distinct signaling motifs[J]. J Biol Chem,1997,272(32): 19777-19784.
[21]Devergne O, Hatzivassiliou E, Izumi KM, et al. Association of TRAF1, TRAF2, and TRAF3 with an Epstein-Barr virus LMP1 domain important for B-lymphocyte transformation:role in NF-kappaB activation[J]. Mol Cell Biol,1996,16(12):7098-9108.
[22]Eliopoulos AG, Blake SM, Floettmann JE, et al. Epstein-Barr virus-encoded latent membrane protein 1 activates the JNK pathway through its extreme C terminus via a mechanism involving TRADD and TRAF2[J]. J Virol,1999,73(2):1023-1035.
[23]Eliopoulos AG, Gallagher NJ, Blake SM, et al. Activation of the p38 mitogen-activated protein kinase pathway by Epstein-Barr virus-encoded latent membrane protein 1 coregulates interleukin-6 and interleukin-8 production[J]. J Biol Chem,1999,274(23):16085-16096.
[24]Eliopoulos AG, Young LS. Activation of the cJun N-terminal kinase (JNK) pathway by the Epstein-Barr virus-encoded latent membrane protein 1 (LMP1). Oncogene,1998,16(13):1731-1742.
[25]Izumi KM, Cahir McFarland ED, Ting AT, et al. The Epstein-Barr virus oncoprotein latent membrane protein 1 engages the tumor necrosis factor receptor-associated proteins TRADD and receptor-interacting protein (RIP) but does not induce apoptosis or require RIP for NF-kappaB activation[J]. Mol Cell Biol,1999,19(8):5759-5767.
[26]Mosialos G, Birkenbach M, Yalamanchili R, et al. The Epstein-Barr virus transforming protein LMP1 engages signaling proteins for the tumor necrosis factor receptor family[J]. Cell,1995,80(3):389-399.
[27]Cahir-McFarland ED, Carter K, Rosenwald A, et al. Role of NF-kappa B in cell survival and transcription of latent membrane protein 1-expressing or Epstein-Barr virus latency Ⅲ-infected cells[J]. J Virol,2004, 78(8):4108-4119.
[28]Cameron JE, Yin QY, Fewell C, et al. Epstein-Barr Virus Latent Membrane Protein 1 Induces Cellular MicroRNA miR-146a, a Modulator of Lymphocyte Signaling Pathways[J]. J of virology,2008,82(4): 1946-1958.
[29]Kluiver J, Poppema S, de Jong D, et al. BIC and miR-155 are highly expressed in Hodgkin, primary mediastinal and diffuse large B cell lymphomas. J Pathol,2005,207(2):243-249.
[30]Li W, Ruan K. MicroRNA detection by microarray[J]. Anal Bioanal Chem, 2009,394(4):1117-1124.
[31]Liu CQ Spizzo R, Calin GA, et al. Expression profiling of microRNA using oligo DNA arrays[J]. Methods,2008,44(1):22-30.
[32]Gaidatzis D, van Nimwegen E, Hausser J, et al. Inference of miRNA targets using evolutionary conservation and pathway analysis[J]. BMC Bioinformatics,2007,8:69.
[33]Gusev Y, Schmittgen TD, Lerner M, et al. Computational analysis of biological functions and pathways collectively targeted by co-expressed microRNAs in cancer[J]. BMC Bioinformatics,2007,8(Suppl7):S16.
[34]Alexiou P, Maragkakis M, Papadopoulos GL, et al. The DIANA-mirExTra web server:from gene expression data to microRNA function[J]. PLoS One,2010,5(2):e9171.
[35]Pushparaj PN, Aarthi JJ, Manikandan J, et al. siRNA, miRNA, and shRNA:in vivo applications[J]. J Dent Res,2008,87(11):992-1003.
[36]Lo AK, Lo KW, Tsao SW, et al. Epstein-Barr virus infection alters cellular signal cascades in human nasopharyngeal epithelial cells[J]. Neoplasia, 2006,8(3):173-80.
[1]Lagos-Quintana M, Rauhut R, Lendeckel W, et al.Identification of novel genes coding for small expressed RNAs[J]. Science,2001,294(5543): 853-858.
[2]Fabbri M. MicroRNAs and cancer epigenetics[J]. Current Opinion in Investigational Drugs,2008,9(6):583-590
[3]Izumi KM, Kaye KM, Kieff ED. The Epstein-Barr virus LMP1 amino acid sequence that engages tumor necrosis factor receptor associated factors is critical for primary B lymphocyte growth transformation[J]. Proc Natl Acad Sci U S A,1997,94(4):1447-1452.
[4]Izumi KM, Kieff ED. The Epstein-Barr virus oncogene product latent membrane protein 1 engages the tumor necrosis factor receptor-associated death domain protein to mediate B lymphocyte growth transformation and activate NF-kappaB[J]. Proc Natl Acad Sci U S A,1997, 94(23):12592-12597.
[5]Brodeur SR, Cheng G, Baltimore D, et al. Localization of the major NF-kappaB-activating site and the sole TRAF3 binding site of LMP-1 defines two distinct signaling motifs[J]. J Biol Chem,1997, 272(32):19777-19784.
[6]Devergne O, Hatzivassiliou E, Izumi KM, et al. Association of TRAF1, TRAF2, and TRAF3 with an Epstein-Barr virus LMP1 domain important for B-lymphocyte transformation:role in NF-kappaB activation[J]. Mol Cell Biol,1996,16(12):7098-9108.
[7]Eliopoulos AG, Blake SM, Floettmann JE, et al. Epstein-Barr virus-encoded latent membrane protein 1 activates the JNK pathway through its extreme C terminus via a mechanism involving TRADD and TRAF2[J]. J Virol,1999,73(2):1023-1035.
[8]Eliopoulos AG, Gallagher NJ, Blake SM, et al. Activation of the p38 mitogen-activated protein kinase pathway by Epstein-Barr virus-encoded latent membrane protein 1 coregulates interleukin-6 and interleukin-8 production[J]. J Biol Chem,1999,274(23):16085-16096.
[9]Eliopoulos AG, Young LS. Activation of the cJun N-terminal kinase (JNK) pathway by the Epstein-Barr virus-encoded latent membrane protein 1 (LMP1). Oncogene,1998,16(13):1731-1742.
[10]Izumi KM, Cahir McFarland ED, Ting AT, et al. The Epstein-Barr virus oncoprotein latent membrane protein 1 engages the tumor necrosis factor receptor-associated proteins TRADD and receptor-interacting protein (RIP) but does not induce apoptosis or require RIP for NF-kappaB activation[J]. Mol Cell Biol,1999,19(8):5759-5767.
[11]Mosialos G, Birkenbach M, Yalamanchili R, et al. The Epstein-Barr virus transforming protein LMP1 engages signaling proteins for the tumor necrosis factor receptor family[J]. Cell,1995,80(3):389-399.
[12]Lo AK, Liu Y, Wang XH, et al. Alterations of biologic properties and gene expression in nasopharyngeal epithelial cells by the Epstein-Barr virus-encoded latent membrane protein 1[J]. Lab Invest,2003, 83(5):697-709.
[13]Gatto G, Rossi A, Rossi D, et al. Epstein-Barr virus latent membrane protein 1 trans-activates miR-155 transcription through the NF-kappaB pathway[J]. Nucleic Acids Res,2008,36(20):6608-19.
[14]Motsch N, Pfuhl T, Mrazek J, et al. Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) induces the expression of the cellular microRNA miR-146a[J]. RNA Biol,2007,4(3):131-137.
[15]Wong QW, Lung RW, Law PT, et al. MicroRNA-223 is commonly repressed in hepatocellular carcinoma and potentiates expression of Stathmin1 [J]. Gastroenterology,2008,135(1):257-269.
[16]Gramantieri L, Ferracin M, Fornari F, et al. Cyclin G1 is a target of miR-122a, a microRNA frequently down-regulated in human hepatocellular carcinoma[J]. Cancer Res,2007,67(13):6092-6099.
[17]Wang X, Lam EK, Zhang J, et al. MicroRNA-122a functions as a novel tumor suppressor downstream of adenomatous polyposis coli in gastrointestinal cancers [J]. Biochem Biophys Res Commun,2009, 387(2):376-380.
[18]Ota A, Tagawa H, Karnan S, et al. Identification and characterization of a novel gene, C13orf25, as a target for 13q31-q32 amplification in malignant lymphoma. Cancer Res,2004,64(9):3087-3095.
[19]Hayashita Y, Osada H, Tatematsu Y, et al. A polycistronic microRNA cluster, miR-17-92, is overexpressed in human lung cancers and enhances cell proliferation[J]. Cancer Res.2005; 65(21):9628-9632.
[20]Takakura S, Mitsutake N, Nakashima M, et al. Oncogenic role of miR-17-92 cluster in anaplastic thyroid cancer cells[J]. Cancer Sci,2008, 99(6):1147-1154.
[21]Pichiorri F, Suh SS, Ladetto M, et al. MicroRNAs regulate critical genes associated with multiple myeloma pathogenesis[J]. Proc Natl Acad Sci USA,2008,105(35):12885-12890.
[22]Connolly E, Melegari M, Landgraf P, et al. Elevated expression of the miR-17-92 polycistron and miR-21 in hepadnavirus-associated hepatocellular carcinoma contributes to the malignant phenotype[J]. Am J Pathol,2008,173(3):856-864.
[23]Shao J, Li Y, Wu Q, et al. High frequency loss of heterozygosity on the long arms of chromosomes 13 and 14 in nasopharyngeal carcinoma in Southern China. Chin Med J (Engl).2002,115(4):571-575.
[24]Chen HC, Chen GH, Chen YH, et al. MicroRNA deregulation and pathway alterations in nasopharyngeal carcinoma. Br J Cancer,2009, 100(6):1002-1011.
[25]Gregory PA, Bert AG, Paterson EL, et al. The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1[J]. Nat Cell Biol.2008,10(5):593-601.
[26]Iorio MV, Casalini P, Piovan C, et al. microRNA-205 regulates HER3 in human breast cancer[J]. Cancer Res,2009,69(6):2195-2200.
[27]Yu J, Ryan DG, Getsios S, et al. MicroRNA-184 antagonizes microRNA-205 to maintain SHIP2 levels in epithelia[J]. Proc Natl Acad Sci USA,2008,105(49):19300-19305.
[28]Wu H, Zhu S, Mo YY. Suppression of cell growth and invasion by miR-205 in breast cancer[J]. Cell Res,2009,19(4):439-448.
[29]Fulci V, Chiaretti S, Goldoni M, et al. Quantitative technologies establish a novel microRNA profile of chronic lymphocytic leukemia[J]. Blood, 2007,109(11):4944-4951.
[30]Wong TS, Liu XB, Wong BY, et al. Mature miR-184 as Potential Oncogenic microRNA of Squamous Cell Carcinoma of Tongue[J]. Clin Cancer Res.2008,14(9):2588-2592.
[31]Mi S, Lu J, Sun M,et al. MicroRNA expression signatures accurately discriminate acute lymphoblastic leukemia from acute myeloid leukemia[J]. Proc Natl Acad Sci U S A.2007,104(50):19971-19976.
[32]李刚,李湘平,彭英,等.应用RNA干扰抑制EB病毒潜伏膜蛋白-1表达对鼻咽癌细胞生长的影响(英文)[J].第一军医大学学报,2004,(03)：241-246
[33]欧小波,陈小毅,吴民华,等.EB病毒LMP1对人鼻咽癌细胞转移能力的影响[J].癌症,2008,27(8)：803-808.
[34]Li W, Ruan K. MicroRNA detection by microarray. Anal Bioanal Chem, 2009,394(4):1117-1124.
[35]Castoldi M, Schmidt S, Benes V, et al. A sensitive array for microRNA expression profiling (miChip) based on locked nucleic acids (LNA)[J]. RNA,2006,12(5):913-920.
[1]Chen HC, Chen GH, Chen YH, et al. MicroRNA deregulation and pathway alterations in nasopharyngeal carcinoma[J]. Br J Cancer,2009, 100(6):1002-1011.
[2]Alexiou P, Maragkakis M, Papadopoulos GL, et al. The DIANA-mirExTra web server:from gene expression data to microRNA function[J]. PLoS One,2010,5(2):e9171.
[3]Gaidatzis D, van Nimwegen E, Hausser J, et al. Inference of miRNA targets using evolutionary conservation and pathway analysis[J]. BMC Bioinformatics,2007,8:69.
[4]Gusev Y, Schmittgen TD, Lerner M, et al. Computational analysis of biological functions and pathways collectively targeted by co-expressed microRNAs in cancer[J]. BMC Bioinformatics,2007,8(Suppl7):S16.
[5]Papadopoulos GL, Alexiou P, Maragkakis M, et al. DIANA-mirPath: Integrating human and mouse microRNAs in pathways[J]. Bioinformatics, 2009,25(15):1991-1993.
[6]Inui M, Martello G, Piccolo S. MicroRNA control of signal transduction[J]. Nat Rev Mol Cell Biol,2010,[Epub ahead of print]
[7]Ota A, Tagawa H, Karnan S, et al. Identification and characterization of a novel gene, C13orf25, as a target for 13q31-q32 amplification in malignant lymphoma[J]. Cancer Res,2004,64(9):3087-3095.
[8]Hayashita Y, Osada H, Tatematsu Y, et al. A polycistronic microRNA cluster, miR-17-92, is overexpressed in human lung cancers and enhances cell proliferation[J]. Cancer Res.2005; 65(21):9628-9632.
[9]Takakura S, Mitsutake N, Nakashima M, et al. Oncogenic role of miR-17-92 cluster in anaplastic thyroid cancer cells[J]. Cancer Sci,2008, 99(6):1147-1154.
[10]Pichiorri F, Suh SS, Ladetto M, et al. MicroRNAs regulate critical genes associated with multiple myeloma pathogenesis[J]. Proc Natl Acad Sci USA,2008,105(35):12885-12890.
[11]Connolly E, Melegari M, Landgraf P, et al. Elevated expression of the miR-17-92 polycistron and miR-21 in hepadnavirus-associated hepatocellular carcinoma contributes to the malignant phenotype[J]. Am J Pathol,2008,173(3):856-864.
[1]Hayashita Y, Osada H, Tatematsu Y, et al. A polycistronic microRNA cluster, miR-17-92, is overexpressed in human lung cancers and enhances cell proliferation[J]. Cancer Res.2005; 65(21):9628-9632.
[2]Alexander WS, Starr R, Metcalf D, et al. Suppressors of cytokine signaling (SOCS):negative regulators of signal transduction[J]. J Leukoc Biol,1999,66(4):588-592.
[3]Manfred W, Marcus S. X-tremeGENE siRNA transfection reagent:a powerful tool for antisense inhibition of miRNA in Human cells with miRCURY knockdown probes[J]. Biochemia,2006,2(1):28-30.
[4]张振武,安洋,滕春波.miR-17-92基因簇microRNAs对哺乳动物器官发育及肿瘤发生的调控[J].遗传,2009,31(11)：1094-1100.
[5]Pichiorri F, Suh SS, Ladetto M, et al. MicroRNAs regulate critical genes associated with multiple myeloma pathogenesis[J]. Proc Natl Acad Sci USA,2008,105(35):12885-12890.
[6]Connolly E, Melegari M, Landgraf P, et al. Elevated expression of the miR-17-92 polycistron and miR-21 in hepadnavirus-associated hepatocellular carcinoma contributes to the malignant phenotype[J]. Am J Pathol,2008,173(3):856-864.
[7]Hackl M, Brunner S, Fortschegger K, et al. miR-17, miR-19b, miR-20a and miR-106a are down-regulated in human aging[J]. Aging Cell.2010 Jan 18. [Epub ahead of print]
[8]Todoerti K, Barbui V, Pedrini O, et al. Pleiotropic anti-myeloma activity of ITF2357:inhibition of interleukin-6 receptor signaling and repression of miR-19a and miR-19b[J]. Haematologica,2010,95(2):260-269.
[9]Smith KA, Griffin JD. Following the cytokine signaling pathway to leukemogenesis:a chronology[J]. J Clin Invest,2008,118(11):3564-3573.
[10]Takeda K, Noguchi K, Shi W, et al. Targeted disruption of the mouse Stat3 gene leads to early embryonic lethality[J]. Proc Natl Acad Sci U S A, 1997,94(8):3801-3804.
[11]Yang XO, Panopoulos AD, Nurieva R, et al. STAT3 regulates cytokine-mediated generation of inflammatory helper T cells[J]. J Biol Chem,2007,282(13):9358-9363.
[12]Alvarez JV, Greulich H, Sellers WR, et al. Signal transducer and activator of transcription 3 is required for the oncogenic effects of non-small-cell lung cancer-associated mutations of the epidermal growth factor receptor[J]. Cancer Res,2006,66(6):3162-3268.
[13]Yin W, Cheepala S, Roberts JN, et al. Active Stat3 is required for survival of human squamous cell carcinoma cells in serum-free conditions [J]. Mol Cancer,2006,5:15.
[14]Kusaba T, Nakayama T, Yamazumi K, et al. Activation of STAT3 is a marker of poor prognosis in human colorectal cancer[J]. Oncol Rep,2006, 15(6):1445-1451.
[15]Akira S, Nishio Y, Inoue M, et al. Molecular cloning of APRF, a novel IFN-stimulated gene factor 3 p91-related transcription factor involved in the gp130-mediated signaling pathway [J]. Cell,1994,77(1):63-71.
[16]Daeipour M, Kumar G, Amaral MC, et al. Recombinant IL-6 activates p42 and p44 mitogen-activated protein kinases in the IL-6 responsive B cell line, AF-10[J]. J Immunol,1993,150(11):4743-4753.
[17]Takeda K, Kaisho T, Yoshida N, et al. Stat3 activation is responsible for IL-6-dependent T cell proliferation through preventing apoptosis:
generation and characterization of T cell-specific Stat3-deficient mice[J]. J Immunol,1998,161(9):4652-4660.
[18]Chen RH, Chang MC, Su YH, et al. Interleukin-6 inhibits transforming growth factor-beta-induced apoptosis through the phosphatidylinositol 3-kinase/Akt and signal transducers and activators of transcription 3 pathways[J]. J Biol Chem,1999,274(33):23013-23019.
[19]Catlett-Falcone R, Landowski TH, Oshiro MM, et al. Constitutive activation of Stat3 signaling confers resistance to apoptosis in human U266 myeloma cells[J]. Immunity,1999,10(1):105-115.
[20]Hilbert DM, Migone TS, Kopf M, et al. Distinct tumorigenic potential of abl and raf in B cell neoplasia:abl activates the IL-6 signaling pathway[J]. Immunity,1996,5(1):81-89.
[21]Mziaut H, Kersting S, Knoch KP, et al. ICA512 signaling enhances pancreatic beta-cell proliferation by regulating cyclins D through STATs[J]. Proc Natl Acad Sci U S A,2008,105(2):674-679.
[22]Endo TA, Masuhara M, Yokouchi M, et al. A new protein containing an SH2 domain that inhibits JAK kinases[J]. Nature,1997,387(6636): 921-924.
[23]Naka T, Narazaki M, Hirata M, et al. Structure and function of a new STAT-induced STAT inhibitor[J]. Nature,1997,387(6636):924-929.
[24]Nicholson SE, Willson TA, Farley A, et al. Mutational analyses of the SOCS proteins suggest a dual domain requirement but distinct mechanisms for inhibition of LIF and IL-6 signal transduction[J]. EMBO J, 1999,18:375-385.
[25]Tischoff I, Hengge UR, Vieth M, et al. Methylation of SOCS-3 and SOCS-1 in the carcinogenesis of Barrett's adenocarcinoma[J]. Gut,2007, 56(8):1047-1053.
[26]Komazaki T, Nagai H, Emi M, et al. Hypermethylation-associated inactivation of the SOCS-1 gene, a JAK/STAT inhibitor, in human pancreatic cancers[J]. Jpn J Clin Oncol,2004,34(4):191-194.
[27]Galm O, Yoshikawa H, Esteller M, et al. SOCS-1, a negative regulator of cytokine signaling, is frequently silenced by methylation in multiple myeloma[J]. Blood,2003,101(7):2784-2788.
[28]Angela KF, Kwok WL, Sai WT, et al. Epstein-Barr virus infection alters cellular signal cascades in human nasopharyngeal epithelial cells[J]. Neoplasia,2006,8(3):173-180.
[29]杨荣宁,吴冬梅.CD62p、IL-6、IL-8的表达与鼻咽癌分期及预后关系的探讨[J].2009,17(2)：231-234
[30]Pichiorri F, Suh SS, Ladetto M, et al. MicroRNAs regulate critical genes associated with multiple myeloma pathogenesis[J]. Proc Natl Acad Sci U S A,2008,105(35):12885-12890.
[31]Papakonstanti EA, Stournaras C. Cell responses regulated by early reorganization of actin cytoskeleton[J]. FEBS Lett.2008 Jun 18; 582(14): 2120-2127
[32]Wendt MK, Allington TM, Schiemann WP. Mechanisms of the epithelial-mesenchymal transition by TGF-beta[J]. Future Oncol,2009, 5(8):1145-1168.
[1]闵华庆,洪旺晃,马骏,等.鼻咽癌新分期的研究[J].癌症,1992,11(4)：290-294.
[2]刘泰福,徐国镇.全国鼻咽癌会议纪要[J].中华放射肿瘤学杂志,1992,1(3)：204.
[3]Stiller CA. International variations in the incidence of childhood carcinomas[J]. Cancer Epidemiol Biomarkers Prev,1994,3(4):305-310.
[4]Lo YM, Chan LY, Lo KW, et al. Quantitative analysis of cell-free Epstein-Barr virus DNA in plasma of patients with nasopharyngeal carcinoma[J]. Cancer Res,1999,59(6):1188-1191.
[5]Lo YM, Leung SF, Chan LY, et al. Kinetics of plasma Epstein-Barr virus DNA during radiation therapy for nasopharyngeal carcinoma[J]. Cancer Res,2000,60(9):2351-2355.
[6]Lo YM, Chan LY, Chan AT, et al. Quantitative and temporal correlation between circulating cell-free Epstein-Barr virus DNA and tumor recurrence in nasopharyngeal carcinoma[J]. Cancer Res,1999,59(21): 5452-5455.
[7]Kim KR, Yoshizake T, Miyamori H, et al. Transformation of Madin darby canine kidney (MDCK) epithelial cell by Epstein-Barr virus latent membrane protein 1 (LMP1) induces expression of Ets1 and invasive growth[J]. Oncogene,2000,19(14):1764-1777.
[8]Wang D, Liebowitz D, Kieff E. An EBV membrane protein expressed in immortalized lymphocytes transforms established rodent cells[J]. Cell, 1985,43(3 Pt 2):831-840.
[9]Izumi KM, Kaye KM, Kieff ED. The Epstein-Barr virus LMP1 amino acid sequence that engages tumor necrosis factor receptor associated factors is critical for primary B lymphocyte growth transformation[J]. Proc Natl Acad Sci U S A,1997,94(4):1447-1452.
[10]Mosialos G, Birkenbach M, Yalamanchili R, et al. The Epstein-Barr virus transforming protein LMP1 engages signaling for the tumor necrosis factor receptor family. Cell,1995,80(3):389-399.
[11]Lo AK, Liu Y, Wang XH, et al. Alterations of biologic properties and gene expression in nasopharyngeal epithelial cells by the Epstein-Barr virus-encoded latent membrane protein 1[J]. Lab Invest,2003, 83(5):697-709.
[12]Li XP, Li G, Peng Y, et al. Suppression of Epstein-Barr virus-encoded latent membrane protein-1 by RNA interference inhibits the metastatic potential of nasopharyngeal carcinoma cells[J]. Biochem Biophys Res Commun,2004,315(1):212-218.
[13]姜武忠,赵素萍,廖遇平,等.鼻咽癌组织中LMP1和VEGF蛋白表达及其临床意义[J].中国耳鼻咽喉颅底外科杂志,2007,13(3)：174-177.
[14]Lagos-Quintana M, Rauhut R, Lendeckel W, et al.Identification of novel genes coding for small expressed RNAs[J]. Science,2001,294(5543): 853-858.
[15]Cho WC. OncomiRs:the discovery and progress of microRNAs in cancers[J]. Mol Cancer,2007,6(1):60-67.
[16]Chen HC, Chen GH, Chen YH, et al. MicroRNA deregulation and pathway alterations in nasopharyngeal carcinoma. Br J Cancer,2009, 100(6):1002-1011.
[17]Sengupta S, den Boon JA, Chen IH, et al. MicroRNA 29c is down-regulated in nasopharyngeal carcinomas, up-regulating mRNAs encoding extracellular matrix proteins[J]. Proc Natl Acad Sci U S A,2008, 105(15):5874-5878.
[18]Hayashita Y, Osada H, Tatematsu Y, et al. A polycistronic microRNA cluster, miR-17-92, is overexpressed in human lung cancers and enhances cell proliferation[J]. Cancer Res.2005; 65(21):9628-9632.
[19]Pichiorri F, Suh SS, Ladetto M, et al. MicroRNAs regulate critical genes associated with multiple myeloma pathogenesis[J]. Proc Natl Acad Sci USA,2008,105(35):12885-12890.
[20]Connolly E, Melegari M, Landgraf P, et al. Elevated expression of the miR-17-92 polycistron and miR-21 in hepadnavirus-associated hepatocellular carcinoma contributes to the malignant phenotype[J]. Am J Pathol,2008,173(3):856-864.
[21]Ma L, Young J, Prabhala H, et al. miR-9, a MYC/MYCN-activated microRNA, regulates E-cadherin and cancer metastasis [J]. Nat Cell Biol, 2010 Feb 21. [Epub ahead of print]
[22]Cameron JE, Yin Q, Fewell C, et al. Epstein-Barr Virus Latent Membrane Protein 1 Induces Cellular MicroRNA miR-146a, a Modulator of Lymphocyte Signaling Pathways[J]. J Virology,2008,82(4):1946-1958.
[1]Bartel DP. MicroRNAs:genomics, biogenesis, mechanism, and function[J]. Cell,2004,116(2):281-297.
[2]Llave C, Xie Z, Kasschau KD, et al. Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA[J]. Science,2002, 297(5589):2053-2056.
[3]Berezikov E, Guryev V, van de Belt J, et al. Phylogenetic shadowing and computational identification of human microRNA genes[J]. Cell,2005, 120(1):21-24.
[4]Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets[J]. Cell,2005,120(1):15-20.
[5]Kloosterman WP, Plasterk RH. The diverse functions of microRNAs in animal development and disease[J]. Dev Cell,2006,11(4):441-450.
[6]Lee RC, Feinbaum RL, Ambros V. The C.elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[J]. Cell,1993,75(5):843-854
[7]Wightman B, Ha I, Ruvkun G. Posttranscriptional regulation of theheterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans[J]. Cell,1993,75(5):855-862.
[8]Reinhart BJ, Frank J, Michael B. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans[J]. Nature,2000, 403(6772):901-906.
[9]Lau NC, Lim LP, Weinstein EG, et al. An abundant class of tiny RNAs with probableregulatory roles in Caenorhabditis elegans[J]. Science,2001, 294(5543):858-862.
[10]Slack FJ, Basson M, Liu Z, et al. The lin-41 RBCC gene acts in the C. elegans heterochronic pathway between the let-7 regulatory RNA and the lin-29 transcription factor[J]. Mol Cell,2000,5(4):659-669.
[11]Pasquinelli AE, Ruvkun G. Control of developmental timing by microRNAs and their targets[J]. Annu Rev Cell Dev Biol,2002,18:495-513.
[12]Abrahante JE, Daul AL, Li M, et al. The Caenorhabditis elegans hunchback-like gene lin-57/hbl-1 controls developmental time and is regulated by microRNAs[J]. Dev Cell,2003,4(5):625-637.
[13]Lin SY, Johnson SM, Abraham M, et al.The C elegans hunchback homolog, hbl-1, controls temporal patterning and is a probable microRNA target[J], Dev Cell,2003,4(5):639-650.
[14]Brennecke J, Hipfner DR, Stark A, et al. bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila[J]. Cell,2003, 113(1):25-36.
[15]Pillai RS, Bhattacharyya SN, Artus CG, et al. Inhibition of translational initiation by Let-7 MicroRNA in human cells[J]. Science,2005, 309(5740):1573-1576.
[16]Bagga S, Bracht J, Hunter S, et al.Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation [J]. Cell,2005,122(4):553-563.
[17]Cai X, Hagedorn CH, Cullen BR. Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs [J]. RNA,2004,10(12):1957-1966.
[18]Denli AM, Tops BB, Plasterk RH, et al. Processing of primary microRNAs by the Microprocessor complex[J]. Nature,2004, 432(7014):231-235.
[19]Lund E, Guttinger S, Calado A, et al. Nuclear export of microRNA precursors[J]. Science,2004,303(5654):95-98.
[20]Sempere LF, Sokol NS, Dubrovsky EB, et al. Temporal regulation of microRNA expression in Drosophila melanogaster mediated by hormonal signals and Broad-Complex gene activity[J]. Dev Biol,2003,259(1):9-18.
[21]Rodriguez A, Griffiths-Jones S, Ashurst JL, et al. Identification of mammalian microRNA host genes and transcription units[J]. Genome Res, 2004,14(1A):1902-1910.
[22]Baskerville S, Bartel DP. Microarray profiling of microRNAs reveals frequent coexpression with neighboring miRNAs and host genes [J]. RNA, 2005, 11(3):241-247.
[23]Khvorova A, Reynolds A, Jayasena SD. Functional siRNAs and miRNAs exhibit strand bias. Cell,2003,115(2):209-216.
[24]Schwarz DS, Hutvagner G, Du T et al. Asymmetry in the assembly of the RNAi enzyme complex[J]. Cell,2003,115(2):199-208.
[25]Lim LP, Lau NC, Weinstein EG, et al. The microRNAs of Caenorhabditis elegans[J]. Genes Dev,2003,17(8):991-1008.
[26]Lim LP, Glasner ME, Yekta S, et al. Vertebrate microRNA genes[J]. Science,2003,299(5612):1540.
[27]Grosshans H, Johnson T, Reinert KL, et al. The temporal patterning microRNA let-7 regulates several transcription factors at the larval to adult transition in C. elegans[J]. Dev Cell,2005,8(3):321-330.
[28]Krek A, Grun D, Poy MN, et al. Combinatorial microRNA target predictions[J]. Nature Genet,2005,37(5):495-500.
[29]Karube Y, Tanaka H, Osada H, et al. Reduced expression of Dicer associated with poor prognosis in lung cancer patients [J]. Cancer Sci, 2005,96(2):111-115.
[30]Kanellopoulou C, Muljo SA, Kung AL, et al. Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing [J]. Genes Dev,2005,19(4):489-501.
[31]Bentwich I, Avniel A, Karov Y, et al. Identification of hundreds of conserved and nonconserved human microRNAs [J]. Nat Genet, 2005,37(7):766-770.
[32]Wienholds E, Kloosterman WP, Miska E, et al. MicroRNA expression in zebrafish embryonic development[J]. Science,2005,309(5732):310-311.
[33]Rajewsky N. microRNA target predictions in animals [J]. Nat Genet, 2006,38(Suppl):S8-13.
[34]Li Y, Wang F, Lee JA, et al. MicroRNA-9a ensures the precise specification of sensory organ precursors in Drosophila[J]. Genes Dev, 2006,20(20):2793-2805.
[35]Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signaling:cell fate control and signal integration in development [J]. Science,1999, 284(5415):770-776.
[36]Vella MC, Choi EY, Lin SY, et al. The C. elegans microRNA let-7 binds to imperfect let-7 complementary sites from the lin-41 3'UTR. Genes Dev, 2004,18(2):132-137.
[37]Yang W, Chendrimada TP, Wang Q, et al. Modulation of microRNA processing and expression through RNA editing by ADAR deaminases[J]. Nat Struct Mol Biol,2006,13(1):13-21.
[38]Grosshans H, Johnson T, Reinert KL, et al. The temporal patterning microRNA let-7 regulates several transcription factors at the larval to adult transition in C. elegans[J]. Dev Cell,2005,8(3):321-330.
[39]Lin SY, Johnson SM, Abraham M, et al. The C elegans hunchback homolog, hb1-1, controls temporal patterning and is a probable microRNA target[J]. Dev Cell,2003,4(5):639-650.
[40]Rosenberg MI, Georges SA, Asawachaicharn A, et al. MyoD inhibits Fstll and Utrn expression by inducing transcription of miR-206[J]. J Cell Biol,2006,175(1):77-85.
[41]Rao PK, Kumar RM, Farkhondeh M, et al. Myogenic factors that regulate expression of muscle-specific microRNAs[J]. Proc Natl Acad Sci USA, 2006,103(23):8721-8726.
[42]Obernosterer G, Leuschner PJ, Alenius M, et al. Post-transcriptional regulation of microRNA expression[J]. RNA,2006,12(7):1161-1167.
[43]Thomson JM, Newman M, Parker JS, et al. Extensive post-transcriptional regulation of microRNAs and its implications for cancer[J]. Genes Dev, 2006,20(16):2202-2207.
[44]Davis BN, Hilyard AC, Lagna G,et al. SMAD proteins control DROSHA-mediated microRNA maturation[J]. Nature,2008,454(7200): 56-61.
[45]Han J, Lee Y, Yeom KH,et al. The Drosha-DGCR8 complex in primary microRNA processing[J]. Genes Dev,2004,18(24):3016-3027.
[46]Lehrbach NJ, Armisen J, Lightfoot HL, et al. LIN-28 and the poly(U) polymerase PUP-2 regulate let-7 microRNA processing in Caenorhabditis elegans[J]. Nat Struct Mol Biol,2009,16(10):1016-1020.
[47]Czech B, Zhou R, Erlich Y, et al. Hierarchical rules for Argonaute loading in Drosophila[J]. Mol Cell,2009,36(3):445-456.
[48]Kennedy S, Wang D, Ruvkun G. A conserved siRNA-degrading RNase negatively regulates RNA interference in C. elegans[J].Nature,2004, 427(6975):645-649.
[49]Ramachandran V, Chen X. Degradation of microRNAs by a family of exoribonucleases in Arabidopsis[J]. Science,2008,321(5895):1490-1492.
[50]Yang W, Chendrimada TP, Wang Q, et al. Modulation of microRNA processing and expression through RNA editing by ADAR deaminases[J]. Nat Struct Mol Biol,2006,13(1):13-21.
[51]Lee CT, Risom T, Strauss WM. MicroRNAs in mammalian development[J]. Birth Defects Res C Embryo Today,2006,78(2): 129-139.
[52]Moss EG. Heterochronic genes and the nature of developmental time[J]. Curr Biol,2007,17(11):R425-34.
[53]Bracken CP, Gregory PA, Kolesnikoff N, et al. A double-negative feedback loop between ZEB1-SIP1 and the microRNA-200 family regulates epithelial-mesenchymal transition[J]. Cancer Res,2008, 68(19):7846-7854.
[54]Calin GA, Croce CM. MicroRNA signatures in human cancers[J]. Nat Rev Cancer,2006,6(11):857-866.
[55]Calin GA, Dumitru CD, Shimizu M, et al. Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia[J]. Proc Natl Acad Sci USA,2002, 99(24):15524-15529.
[56]Calin GA, Sevignani C, Dumitru CD, et al. Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers[J]. Proc Natl Acad Sci USA,2004,101(9):2999-3004.
[57]Bottoni A, Piccin D, Tagliati F, et al. miR-15a and miR-16-1 down-regulation in pituitary adenomas[J]. J Cell Physiol,2005, 204(1):280-285.
[58]Cimmino A, Calin GA, Fabbri M, et al. miR-15 and miR-16 induce apoptosis by targeting BCL2[J]. Proc Natl Acad Sci USA,2005, 102(39):13944-13949.
[59]He L, Thomson JM, Hemann MT, et al. A microRNA polycistron as a potential human oncogene[J]. Nature,2005,435(7043):828-833.
[60]O'Donnell KA, Wentzel EA, Zeller KI, et al. c-Myc regulated microRNAs modulate E2F1 expression[J]. Nature,2005,435(7043):839-843.
[61]Woods K, Thomson JM, Hammond SM. Direct regulation of an oncogenic micro-RNA cluster by E2F transcription factors[J]. J Biol Chem,2007,282(4):2130-2134.
[62]Hayashita Y, Osada H, Tatematsu Y, et al. A polycistronic microRNA cluster, miR-17-92, is overexpressed in human lung cancers and enhances cell proliferation[J]. Cancer Res,2005,65(21):9628-9632.
[63]Matsubara H, Takeuchi T, Nishikawa E, et al. Apoptosis induction by antisense oligonucleotides against miR-17-5p and miR-20a in lung cancers overexpressing miR-17-92[J]. Oncogene,2007,26(41):6099- 6105.
[64]Iorio MV, Ferracin M, Liu CG, et al. MicroRNA gene expression deregulation in human breast cancer[J]. Cancer Res,2005,65(16): 7065-7070.
[65]Si ML, Zhu S, Wu H, et al. miR-21-mediated tumor growth[J]. Oncogene, 2007,26(19):2799-2803.
[66]Lu J, Getz G, Miska EA, et al. MicroRNA expression profiles classify human cancers[J]. Nature,2005,435(7043):834-838.
[67]Bottoni A, Zatelli MC, Ferracin M, et al. Identification of differentially expressed microRNAs by microarray:a possible role for microRNA genes in pituitary adenomas[J]. J Cell Physiol,2007,210(2):370-377.
[68]Fabbri M, Croce CM, Calin GA. MicroRNAs[J]. Cancer J,2008, 14(1):1-6.
[69]Takamizawa J, Konishi H, Yanagisawa K, et al. Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival[J]. Cancer Res,2004,64(11):3753-3756.
[70]Roldo C, Missiaglia E, Hagan JP, et al. MicroRNA expression abnormalities in pancreatic endocrine and acinar tumors are associated with distinctive pathologic features and clinical behavior[J]. J Clin Oncol, 2006,24(29):4677-4684.
[71]Bloomston M, Frankel WL, Petrocca F, et al. MicroRNA expression patterns to differentiate pancreatic adenocarcinoma from normal pancreas and chronic pancreatitis[J]. JAMA,2007,297(17):1901-1908.
[72]Raveche ES, Salerno E, Scaglione BJ, et al. Abnormal microRNA-16 locus with synteny to human 13q14 linked to CLL in NZB mice[J]. Blood, 2007,109(12):5079-5086.
[73]Volinia S, Calin GA, Liu CG, et al. A microRNA expression signature of human solid tumors defines cancer gene targets[J]. Proc Natl Acad Sci USA,2006,103(7):2257-2261.
[74]Ciafre SA, Galardi S, Mangiola A, et al. Extensive modulation of a set of microRNAs in primary glioblastoma[J]. Biochem Biophys Res Commun, 2005,334(4):1351-1358.
[75]Krichevsky AM, King KS, Donahue CP, et al. A microRNA array reveals extensive regulation of microRNAs during brain development[J]. RNA, 2003,9(),1274-1281.
[76]Wang T, Zhang X, Obijuru L, et al. A micro-RNA signature associated with race, tumor size, and target gene activity in human uterine leiomyomas[J]. Genes Chromosomes Cancer,2007,46(4):336-347.
[77]Meng F, Henson R, Lang M, et al. Involvement of human micro-RNA in growth and response to chemotherapy in human cholangiocarcinoma cell lines[J]. Gastroenterology,2007,130(7):2113-2129.
[78]Zhu S, Si ML, Wu H, et al. MicroRNA-21 targets the tumor suppressor gene tropomyosin 1 (TPM1). J Biol Chem,2007,282(19:14328-14336.
[2]Llave C, Xie Z, Kasschau KD, et al. Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA[J]. Science,2002, 297(5589):2053-2056.
[3]Aurora EK, Frank JS. Oncomirs-microRNAs with a role in cancer[J]. Nature Reviews Cancer,2006,6(4):259-269
[4]Cimmino A, Calin GA, Fabbri M, et al. miR-15 and miR-16 induce apoptosis by targeting BCL2[J]. Proc Natl Acad Sci USA,2005, 102(39):13944-13949.
[5]O'Donnell KA, Wentzel EA, Zeller KI, et al. c-Myc-regulated microRNAs modulate E2F1 expression[J]. Nature,2005,435(7043), 839-843.
[6]Si ML, Zhu S, Wu H, et al. miR-21-mediated tumor growth[J]. Oncogene, 2007,26(19):2799-2803.
[7]Liu CG, Calin GA, Meloon B, et al. An oligonucleotide microchip for genome-wide microRNA profiling in human and mouse tissues[J]. Proc Natl Acad Sci USA,2004,101(26):9740-9744.
[8]Calin GA, Dumitru CD, Shimizu M, et al. Frequent deletions and down-regulation of micro-RNA genes miR-15 and miR-16 at 13q14 in chronic lymphocytic leukemia[J]. Proc Natl Acad Sci USA,2002, 99(24):15524-15529.
[9]Raver-Shapira N, Marciano E, Meiri E, et al. Transcriptional activation of miR-34a contributes to p53-mediated apoptosis[J]. Mol Cell,2007, 26(5):731-743.
[10]Knipe DM, Howley PM, Griffin DE, et al. Fields virology[M]. Philadelphia PA:Lippincott Williams & Wilkins.2007,5th ed, vol 2: 2603-2654.
[11]Knipe DM, Howley PM, Griffin DE, et al. Fields virology[M]. Philadelphia PA:Lippincott Williams & Wilkins.2007,5th ed, vol 2: 2655-2700.
[12]Young LS, Murray PG. Epstein-Barr virus and oncogenesis:from latent genes to tumours[J]. Oncogene,2003,22(33):5108-5121.
[13]Kulwichit W, Edwards RH, Davenport EM, et al. Expression of the Epstein-Barr virus latent membrane protein 1 induces B cell lymphoma in transgenic mice[J]. Proc Natl Acad Sci U S A,1998,95(20):11963-11968.
[14]Gires O, Zimber-Strobl U, Gonnella R, et al. Latent membrane protein 1 of Epstein-Barr virus mimics a constitutively active receptor molecule[J]. EMBO J,1997,16(20):6131-6140.
[15]Hatzivassiliou E, Miller WE, Raab-Traub N, et al. A fusion of the EBV latent membrane protein-1 (LMP1) transmembrane domains to the CD40 cytoplasmic domain is similar to LMP1 in constitutive activation of epidermal growth factor receptor expression, nuclear factor-kappa B, and stress-activated protein kinase[J]. J Immunol,1998,160(3):1116-1121.
[16]Liebowitz D, Wang D, Kieff E. Orientation and patching of the latent infection membrane protein encoded by Epstein-Barr virus[J]. J Virol, 1986,58(1):233-237.
[17]Izumi KM, Kaye KM, Kieff ED. The Epstein-Barr virus LMP1 amino acid sequence that engages tumor necrosis factor receptor associated factors is critical for primary B lymphocyte growth transformation[J]. Proc Natl Acad Sci U S A,1997,94(4):1447-1452.
[18]Izumi KM, Kieff ED. The Epstein-Barr virus oncogene product latent membrane protein 1 engages the tumor necrosis factor receptor-associated death domain protein to mediate B lymphocyte growth transformation and activate NF-kappaB[J]. Proc Natl Acad Sci U S A,1997,94(23): 12592-12597.
[19]Kaye KM, Izumi KM, Mosialos G, et al. The Epstein-Barr virus LMP1 cytoplasmic carboxy terminus is essential for B-lymphocyte transformation; fibroblast cocultivation complements a critical function within the terminal 155 residues. J Virol,1995,69(2):675-683.
[20]Brodeur SR, Cheng G, Baltimore D, et al. Localization of the major NF-kappaB-activating site and the sole TRAF3 binding site of LMP-1 defines two distinct signaling motifs[J]. J Biol Chem,1997,272(32): 19777-19784.
[21]Devergne O, Hatzivassiliou E, Izumi KM, et al. Association of TRAF1, TRAF2, and TRAF3 with an Epstein-Barr virus LMP1 domain important for B-lymphocyte transformation:role in NF-kappaB activation[J]. Mol Cell Biol,1996,16(12):7098-9108.
[22]Eliopoulos AG, Blake SM, Floettmann JE, et al. Epstein-Barr virus-encoded latent membrane protein 1 activates the JNK pathway through its extreme C terminus via a mechanism involving TRADD and TRAF2[J]. J Virol,1999,73(2):1023-1035.
[23]Eliopoulos AG, Gallagher NJ, Blake SM, et al. Activation of the p38 mitogen-activated protein kinase pathway by Epstein-Barr virus-encoded latent membrane protein 1 coregulates interleukin-6 and interleukin-8 production[J]. J Biol Chem,1999,274(23):16085-16096.
[24]Eliopoulos AG, Young LS. Activation of the cJun N-terminal kinase (JNK) pathway by the Epstein-Barr virus-encoded latent membrane protein 1 (LMP1). Oncogene,1998,16(13):1731-1742.
[25]Izumi KM, Cahir McFarland ED, Ting AT, et al. The Epstein-Barr virus oncoprotein latent membrane protein 1 engages the tumor necrosis factor receptor-associated proteins TRADD and receptor-interacting protein (RIP) but does not induce apoptosis or require RIP for NF-kappaB activation[J]. Mol Cell Biol,1999,19(8):5759-5767.
[26]Mosialos G, Birkenbach M, Yalamanchili R, et al. The Epstein-Barr virus transforming protein LMP1 engages signaling proteins for the tumor necrosis factor receptor family[J]. Cell,1995,80(3):389-399.
[27]Cahir-McFarland ED, Carter K, Rosenwald A, et al. Role of NF-kappa B in cell survival and transcription of latent membrane protein 1-expressing or Epstein-Barr virus latency Ⅲ-infected cells[J]. J Virol,2004, 78(8):4108-4119.
[28]Cameron JE, Yin QY, Fewell C, et al. Epstein-Barr Virus Latent Membrane Protein 1 Induces Cellular MicroRNA miR-146a, a Modulator of Lymphocyte Signaling Pathways[J]. J of virology,2008,82(4): 1946-1958.
[29]Kluiver J, Poppema S, de Jong D, et al. BIC and miR-155 are highly expressed in Hodgkin, primary mediastinal and diffuse large B cell lymphomas. J Pathol,2005,207(2):243-249.
[30]Li W, Ruan K. MicroRNA detection by microarray[J]. Anal Bioanal Chem, 2009,394(4):1117-1124.
[31]Liu CQ Spizzo R, Calin GA, et al. Expression profiling of microRNA using oligo DNA arrays[J]. Methods,2008,44(1):22-30.
[32]Gaidatzis D, van Nimwegen E, Hausser J, et al. Inference of miRNA targets using evolutionary conservation and pathway analysis[J]. BMC Bioinformatics,2007,8:69.
[33]Gusev Y, Schmittgen TD, Lerner M, et al. Computational analysis of biological functions and pathways collectively targeted by co-expressed microRNAs in cancer[J]. BMC Bioinformatics,2007,8(Suppl7):S16.
[34]Alexiou P, Maragkakis M, Papadopoulos GL, et al. The DIANA-mirExTra web server:from gene expression data to microRNA function[J]. PLoS One,2010,5(2):e9171.
[35]Pushparaj PN, Aarthi JJ, Manikandan J, et al. siRNA, miRNA, and shRNA:in vivo applications[J]. J Dent Res,2008,87(11):992-1003.
[36]Lo AK, Lo KW, Tsao SW, et al. Epstein-Barr virus infection alters cellular signal cascades in human nasopharyngeal epithelial cells[J]. Neoplasia, 2006,8(3):173-80.
[1]Lagos-Quintana M, Rauhut R, Lendeckel W, et al.Identification of novel genes coding for small expressed RNAs[J]. Science,2001,294(5543): 853-858.
[2]Fabbri M. MicroRNAs and cancer epigenetics[J]. Current Opinion in Investigational Drugs,2008,9(6):583-590
[3]Izumi KM, Kaye KM, Kieff ED. The Epstein-Barr virus LMP1 amino acid sequence that engages tumor necrosis factor receptor associated factors is critical for primary B lymphocyte growth transformation[J]. Proc Natl Acad Sci U S A,1997,94(4):1447-1452.
[4]Izumi KM, Kieff ED. The Epstein-Barr virus oncogene product latent membrane protein 1 engages the tumor necrosis factor receptor-associated death domain protein to mediate B lymphocyte growth transformation and activate NF-kappaB[J]. Proc Natl Acad Sci U S A,1997, 94(23):12592-12597.
[5]Brodeur SR, Cheng G, Baltimore D, et al. Localization of the major NF-kappaB-activating site and the sole TRAF3 binding site of LMP-1 defines two distinct signaling motifs[J]. J Biol Chem,1997, 272(32):19777-19784.
[6]Devergne O, Hatzivassiliou E, Izumi KM, et al. Association of TRAF1, TRAF2, and TRAF3 with an Epstein-Barr virus LMP1 domain important for B-lymphocyte transformation:role in NF-kappaB activation[J]. Mol Cell Biol,1996,16(12):7098-9108.
[7]Eliopoulos AG, Blake SM, Floettmann JE, et al. Epstein-Barr virus-encoded latent membrane protein 1 activates the JNK pathway through its extreme C terminus via a mechanism involving TRADD and TRAF2[J]. J Virol,1999,73(2):1023-1035.
[8]Eliopoulos AG, Gallagher NJ, Blake SM, et al. Activation of the p38 mitogen-activated protein kinase pathway by Epstein-Barr virus-encoded latent membrane protein 1 coregulates interleukin-6 and interleukin-8 production[J]. J Biol Chem,1999,274(23):16085-16096.
[9]Eliopoulos AG, Young LS. Activation of the cJun N-terminal kinase (JNK) pathway by the Epstein-Barr virus-encoded latent membrane protein 1 (LMP1). Oncogene,1998,16(13):1731-1742.
[10]Izumi KM, Cahir McFarland ED, Ting AT, et al. The Epstein-Barr virus oncoprotein latent membrane protein 1 engages the tumor necrosis factor receptor-associated proteins TRADD and receptor-interacting protein (RIP) but does not induce apoptosis or require RIP for NF-kappaB activation[J]. Mol Cell Biol,1999,19(8):5759-5767.
[11]Mosialos G, Birkenbach M, Yalamanchili R, et al. The Epstein-Barr virus transforming protein LMP1 engages signaling proteins for the tumor necrosis factor receptor family[J]. Cell,1995,80(3):389-399.
[12]Lo AK, Liu Y, Wang XH, et al. Alterations of biologic properties and gene expression in nasopharyngeal epithelial cells by the Epstein-Barr virus-encoded latent membrane protein 1[J]. Lab Invest,2003, 83(5):697-709.
[13]Gatto G, Rossi A, Rossi D, et al. Epstein-Barr virus latent membrane protein 1 trans-activates miR-155 transcription through the NF-kappaB pathway[J]. Nucleic Acids Res,2008,36(20):6608-19.
[14]Motsch N, Pfuhl T, Mrazek J, et al. Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) induces the expression of the cellular microRNA miR-146a[J]. RNA Biol,2007,4(3):131-137.
[15]Wong QW, Lung RW, Law PT, et al. MicroRNA-223 is commonly repressed in hepatocellular carcinoma and potentiates expression of Stathmin1 [J]. Gastroenterology,2008,135(1):257-269.
[16]Gramantieri L, Ferracin M, Fornari F, et al. Cyclin G1 is a target of miR-122a, a microRNA frequently down-regulated in human hepatocellular carcinoma[J]. Cancer Res,2007,67(13):6092-6099.
[17]Wang X, Lam EK, Zhang J, et al. MicroRNA-122a functions as a novel tumor suppressor downstream of adenomatous polyposis coli in gastrointestinal cancers [J]. Biochem Biophys Res Commun,2009, 387(2):376-380.
[18]Ota A, Tagawa H, Karnan S, et al. Identification and characterization of a novel gene, C13orf25, as a target for 13q31-q32 amplification in malignant lymphoma. Cancer Res,2004,64(9):3087-3095.
[19]Hayashita Y, Osada H, Tatematsu Y, et al. A polycistronic microRNA cluster, miR-17-92, is overexpressed in human lung cancers and enhances cell proliferation[J]. Cancer Res.2005; 65(21):9628-9632.
[20]Takakura S, Mitsutake N, Nakashima M, et al. Oncogenic role of miR-17-92 cluster in anaplastic thyroid cancer cells[J]. Cancer Sci,2008, 99(6):1147-1154.
[21]Pichiorri F, Suh SS, Ladetto M, et al. MicroRNAs regulate critical genes associated with multiple myeloma pathogenesis[J]. Proc Natl Acad Sci USA,2008,105(35):12885-12890.
[22]Connolly E, Melegari M, Landgraf P, et al. Elevated expression of the miR-17-92 polycistron and miR-21 in hepadnavirus-associated hepatocellular carcinoma contributes to the malignant phenotype[J]. Am J Pathol,2008,173(3):856-864.
[23]Shao J, Li Y, Wu Q, et al. High frequency loss of heterozygosity on the long arms of chromosomes 13 and 14 in nasopharyngeal carcinoma in Southern China. Chin Med J (Engl).2002,115(4):571-575.
[24]Chen HC, Chen GH, Chen YH, et al. MicroRNA deregulation and pathway alterations in nasopharyngeal carcinoma. Br J Cancer,2009, 100(6):1002-1011.
[25]Gregory PA, Bert AG, Paterson EL, et al. The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1[J]. Nat Cell Biol.2008,10(5):593-601.
[26]Iorio MV, Casalini P, Piovan C, et al. microRNA-205 regulates HER3 in human breast cancer[J]. Cancer Res,2009,69(6):2195-2200.
[27]Yu J, Ryan DG, Getsios S, et al. MicroRNA-184 antagonizes microRNA-205 to maintain SHIP2 levels in epithelia[J]. Proc Natl Acad Sci USA,2008,105(49):19300-19305.
[28]Wu H, Zhu S, Mo YY. Suppression of cell growth and invasion by miR-205 in breast cancer[J]. Cell Res,2009,19(4):439-448.
[29]Fulci V, Chiaretti S, Goldoni M, et al. Quantitative technologies establish a novel microRNA profile of chronic lymphocytic leukemia[J]. Blood, 2007,109(11):4944-4951.
[30]Wong TS, Liu XB, Wong BY, et al. Mature miR-184 as Potential Oncogenic microRNA of Squamous Cell Carcinoma of Tongue[J]. Clin Cancer Res.2008,14(9):2588-2592.
[31]Mi S, Lu J, Sun M,et al. MicroRNA expression signatures accurately discriminate acute lymphoblastic leukemia from acute myeloid leukemia[J]. Proc Natl Acad Sci U S A.2007,104(50):19971-19976.
[32]李刚,李湘平,彭英,等.应用RNA干扰抑制EB病毒潜伏膜蛋白-1表达对鼻咽癌细胞生长的影响(英文)[J].第一军医大学学报,2004,(03)：241-246
[33]欧小波,陈小毅,吴民华,等.EB病毒LMP1对人鼻咽癌细胞转移能力的影响[J].癌症,2008,27(8)：803-808.
[34]Li W, Ruan K. MicroRNA detection by microarray. Anal Bioanal Chem, 2009,394(4):1117-1124.
[35]Castoldi M, Schmidt S, Benes V, et al. A sensitive array for microRNA expression profiling (miChip) based on locked nucleic acids (LNA)[J]. RNA,2006,12(5):913-920.
[1]Chen HC, Chen GH, Chen YH, et al. MicroRNA deregulation and pathway alterations in nasopharyngeal carcinoma[J]. Br J Cancer,2009, 100(6):1002-1011.
[2]Alexiou P, Maragkakis M, Papadopoulos GL, et al. The DIANA-mirExTra web server:from gene expression data to microRNA function[J]. PLoS One,2010,5(2):e9171.
[3]Gaidatzis D, van Nimwegen E, Hausser J, et al. Inference of miRNA targets using evolutionary conservation and pathway analysis[J]. BMC Bioinformatics,2007,8:69.
[4]Gusev Y, Schmittgen TD, Lerner M, et al. Computational analysis of biological functions and pathways collectively targeted by co-expressed microRNAs in cancer[J]. BMC Bioinformatics,2007,8(Suppl7):S16.
[5]Papadopoulos GL, Alexiou P, Maragkakis M, et al. DIANA-mirPath: Integrating human and mouse microRNAs in pathways[J]. Bioinformatics, 2009,25(15):1991-1993.
[6]Inui M, Martello G, Piccolo S. MicroRNA control of signal transduction[J]. Nat Rev Mol Cell Biol,2010,[Epub ahead of print]
[7]Ota A, Tagawa H, Karnan S, et al. Identification and characterization of a novel gene, C13orf25, as a target for 13q31-q32 amplification in malignant lymphoma[J]. Cancer Res,2004,64(9):3087-3095.
[8]Hayashita Y, Osada H, Tatematsu Y, et al. A polycistronic microRNA cluster, miR-17-92, is overexpressed in human lung cancers and enhances cell proliferation[J]. Cancer Res.2005; 65(21):9628-9632.
[9]Takakura S, Mitsutake N, Nakashima M, et al. Oncogenic role of miR-17-92 cluster in anaplastic thyroid cancer cells[J]. Cancer Sci,2008, 99(6):1147-1154.
[10]Pichiorri F, Suh SS, Ladetto M, et al. MicroRNAs regulate critical genes associated with multiple myeloma pathogenesis[J]. Proc Natl Acad Sci USA,2008,105(35):12885-12890.
[11]Connolly E, Melegari M, Landgraf P, et al. Elevated expression of the miR-17-92 polycistron and miR-21 in hepadnavirus-associated hepatocellular carcinoma contributes to the malignant phenotype[J]. Am J Pathol,2008,173(3):856-864.
[1]Hayashita Y, Osada H, Tatematsu Y, et al. A polycistronic microRNA cluster, miR-17-92, is overexpressed in human lung cancers and enhances cell proliferation[J]. Cancer Res.2005; 65(21):9628-9632.
[2]Alexander WS, Starr R, Metcalf D, et al. Suppressors of cytokine signaling (SOCS):negative regulators of signal transduction[J]. J Leukoc Biol,1999,66(4):588-592.
[3]Manfred W, Marcus S. X-tremeGENE siRNA transfection reagent:a powerful tool for antisense inhibition of miRNA in Human cells with miRCURY knockdown probes[J]. Biochemia,2006,2(1):28-30.
[4]张振武,安洋,滕春波.miR-17-92基因簇microRNAs对哺乳动物器官发育及肿瘤发生的调控[J].遗传,2009,31(11)：1094-1100.
[5]Pichiorri F, Suh SS, Ladetto M, et al. MicroRNAs regulate critical genes associated with multiple myeloma pathogenesis[J]. Proc Natl Acad Sci USA,2008,105(35):12885-12890.
[6]Connolly E, Melegari M, Landgraf P, et al. Elevated expression of the miR-17-92 polycistron and miR-21 in hepadnavirus-associated hepatocellular carcinoma contributes to the malignant phenotype[J]. Am J Pathol,2008,173(3):856-864.
[7]Hackl M, Brunner S, Fortschegger K, et al. miR-17, miR-19b, miR-20a and miR-106a are down-regulated in human aging[J]. Aging Cell.2010 Jan 18. [Epub ahead of print]
[8]Todoerti K, Barbui V, Pedrini O, et al. Pleiotropic anti-myeloma activity of ITF2357:inhibition of interleukin-6 receptor signaling and repression of miR-19a and miR-19b[J]. Haematologica,2010,95(2):260-269.
[9]Smith KA, Griffin JD. Following the cytokine signaling pathway to leukemogenesis:a chronology[J]. J Clin Invest,2008,118(11):3564-3573.
[10]Takeda K, Noguchi K, Shi W, et al. Targeted disruption of the mouse Stat3 gene leads to early embryonic lethality[J]. Proc Natl Acad Sci U S A, 1997,94(8):3801-3804.
[11]Yang XO, Panopoulos AD, Nurieva R, et al. STAT3 regulates cytokine-mediated generation of inflammatory helper T cells[J]. J Biol Chem,2007,282(13):9358-9363.
[12]Alvarez JV, Greulich H, Sellers WR, et al. Signal transducer and activator of transcription 3 is required for the oncogenic effects of non-small-cell lung cancer-associated mutations of the epidermal growth factor receptor[J]. Cancer Res,2006,66(6):3162-3268.
[13]Yin W, Cheepala S, Roberts JN, et al. Active Stat3 is required for survival of human squamous cell carcinoma cells in serum-free conditions [J]. Mol Cancer,2006,5:15.
[14]Kusaba T, Nakayama T, Yamazumi K, et al. Activation of STAT3 is a marker of poor prognosis in human colorectal cancer[J]. Oncol Rep,2006, 15(6):1445-1451.
[15]Akira S, Nishio Y, Inoue M, et al. Molecular cloning of APRF, a novel IFN-stimulated gene factor 3 p91-related transcription factor involved in the gp130-mediated signaling pathway [J]. Cell,1994,77(1):63-71.
[16]Daeipour M, Kumar G, Amaral MC, et al. Recombinant IL-6 activates p42 and p44 mitogen-activated protein kinases in the IL-6 responsive B cell line, AF-10[J]. J Immunol,1993,150(11):4743-4753.
[17]Takeda K, Kaisho T, Yoshida N, et al. Stat3 activation is responsible for IL-6-dependent T cell proliferation through preventing apoptosis:
generation and characterization of T cell-specific Stat3-deficient mice[J]. J Immunol,1998,161(9):4652-4660.
[18]Chen RH, Chang MC, Su YH, et al. Interleukin-6 inhibits transforming growth factor-beta-induced apoptosis through the phosphatidylinositol 3-kinase/Akt and signal transducers and activators of transcription 3 pathways[J]. J Biol Chem,1999,274(33):23013-23019.
[19]Catlett-Falcone R, Landowski TH, Oshiro MM, et al. Constitutive activation of Stat3 signaling confers resistance to apoptosis in human U266 myeloma cells[J]. Immunity,1999,10(1):105-115.
[20]Hilbert DM, Migone TS, Kopf M, et al. Distinct tumorigenic potential of abl and raf in B cell neoplasia:abl activates the IL-6 signaling pathway[J]. Immunity,1996,5(1):81-89.
[21]Mziaut H, Kersting S, Knoch KP, et al. ICA512 signaling enhances pancreatic beta-cell proliferation by regulating cyclins D through STATs[J]. Proc Natl Acad Sci U S A,2008,105(2):674-679.
[22]Endo TA, Masuhara M, Yokouchi M, et al. A new protein containing an SH2 domain that inhibits JAK kinases[J]. Nature,1997,387(6636): 921-924.
[23]Naka T, Narazaki M, Hirata M, et al. Structure and function of a new STAT-induced STAT inhibitor[J]. Nature,1997,387(6636):924-929.
[24]Nicholson SE, Willson TA, Farley A, et al. Mutational analyses of the SOCS proteins suggest a dual domain requirement but distinct mechanisms for inhibition of LIF and IL-6 signal transduction[J]. EMBO J, 1999,18:375-385.
[25]Tischoff I, Hengge UR, Vieth M, et al. Methylation of SOCS-3 and SOCS-1 in the carcinogenesis of Barrett's adenocarcinoma[J]. Gut,2007, 56(8):1047-1053.
[26]Komazaki T, Nagai H, Emi M, et al. Hypermethylation-associated inactivation of the SOCS-1 gene, a JAK/STAT inhibitor, in human pancreatic cancers[J]. Jpn J Clin Oncol,2004,34(4):191-194.
[27]Galm O, Yoshikawa H, Esteller M, et al. SOCS-1, a negative regulator of cytokine signaling, is frequently silenced by methylation in multiple myeloma[J]. Blood,2003,101(7):2784-2788.
[28]Angela KF, Kwok WL, Sai WT, et al. Epstein-Barr virus infection alters cellular signal cascades in human nasopharyngeal epithelial cells[J]. Neoplasia,2006,8(3):173-180.
[29]杨荣宁,吴冬梅.CD62p、IL-6、IL-8的表达与鼻咽癌分期及预后关系的探讨[J].2009,17(2)：231-234
[30]Pichiorri F, Suh SS, Ladetto M, et al. MicroRNAs regulate critical genes associated with multiple myeloma pathogenesis[J]. Proc Natl Acad Sci U S A,2008,105(35):12885-12890.
[31]Papakonstanti EA, Stournaras C. Cell responses regulated by early reorganization of actin cytoskeleton[J]. FEBS Lett.2008 Jun 18; 582(14): 2120-2127
[32]Wendt MK, Allington TM, Schiemann WP. Mechanisms of the epithelial-mesenchymal transition by TGF-beta[J]. Future Oncol,2009, 5(8):1145-1168.
[1]闵华庆,洪旺晃,马骏,等.鼻咽癌新分期的研究[J].癌症,1992,11(4)：290-294.
[2]刘泰福,徐国镇.全国鼻咽癌会议纪要[J].中华放射肿瘤学杂志,1992,1(3)：204.
[3]Stiller CA. International variations in the incidence of childhood carcinomas[J]. Cancer Epidemiol Biomarkers Prev,1994,3(4):305-310.
[4]Lo YM, Chan LY, Lo KW, et al. Quantitative analysis of cell-free Epstein-Barr virus DNA in plasma of patients with nasopharyngeal carcinoma[J]. Cancer Res,1999,59(6):1188-1191.
[5]Lo YM, Leung SF, Chan LY, et al. Kinetics of plasma Epstein-Barr virus DNA during radiation therapy for nasopharyngeal carcinoma[J]. Cancer Res,2000,60(9):2351-2355.
[6]Lo YM, Chan LY, Chan AT, et al. Quantitative and temporal correlation between circulating cell-free Epstein-Barr virus DNA and tumor recurrence in nasopharyngeal carcinoma[J]. Cancer Res,1999,59(21): 5452-5455.
[7]Kim KR, Yoshizake T, Miyamori H, et al. Transformation of Madin darby canine kidney (MDCK) epithelial cell by Epstein-Barr virus latent membrane protein 1 (LMP1) induces expression of Ets1 and invasive growth[J]. Oncogene,2000,19(14):1764-1777.
[8]Wang D, Liebowitz D, Kieff E. An EBV membrane protein expressed in immortalized lymphocytes transforms established rodent cells[J]. Cell, 1985,43(3 Pt 2):831-840.
[9]Izumi KM, Kaye KM, Kieff ED. The Epstein-Barr virus LMP1 amino acid sequence that engages tumor necrosis factor receptor associated factors is critical for primary B lymphocyte growth transformation[J]. Proc Natl Acad Sci U S A,1997,94(4):1447-1452.
[10]Mosialos G, Birkenbach M, Yalamanchili R, et al. The Epstein-Barr virus transforming protein LMP1 engages signaling for the tumor necrosis factor receptor family. Cell,1995,80(3):389-399.
[11]Lo AK, Liu Y, Wang XH, et al. Alterations of biologic properties and gene expression in nasopharyngeal epithelial cells by the Epstein-Barr virus-encoded latent membrane protein 1[J]. Lab Invest,2003, 83(5):697-709.
[12]Li XP, Li G, Peng Y, et al. Suppression of Epstein-Barr virus-encoded latent membrane protein-1 by RNA interference inhibits the metastatic potential of nasopharyngeal carcinoma cells[J]. Biochem Biophys Res Commun,2004,315(1):212-218.
[13]姜武忠,赵素萍,廖遇平,等.鼻咽癌组织中LMP1和VEGF蛋白表达及其临床意义[J].中国耳鼻咽喉颅底外科杂志,2007,13(3)：174-177.
[14]Lagos-Quintana M, Rauhut R, Lendeckel W, et al.Identification of novel genes coding for small expressed RNAs[J]. Science,2001,294(5543): 853-858.
[15]Cho WC. OncomiRs:the discovery and progress of microRNAs in cancers[J]. Mol Cancer,2007,6(1):60-67.
[16]Chen HC, Chen GH, Chen YH, et al. MicroRNA deregulation and pathway alterations in nasopharyngeal carcinoma. Br J Cancer,2009, 100(6):1002-1011.
[17]Sengupta S, den Boon JA, Chen IH, et al. MicroRNA 29c is down-regulated in nasopharyngeal carcinomas, up-regulating mRNAs encoding extracellular matrix proteins[J]. Proc Natl Acad Sci U S A,2008, 105(15):5874-5878.
[18]Hayashita Y, Osada H, Tatematsu Y, et al. A polycistronic microRNA cluster, miR-17-92, is overexpressed in human lung cancers and enhances cell proliferation[J]. Cancer Res.2005; 65(21):9628-9632.
[19]Pichiorri F, Suh SS, Ladetto M, et al. MicroRNAs regulate critical genes associated with multiple myeloma pathogenesis[J]. Proc Natl Acad Sci USA,2008,105(35):12885-12890.
[20]Connolly E, Melegari M, Landgraf P, et al. Elevated expression of the miR-17-92 polycistron and miR-21 in hepadnavirus-associated hepatocellular carcinoma contributes to the malignant phenotype[J]. Am J Pathol,2008,173(3):856-864.
[21]Ma L, Young J, Prabhala H, et al. miR-9, a MYC/MYCN-activated microRNA, regulates E-cadherin and cancer metastasis [J]. Nat Cell Biol, 2010 Feb 21. [Epub ahead of print]
[22]Cameron JE, Yin Q, Fewell C, et al. Epstein-Barr Virus Latent Membrane Protein 1 Induces Cellular MicroRNA miR-146a, a Modulator of Lymphocyte Signaling Pathways[J]. J Virology,2008,82(4):1946-1958.
[1]Bartel DP. MicroRNAs:genomics, biogenesis, mechanism, and function[J]. Cell,2004,116(2):281-297.
[2]Llave C, Xie Z, Kasschau KD, et al. Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA[J]. Science,2002, 297(5589):2053-2056.
[3]Berezikov E, Guryev V, van de Belt J, et al. Phylogenetic shadowing and computational identification of human microRNA genes[J]. Cell,2005, 120(1):21-24.
[4]Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets[J]. Cell,2005,120(1):15-20.
[5]Kloosterman WP, Plasterk RH. The diverse functions of microRNAs in animal development and disease[J]. Dev Cell,2006,11(4):441-450.
[6]Lee RC, Feinbaum RL, Ambros V. The C.elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[J]. Cell,1993,75(5):843-854
[7]Wightman B, Ha I, Ruvkun G. Posttranscriptional regulation of theheterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans[J]. Cell,1993,75(5):855-862.
[8]Reinhart BJ, Frank J, Michael B. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans[J]. Nature,2000, 403(6772):901-906.
[9]Lau NC, Lim LP, Weinstein EG, et al. An abundant class of tiny RNAs with probableregulatory roles in Caenorhabditis elegans[J]. Science,2001, 294(5543):858-862.
[10]Slack FJ, Basson M, Liu Z, et al. The lin-41 RBCC gene acts in the C. elegans heterochronic pathway between the let-7 regulatory RNA and the lin-29 transcription factor[J]. Mol Cell,2000,5(4):659-669.
[11]Pasquinelli AE, Ruvkun G. Control of developmental timing by microRNAs and their targets[J]. Annu Rev Cell Dev Biol,2002,18:495-513.
[12]Abrahante JE, Daul AL, Li M, et al. The Caenorhabditis elegans hunchback-like gene lin-57/hbl-1 controls developmental time and is regulated by microRNAs[J]. Dev Cell,2003,4(5):625-637.
[13]Lin SY, Johnson SM, Abraham M, et al.The C elegans hunchback homolog, hbl-1, controls temporal patterning and is a probable microRNA target[J], Dev Cell,2003,4(5):639-650.
[14]Brennecke J, Hipfner DR, Stark A, et al. bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila[J]. Cell,2003, 113(1):25-36.
[15]Pillai RS, Bhattacharyya SN, Artus CG, et al. Inhibition of translational initiation by Let-7 MicroRNA in human cells[J]. Science,2005, 309(5740):1573-1576.
[16]Bagga S, Bracht J, Hunter S, et al.Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation [J]. Cell,2005,122(4):553-563.
[17]Cai X, Hagedorn CH, Cullen BR. Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs [J]. RNA,2004,10(12):1957-1966.
[18]Denli AM, Tops BB, Plasterk RH, et al. Processing of primary microRNAs by the Microprocessor complex[J]. Nature,2004, 432(7014):231-235.
[19]Lund E, Guttinger S, Calado A, et al. Nuclear export of microRNA precursors[J]. Science,2004,303(5654):95-98.
[20]Sempere LF, Sokol NS, Dubrovsky EB, et al. Temporal regulation of microRNA expression in Drosophila melanogaster mediated by hormonal signals and Broad-Complex gene activity[J]. Dev Biol,2003,259(1):9-18.
[21]Rodriguez A, Griffiths-Jones S, Ashurst JL, et al. Identification of mammalian microRNA host genes and transcription units[J]. Genome Res, 2004,14(1A):1902-1910.
[22]Baskerville S, Bartel DP. Microarray profiling of microRNAs reveals frequent coexpression with neighboring miRNAs and host genes [J]. RNA, 2005, 11(3):241-247.
[23]Khvorova A, Reynolds A, Jayasena SD. Functional siRNAs and miRNAs exhibit strand bias. Cell,2003,115(2):209-216.
[24]Schwarz DS, Hutvagner G, Du T et al. Asymmetry in the assembly of the RNAi enzyme complex[J]. Cell,2003,115(2):199-208.
[25]Lim LP, Lau NC, Weinstein EG, et al. The microRNAs of Caenorhabditis elegans[J]. Genes Dev,2003,17(8):991-1008.
[26]Lim LP, Glasner ME, Yekta S, et al. Vertebrate microRNA genes[J]. Science,2003,299(5612):1540.
[27]Grosshans H, Johnson T, Reinert KL, et al. The temporal patterning microRNA let-7 regulates several transcription factors at the larval to adult transition in C. elegans[J]. Dev Cell,2005,8(3):321-330.
[28]Krek A, Grun D, Poy MN, et al. Combinatorial microRNA target predictions[J]. Nature Genet,2005,37(5):495-500.
[29]Karube Y, Tanaka H, Osada H, et al. Reduced expression of Dicer associated with poor prognosis in lung cancer patients [J]. Cancer Sci, 2005,96(2):111-115.
[30]Kanellopoulou C, Muljo SA, Kung AL, et al. Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing [J]. Genes Dev,2005,19(4):489-501.
[31]Bentwich I, Avniel A, Karov Y, et al. Identification of hundreds of conserved and nonconserved human microRNAs [J]. Nat Genet, 2005,37(7):766-770.
[32]Wienholds E, Kloosterman WP, Miska E, et al. MicroRNA expression in zebrafish embryonic development[J]. Science,2005,309(5732):310-311.
[33]Rajewsky N. microRNA target predictions in animals [J]. Nat Genet, 2006,38(Suppl):S8-13.
[34]Li Y, Wang F, Lee JA, et al. MicroRNA-9a ensures the precise specification of sensory organ precursors in Drosophila[J]. Genes Dev, 2006,20(20):2793-2805.
[35]Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signaling:cell fate control and signal integration in development [J]. Science,1999, 284(5415):770-776.
[36]Vella MC, Choi EY, Lin SY, et al. The C. elegans microRNA let-7 binds to imperfect let-7 complementary sites from the lin-41 3'UTR. Genes Dev, 2004,18(2):132-137.
[37]Yang W, Chendrimada TP, Wang Q, et al. Modulation of microRNA processing and expression through RNA editing by ADAR deaminases[J]. Nat Struct Mol Biol,2006,13(1):13-21.
[38]Grosshans H, Johnson T, Reinert KL, et al. The temporal patterning microRNA let-7 regulates several transcription factors at the larval to adult transition in C. elegans[J]. Dev Cell,2005,8(3):321-330.
[39]Lin SY, Johnson SM, Abraham M, et al. The C elegans hunchback homolog, hb1-1, controls temporal patterning and is a probable microRNA target[J]. Dev Cell,2003,4(5):639-650.
[40]Rosenberg MI, Georges SA, Asawachaicharn A, et al. MyoD inhibits Fstll and Utrn expression by inducing transcription of miR-206[J]. J Cell Biol,2006,175(1):77-85.
[41]Rao PK, Kumar RM, Farkhondeh M, et al. Myogenic factors that regulate expression of muscle-specific microRNAs[J]. Proc Natl Acad Sci USA, 2006,103(23):8721-8726.
[42]Obernosterer G, Leuschner PJ, Alenius M, et al. Post-transcriptional regulation of microRNA expression[J]. RNA,2006,12(7):1161-1167.
[43]Thomson JM, Newman M, Parker JS, et al. Extensive post-transcriptional regulation of microRNAs and its implications for cancer[J]. Genes Dev, 2006,20(16):2202-2207.
[44]Davis BN, Hilyard AC, Lagna G,et al. SMAD proteins control DROSHA-mediated microRNA maturation[J]. Nature,2008,454(7200): 56-61.
[45]Han J, Lee Y, Yeom KH,et al. The Drosha-DGCR8 complex in primary microRNA processing[J]. Genes Dev,2004,18(24):3016-3027.
[46]Lehrbach NJ, Armisen J, Lightfoot HL, et al. LIN-28 and the poly(U) polymerase PUP-2 regulate let-7 microRNA processing in Caenorhabditis elegans[J]. Nat Struct Mol Biol,2009,16(10):1016-1020.
[47]Czech B, Zhou R, Erlich Y, et al. Hierarchical rules for Argonaute loading in Drosophila[J]. Mol Cell,2009,36(3):445-456.
[48]Kennedy S, Wang D, Ruvkun G. A conserved siRNA-degrading RNase negatively regulates RNA interference in C. elegans[J].Nature,2004, 427(6975):645-649.
[49]Ramachandran V, Chen X. Degradation of microRNAs by a family of exoribonucleases in Arabidopsis[J]. Science,2008,321(5895):1490-1492.
[50]Yang W, Chendrimada TP, Wang Q, et al. Modulation of microRNA processing and expression through RNA editing by ADAR deaminases[J]. Nat Struct Mol Biol,2006,13(1):13-21.
[51]Lee CT, Risom T, Strauss WM. MicroRNAs in mammalian development[J]. Birth Defects Res C Embryo Today,2006,78(2): 129-139.
[52]Moss EG. Heterochronic genes and the nature of developmental time[J]. Curr Biol,2007,17(11):R425-34.
[53]Bracken CP, Gregory PA, Kolesnikoff N, et al. A double-negative feedback loop between ZEB1-SIP1 and the microRNA-200 family regulates epithelial-mesenchymal transition[J]. Cancer Res,2008, 68(19):7846-7854.
[54]Calin GA, Croce CM. MicroRNA signatures in human cancers[J]. Nat Rev Cancer,2006,6(11):857-866.
[55]Calin GA, Dumitru CD, Shimizu M, et al. Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia[J]. Proc Natl Acad Sci USA,2002, 99(24):15524-15529.
[56]Calin GA, Sevignani C, Dumitru CD, et al. Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers[J]. Proc Natl Acad Sci USA,2004,101(9):2999-3004.
[57]Bottoni A, Piccin D, Tagliati F, et al. miR-15a and miR-16-1 down-regulation in pituitary adenomas[J]. J Cell Physiol,2005, 204(1):280-285.
[58]Cimmino A, Calin GA, Fabbri M, et al. miR-15 and miR-16 induce apoptosis by targeting BCL2[J]. Proc Natl Acad Sci USA,2005, 102(39):13944-13949.
[59]He L, Thomson JM, Hemann MT, et al. A microRNA polycistron as a potential human oncogene[J]. Nature,2005,435(7043):828-833.
[60]O'Donnell KA, Wentzel EA, Zeller KI, et al. c-Myc regulated microRNAs modulate E2F1 expression[J]. Nature,2005,435(7043):839-843.
[61]Woods K, Thomson JM, Hammond SM. Direct regulation of an oncogenic micro-RNA cluster by E2F transcription factors[J]. J Biol Chem,2007,282(4):2130-2134.
[62]Hayashita Y, Osada H, Tatematsu Y, et al. A polycistronic microRNA cluster, miR-17-92, is overexpressed in human lung cancers and enhances cell proliferation[J]. Cancer Res,2005,65(21):9628-9632.
[63]Matsubara H, Takeuchi T, Nishikawa E, et al. Apoptosis induction by antisense oligonucleotides against miR-17-5p and miR-20a in lung cancers overexpressing miR-17-92[J]. Oncogene,2007,26(41):6099- 6105.
[64]Iorio MV, Ferracin M, Liu CG, et al. MicroRNA gene expression deregulation in human breast cancer[J]. Cancer Res,2005,65(16): 7065-7070.
[65]Si ML, Zhu S, Wu H, et al. miR-21-mediated tumor growth[J]. Oncogene, 2007,26(19):2799-2803.
[66]Lu J, Getz G, Miska EA, et al. MicroRNA expression profiles classify human cancers[J]. Nature,2005,435(7043):834-838.
[67]Bottoni A, Zatelli MC, Ferracin M, et al. Identification of differentially expressed microRNAs by microarray:a possible role for microRNA genes in pituitary adenomas[J]. J Cell Physiol,2007,210(2):370-377.
[68]Fabbri M, Croce CM, Calin GA. MicroRNAs[J]. Cancer J,2008, 14(1):1-6.
[69]Takamizawa J, Konishi H, Yanagisawa K, et al. Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival[J]. Cancer Res,2004,64(11):3753-3756.
[70]Roldo C, Missiaglia E, Hagan JP, et al. MicroRNA expression abnormalities in pancreatic endocrine and acinar tumors are associated with distinctive pathologic features and clinical behavior[J]. J Clin Oncol, 2006,24(29):4677-4684.
[71]Bloomston M, Frankel WL, Petrocca F, et al. MicroRNA expression patterns to differentiate pancreatic adenocarcinoma from normal pancreas and chronic pancreatitis[J]. JAMA,2007,297(17):1901-1908.
[72]Raveche ES, Salerno E, Scaglione BJ, et al. Abnormal microRNA-16 locus with synteny to human 13q14 linked to CLL in NZB mice[J]. Blood, 2007,109(12):5079-5086.
[73]Volinia S, Calin GA, Liu CG, et al. A microRNA expression signature of human solid tumors defines cancer gene targets[J]. Proc Natl Acad Sci USA,2006,103(7):2257-2261.
[74]Ciafre SA, Galardi S, Mangiola A, et al. Extensive modulation of a set of microRNAs in primary glioblastoma[J]. Biochem Biophys Res Commun, 2005,334(4):1351-1358.
[75]Krichevsky AM, King KS, Donahue CP, et al. A microRNA array reveals extensive regulation of microRNAs during brain development[J]. RNA, 2003,9(),1274-1281.
[76]Wang T, Zhang X, Obijuru L, et al. A micro-RNA signature associated with race, tumor size, and target gene activity in human uterine leiomyomas[J]. Genes Chromosomes Cancer,2007,46(4):336-347.
[77]Meng F, Henson R, Lang M, et al. Involvement of human micro-RNA in growth and response to chemotherapy in human cholangiocarcinoma cell lines[J]. Gastroenterology,2007,130(7):2113-2129.
[78]Zhu S, Si ML, Wu H, et al. MicroRNA-21 targets the tumor suppressor gene tropomyosin 1 (TPM1). J Biol Chem,2007,282(19:14328-14336.