RNA加尾和引物延伸RT-PCR法实时定量检测胃癌中miRNAs的表达
摘要
目的
胃癌是全世界范围内处于发病率第二位的恶性肿瘤,导致每年约有100百万人死亡。在中国,胃癌的发病率高居各种恶性肿瘤之首,每年新确诊患者达40万,大约死亡26万/年,占所有恶性肿瘤死亡的23.24%,同时仍然呈上升趋势。由于胃癌患者经常处于晚期才被确诊,因而预后较差,五年生存率低于20%。导致胃癌发生的因素很多,包括胃幽门螺杆菌感染,饮食,环境和遗传因素等,其中幽门螺杆菌是80%或更多胃癌患者的主要危险因素,已有研究提出其在胃十二指肠疾病中的作用。
近来,尽管胃癌的诊断和治疗方面取得了一些进展,但是胃癌病人的预后仍然很差,尤其是晚期病人。尽管发现了一系列诸如p53,APC,C-met等胃癌相关的癌基因和抑癌基因,然而胃癌发生的分子机制依然不是十分清楚。目前microRNA与肿瘤发生发展的关系及潜在的诊断价值已成为肿瘤研究的新热点。
微小RNA(miRNAs)是一种小分子非编码RNA,通过抑制基因mRNA的翻译或诱导mRNA的降解来调节大约30%人类基因的表达。近年来,大量的研究表明miRNA在细胞增殖、凋亡、发育、分化和代谢等过程中发挥了重要的作用。最近,miRNA的表达被证明与肿瘤的发生发展有密切的关系。如果一个miRNA在某种肿瘤中表达下调并且其靶基因是癌基因,那么这个miRNA就发挥抑癌基因的作用;同理,如果一个miRNA在某种肿瘤中表达上调并且其靶基因是抑癌基因或重要的控制分化基因,那么它就发挥癌基因的作用。因此,通过检测胃癌组织中表达失调的miRNA,并进一步确定其靶基因及其参与的信号调节通路,将对阐明胃癌发生的分子机制具有重要的理论意义。而且miRNA作为重要的生长调节和细胞周期调控分子,具有成为新一代肿瘤标记物的潜力,对于胃癌的早期诊断、分期和预后提示均具有重要的临床意义。
本研究旨在通过RNA加尾引物延RT-PCR法实时定量检测miR-18b、miR-21、miR-148b、miR-365在胃癌中的表达水平。我们首先将总RNA加poly(A)尾后进行反转录,之后再用特异引物进行PCR扩增特异的miRNA,经凝胶电泳检测扩增产物的完整性和特异性后进行实时定量PCR,最后采用2~(-ΔΔCt)法对检测结果进行分析。
材料与方法
实验用组织均取自于中国医科大学附属第一医院肿瘤外科2007年11月至2008年7月间的12例胃癌患者经外科手术切除的标本(取前均取得患者家属同意,并与之签订知情同意书)。所取癌组织及相对应正常组织均在标离体后30分钟内取得,并在液氮冷冻30分钟后放入-80℃冰箱保存。其中男性8例,女性4例,年龄(43-72)(平均56)岁,术前均未经放化疗。
提取12例胃癌手术切除标本中癌组织及相应正常组织中的总RNA,在3′末端加尾并抽提后用5′端带40nt延伸序列的单碱基锚定Olig-dT引物进行反转录,得到约80nt的cDNA,然后用特异miRNA引物进行SYBR Green Real-time PCR扩增,并用2~(-ΔΔCt)法对检测结果进行分析,计算其在癌组织与相应正常组织中表达水平的相对比值。
实验结果
一、相对定量2~(-ΔΔCt)分析方法的优化
用相对定量2~(—ΔΔCt)法对SYBR Green Real-time PCR检测的结果进行分析。该实验要求对目的基因(miR-18b、miR-21、miR-148b及miR-365)和管家基因(U6SnRNA)分别做标准曲线,且其标准曲线的斜率之差M＜0.1.miR-18b、miR-21、miR-148b、miR-365和U6在260nm波长UV下定量化,并被10倍梯度稀释:10~5,10~4,10~3,10~2,10~1。结果显示miR-18b、miR-21、miR-148b、miR-365与U6标准曲线斜率之差M分别为0.062,0.003,0.004,0.093,均小于0.1,符合该分析方法的要求。
二、miR-18b、miR-21、miR-148b及miR-365在胃癌中的表达
为了证实4种miRNAs在胃癌中的表达水平,我们用SYBR Green Real-time PCR从12例胃癌患者手术切除标本的癌组织及相应正常组织中检测出成熟miRNAs和U6 sn RNA的表达水平。结果显示miR-18b、miR-21、miR-365分别在其中的6例、7例、8例标本的胃癌组织中呈高表达,而miR-148b在其中的7例标本的胃癌组织中呈低表达。
结论
RNA加尾和引物延伸RT-PCR法是一种可行的检测miRNAs表达的方法。
miR-148b在胃癌组织中较在正组织中表达有下调趋势,miR-18b、miR-21、miR-365表达则有上调的趋势,提示mi R-148b与miR-18b、miR-21、miR-365可能是在胃癌发生发展过程中分别起抑癌基因和癌基因作用的重要分子。
Objective
Gastric cancer,the second most common cancer in the world,causes nearly one million deaths worldwide per year.In china,gastric cancer ranked the top common cancer with 4000,000 new patients and 260,000 death per year.Unfortunately,gastric cancer is usually diagnosed at an advanced staged and the prognosis is poorthe overall relative 5—rear survival rate is currently less than 20%.Many etiological factors can increase the risks of gastric cancer,including H.pylori infection,food,environmental and genetic factors.H.pylori is the main risk factor in about 80%or more of gastric cancers.The role of H.pylori on gastroduodenal diseases has been proposed,but the detailedmolecular pathway remains unclear.
Recently years,several oncogenes and tumer supressors have been found in gastric cancer,such as p53,APC,C-met,FGF2,CDH1 and heparinase.However,the molecular events leading to gastric malignancy and the genetic components that are altered at the inception and the course of the neoplasm are largely unknown.At present, the relationship between microRNA and the development of the tumor and the potential diagnostic value of cancer research has become the new hot spots.
MicroRNAs are a family of 21-25 nucleotide small RNAs that,at least for those few that have characterized targets,negatively regulate gene expression at the post-transcription level.Recently,many studies revealed miRNAs play key roles in cell proliferation,apoptosis,development,differentiation and metabolism.Indeed,miRNA abrrent expression has been previously found in various types of hunman cancers,and miRNA signatures were associated with specific clinicobiological features.In addition, miRNAs are proved to function as oncogenes and tumor suppressor genes through post-transcriptional regulation on their target genes.Therefore,it is necessary to indentify deregulation miRNAs as new biological makers that can be used to screen high-risk pations in order to allow better gastric cancer detection,earlier intervention and increase the likelihood of successful treatment.In this study,we investigate the miR-18b、miR-21、miR-148b and miR-365 expression levels in gastric cancer,and try to unravel the biological pathways of miRNAs on gastric tumorigenesis.However,as an important growth regulator of cell cycle regulation and molecular,miRNA has become a new generation of potential tumor marker.
The purpose of this study through SYBR Green Real-time quantitative RT-PCR detection of miR-18b、miR-21、miR-148b and miR-365 express levels in gastric cancer. we have established a simple method to detect the miRNAs expresion levels.Total RNA was polyadenylated by poly(A)polymerase,and then cDNA was synthesized by a specific reverse transcriptase primer and reverse transcriptase using the poly(A)-tailed totoal RNA as templates.And then the results of SYBR Green real-time PCR were analysis by the Relatively quantitative 2~(-ΔΔCt)method.
Materials And Methods
All hunman tissue samples were obtained from surgical specimens of 12 patients with gastic carcinoma from 2007 to 2008 at Department of Surgerical Oncolgy,The First Hospital of China Medical University,china.All tissues,including gastric carcinoma and corresponding adjacent normal tissue,were divided into two parts and preserved in liquid nitrogen for 30 min after removing from the body,then stored at -80℃.Informed cosent was takenen from all subjects.
Small-sized RNAs(<200bp)were extracted and polyadenylated by poy(A) polymerase.One-base anchored olig-dT primers with 40nt nt extension at their 5'-ends were used to reverse transcribe the poly(A)-tailed miRNA,then were amplified by SRBR Green real-time PCR using miRNA specific primers.And then the results were analysised By 2~(-ΔΔCt)method.
Results
一、The optimized of 2~(-ΔΔCt) analysis methods
The results of SYBR Green real-time PCR were analysis by the Relatively quantitative 2~(-ΔΔCt) method.This analysis methods requires that the difference of the standard curve slope between the target gene(miR-18b,miR-21,miR-148b and miR-365)and housekeeping gene(U6 SnRNA)should be less than 0.01.miR-18b, miR-21,miR-148b,miR-365 and U6 snRNA were quantified at 260nm wavelength UV and were diluted 10-fold gradient:105,104,103,102,101,respectively.The results showed that the difference between the slope 0.062,0.003,0.004,0.093 respectively, were less than 0.01,consistent with the analysis conditions.
二、Expression of miRNAs in gastric carcinoma
In order to confirm the level of miRNAs in human gastric cancer,we examined using SYBR Green real-time RT-PCR,the expression of miR-18b、miR-21、miR-148b、miR-365 and U6 snRNA in 12 matched paired of gastric tumoral and non-tumoral tissuses from patients.The results showed that miR-18b,miR-21 and miR-365 was up-regulated in 6,7,8of 12 Samples with gastric cancer respectively.And miR-148b was down-regulated in 7of 12 Samples with gastric cancer.
Conclusions
1.RNA-tailing and primer-extension RT-PCR is a convenient and reliable method to detect miRNAs which may play an important role in the development of gastric carcinoma.
2.The expression levels of miR-148b in gastric cancer tissues was lower compared to normal tissues which forecast miR-148b may play a key role as tumor suppressor gene.The expression levels of miR-18b,miR-21,miR-365 in gastric cancer tissues was higher compared to normal tissues which forecast miR-18b,miR-21,miR-365 may play a key roles as tumor oncogenes.
胃癌是全世界范围内处于发病率第二位的恶性肿瘤,导致每年约有100百万人死亡。在中国,胃癌的发病率高居各种恶性肿瘤之首,每年新确诊患者达40万,大约死亡26万/年,占所有恶性肿瘤死亡的23.24%,同时仍然呈上升趋势。由于胃癌患者经常处于晚期才被确诊,因而预后较差,五年生存率低于20%。导致胃癌发生的因素很多,包括胃幽门螺杆菌感染,饮食,环境和遗传因素等,其中幽门螺杆菌是80%或更多胃癌患者的主要危险因素,已有研究提出其在胃十二指肠疾病中的作用。
近来,尽管胃癌的诊断和治疗方面取得了一些进展,但是胃癌病人的预后仍然很差,尤其是晚期病人。尽管发现了一系列诸如p53,APC,C-met等胃癌相关的癌基因和抑癌基因,然而胃癌发生的分子机制依然不是十分清楚。目前microRNA与肿瘤发生发展的关系及潜在的诊断价值已成为肿瘤研究的新热点。
微小RNA(miRNAs)是一种小分子非编码RNA,通过抑制基因mRNA的翻译或诱导mRNA的降解来调节大约30%人类基因的表达。近年来,大量的研究表明miRNA在细胞增殖、凋亡、发育、分化和代谢等过程中发挥了重要的作用。最近,miRNA的表达被证明与肿瘤的发生发展有密切的关系。如果一个miRNA在某种肿瘤中表达下调并且其靶基因是癌基因,那么这个miRNA就发挥抑癌基因的作用;同理,如果一个miRNA在某种肿瘤中表达上调并且其靶基因是抑癌基因或重要的控制分化基因,那么它就发挥癌基因的作用。因此,通过检测胃癌组织中表达失调的miRNA,并进一步确定其靶基因及其参与的信号调节通路,将对阐明胃癌发生的分子机制具有重要的理论意义。而且miRNA作为重要的生长调节和细胞周期调控分子,具有成为新一代肿瘤标记物的潜力,对于胃癌的早期诊断、分期和预后提示均具有重要的临床意义。
本研究旨在通过RNA加尾引物延RT-PCR法实时定量检测miR-18b、miR-21、miR-148b、miR-365在胃癌中的表达水平。我们首先将总RNA加poly(A)尾后进行反转录,之后再用特异引物进行PCR扩增特异的miRNA,经凝胶电泳检测扩增产物的完整性和特异性后进行实时定量PCR,最后采用2~(-ΔΔCt)法对检测结果进行分析。
材料与方法
实验用组织均取自于中国医科大学附属第一医院肿瘤外科2007年11月至2008年7月间的12例胃癌患者经外科手术切除的标本(取前均取得患者家属同意,并与之签订知情同意书)。所取癌组织及相对应正常组织均在标离体后30分钟内取得,并在液氮冷冻30分钟后放入-80℃冰箱保存。其中男性8例,女性4例,年龄(43-72)(平均56)岁,术前均未经放化疗。
提取12例胃癌手术切除标本中癌组织及相应正常组织中的总RNA,在3′末端加尾并抽提后用5′端带40nt延伸序列的单碱基锚定Olig-dT引物进行反转录,得到约80nt的cDNA,然后用特异miRNA引物进行SYBR Green Real-time PCR扩增,并用2~(-ΔΔCt)法对检测结果进行分析,计算其在癌组织与相应正常组织中表达水平的相对比值。
实验结果
一、相对定量2~(-ΔΔCt)分析方法的优化
用相对定量2~(—ΔΔCt)法对SYBR Green Real-time PCR检测的结果进行分析。该实验要求对目的基因(miR-18b、miR-21、miR-148b及miR-365)和管家基因(U6SnRNA)分别做标准曲线,且其标准曲线的斜率之差M＜0.1.miR-18b、miR-21、miR-148b、miR-365和U6在260nm波长UV下定量化,并被10倍梯度稀释:10~5,10~4,10~3,10~2,10~1。结果显示miR-18b、miR-21、miR-148b、miR-365与U6标准曲线斜率之差M分别为0.062,0.003,0.004,0.093,均小于0.1,符合该分析方法的要求。
二、miR-18b、miR-21、miR-148b及miR-365在胃癌中的表达
为了证实4种miRNAs在胃癌中的表达水平,我们用SYBR Green Real-time PCR从12例胃癌患者手术切除标本的癌组织及相应正常组织中检测出成熟miRNAs和U6 sn RNA的表达水平。结果显示miR-18b、miR-21、miR-365分别在其中的6例、7例、8例标本的胃癌组织中呈高表达,而miR-148b在其中的7例标本的胃癌组织中呈低表达。
结论
RNA加尾和引物延伸RT-PCR法是一种可行的检测miRNAs表达的方法。
miR-148b在胃癌组织中较在正组织中表达有下调趋势,miR-18b、miR-21、miR-365表达则有上调的趋势,提示mi R-148b与miR-18b、miR-21、miR-365可能是在胃癌发生发展过程中分别起抑癌基因和癌基因作用的重要分子。
Objective
Gastric cancer,the second most common cancer in the world,causes nearly one million deaths worldwide per year.In china,gastric cancer ranked the top common cancer with 4000,000 new patients and 260,000 death per year.Unfortunately,gastric cancer is usually diagnosed at an advanced staged and the prognosis is poorthe overall relative 5—rear survival rate is currently less than 20%.Many etiological factors can increase the risks of gastric cancer,including H.pylori infection,food,environmental and genetic factors.H.pylori is the main risk factor in about 80%or more of gastric cancers.The role of H.pylori on gastroduodenal diseases has been proposed,but the detailedmolecular pathway remains unclear.
Recently years,several oncogenes and tumer supressors have been found in gastric cancer,such as p53,APC,C-met,FGF2,CDH1 and heparinase.However,the molecular events leading to gastric malignancy and the genetic components that are altered at the inception and the course of the neoplasm are largely unknown.At present, the relationship between microRNA and the development of the tumor and the potential diagnostic value of cancer research has become the new hot spots.
MicroRNAs are a family of 21-25 nucleotide small RNAs that,at least for those few that have characterized targets,negatively regulate gene expression at the post-transcription level.Recently,many studies revealed miRNAs play key roles in cell proliferation,apoptosis,development,differentiation and metabolism.Indeed,miRNA abrrent expression has been previously found in various types of hunman cancers,and miRNA signatures were associated with specific clinicobiological features.In addition, miRNAs are proved to function as oncogenes and tumor suppressor genes through post-transcriptional regulation on their target genes.Therefore,it is necessary to indentify deregulation miRNAs as new biological makers that can be used to screen high-risk pations in order to allow better gastric cancer detection,earlier intervention and increase the likelihood of successful treatment.In this study,we investigate the miR-18b、miR-21、miR-148b and miR-365 expression levels in gastric cancer,and try to unravel the biological pathways of miRNAs on gastric tumorigenesis.However,as an important growth regulator of cell cycle regulation and molecular,miRNA has become a new generation of potential tumor marker.
The purpose of this study through SYBR Green Real-time quantitative RT-PCR detection of miR-18b、miR-21、miR-148b and miR-365 express levels in gastric cancer. we have established a simple method to detect the miRNAs expresion levels.Total RNA was polyadenylated by poly(A)polymerase,and then cDNA was synthesized by a specific reverse transcriptase primer and reverse transcriptase using the poly(A)-tailed totoal RNA as templates.And then the results of SYBR Green real-time PCR were analysis by the Relatively quantitative 2~(-ΔΔCt)method.
Materials And Methods
All hunman tissue samples were obtained from surgical specimens of 12 patients with gastic carcinoma from 2007 to 2008 at Department of Surgerical Oncolgy,The First Hospital of China Medical University,china.All tissues,including gastric carcinoma and corresponding adjacent normal tissue,were divided into two parts and preserved in liquid nitrogen for 30 min after removing from the body,then stored at -80℃.Informed cosent was takenen from all subjects.
Small-sized RNAs(<200bp)were extracted and polyadenylated by poy(A) polymerase.One-base anchored olig-dT primers with 40nt nt extension at their 5'-ends were used to reverse transcribe the poly(A)-tailed miRNA,then were amplified by SRBR Green real-time PCR using miRNA specific primers.And then the results were analysised By 2~(-ΔΔCt)method.
Results
一、The optimized of 2~(-ΔΔCt) analysis methods
The results of SYBR Green real-time PCR were analysis by the Relatively quantitative 2~(-ΔΔCt) method.This analysis methods requires that the difference of the standard curve slope between the target gene(miR-18b,miR-21,miR-148b and miR-365)and housekeeping gene(U6 SnRNA)should be less than 0.01.miR-18b, miR-21,miR-148b,miR-365 and U6 snRNA were quantified at 260nm wavelength UV and were diluted 10-fold gradient:105,104,103,102,101,respectively.The results showed that the difference between the slope 0.062,0.003,0.004,0.093 respectively, were less than 0.01,consistent with the analysis conditions.
二、Expression of miRNAs in gastric carcinoma
In order to confirm the level of miRNAs in human gastric cancer,we examined using SYBR Green real-time RT-PCR,the expression of miR-18b、miR-21、miR-148b、miR-365 and U6 snRNA in 12 matched paired of gastric tumoral and non-tumoral tissuses from patients.The results showed that miR-18b,miR-21 and miR-365 was up-regulated in 6,7,8of 12 Samples with gastric cancer respectively.And miR-148b was down-regulated in 7of 12 Samples with gastric cancer.
Conclusions
1.RNA-tailing and primer-extension RT-PCR is a convenient and reliable method to detect miRNAs which may play an important role in the development of gastric carcinoma.
2.The expression levels of miR-148b in gastric cancer tissues was lower compared to normal tissues which forecast miR-148b may play a key role as tumor suppressor gene.The expression levels of miR-18b,miR-21,miR-365 in gastric cancer tissues was higher compared to normal tissues which forecast miR-18b,miR-21,miR-365 may play a key roles as tumor oncogenes.
引文
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27 Sempere LF,Christensen M,Silahtaroglu A,et al.Altered microRNA expression confined to specific epithelial cell subpopulations in breast cancer[J].Caner Res,2007,67:11612-11620.
28 Slaby O,Svoboda M,Fabian P,et al.Alter expression of miR-21,miR-31,miR-143 and miR-145 is related to clinicopathologic features of colorectal cancer[J].Oncology,2007,72:397-402.
29 Jiang J,Gusev Y,Aderca L,et al.Association of microRNA expression in hepatocellular carcinomas with hepatitis infection,cirrhosis,and patient survival[J].Clin Cancer Res,2008,14:419-427.
30 Guo Y,Chen Z,Zhang L,et al.Distinctive microRNA profiles relating to patient survival in esophageal squamous cell carcinoma[J].Cancer Res,2008,68:26-33.
31 Chan JA,Krichevsky AM and Kosik KS:MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells[J].Cancer Res,2005,65:6025-6029.
32 Meng F,Henson F,Lang M,et al.Involvement of human microRNA in growth and response to chemotherapy in human cholangiocarcinoma cell lines[J].Gastroemerology,2006,130:2113-2129.
33 Chen C,Ridzon DA,BroomerAJ.Real-time quantification of microRNAs by stem-loop RT-PCR.[J].Nucleic Acids Res,2005,33:791.
34 Raymond CK,Roberts BS,Garrett2Engele P,et al.Simple,quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs[J].RNA,2005,11:1737-1744.
35 Shi R,Chiang VL.Facile means for quantifying microRNA expression by real-time PCR[J].Biotechniques,2005,39:519 -525.
36 Liang P,Zhu W,Zhang X,et al.Differential display using onebase anchored olig-dT primers[J].Nucleic Acids Res,1994,22:563-5764.
37 Shi R,Chiang VL.Facile means for quantifying microRNA expression by real-ti-me PCR[J].Biotechniques,2005,39(4):519-525.
38 张旗,何湘君,潘秀英.RNA加尾和引物延伸RT-PCR法实时定量检测microRNA[J].北京大学学报,2007,39(1):87-91.
39 Shih-hsuan C,Chew-wun wu,Anna F-Y Li,et al.miR-21 microRNA expression in hunman gastric carcinoma and its clinical association[J].Anticancer Research,2008,28: 907-912.
40 Zhiyu Z,Zejuan Li,Caiping G,et al.miRNA plays a pivotal role in gastric cancer pathogenesis and progression [J],Laboratory Investigation,2008,12:1-9.
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6 Pasquinelli A,Reinhart B,Slack F,et al.Conservation of the sequence and temporal expressionof let-7 heterochronic regulatory RNA [J].Nature,2000,4 08: 8689.
7 Yin JQ,Wang Y.siRNA-mediated gene regulation system: Now and the future [J].Int J Mol Med,2002,10: 355-365.
8 Tan FL,inJQ.Application of RNAi to cancer research and therapy [J].Front Biosci,2005,10:1946-1960.
9 Bentwich I,Avniel A,Karov Y,et al.Identification of hundreds of conserved and nonconserved human microRNAs [J].Nat Genet,2005,37: 766-770.
10 Zhang W,Dahiberg JE,Tam W,et al.MicroRNAs in Tumorigenesis,A primer [J].Am J Pathol,2007,171(3 ): 728-738.
11 Sempere LF,Freemantle S,Pitha-Rowe I,et al.Expression profiling of mammalian microRNAs uncovers a subset of brain-expressed microRNAs with possible roles in murine and human neuronal differentiation [J].Genome Biol,2004,5(3): R13.
12 Lim LP,Lau NC,Weinstein EG,et al.The microRNAs of Caenorhabditis elegans[J].Genes Dev,2003,17(8): 991-1008.
13 Hayasbita 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.
14 Weinholds E,Kloosterman W.P,Miska,E,et al.MicroRNA expression in zebrafish embryonic development [J].Science,2005,309,310-311.
15 Kloosterman W.P,Weinholds E,Debruun,E,et al.In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes [J].Nat Methods,2006,3(l):27-29.
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17 Liu CG,Calin GA,Meloon B,et al.An oligonucleotide micro-chip for genomewide microRNA profiling in human and mouse tissues [J].Proc Natl Acad Sci USA,2004,101(26): 9740-9744.
18 Calin GA,Liu CG,Sevignani C,et al.MicroRNA profiling reveals Distinct signatures in B cell chronic lymphocytic leukemias [J].Proc Natl Acad Sci USA,2004,101(32): 11755-11760.
19 Murakami Y,Yasuda T,Saigo K,et al.Comprehensivea nalysis of microRNA expression pattenrs in hepatocellularc arcinoma and nontumorous tissues [J].Oncogene,2006,25(17): 2537-2545
20 Nelson PT,Baldwin DA,Scearce LM,et al.Microarray-based,high-Throughput gene expression profiling of microRNAs [J].Nat Methods,2004,1(2): 155-161.
21 Castoldi M,Schmidt S,Benes V,et al.A sensitive array for microRNA expression profiling (miChip) based on locked nucleic acids (LNA).RNA,2006,12: 913-920.
22 Akao Y,Nakagawa Y,Naoe T.MicroRNA-143 and-145 in colon cancer [J].DNA and Cell Biology,2007,26(5): 311-320.
23 Akao Y,Nakagawa Y,Naoe T.let-7 MicroRNA Functions as a potential growth Suppressor in Human Colon Cancer Cells [J].Biol Pharm Bull,2006,29(5): 903-906.
24 Fu HJ,Zhu J,Yang M,et al.A novel method to monitor the expression of microRNAs [J].Mol Biotechnol,2006,32(3): 197-204.
25 Yan Z,Bryan R,Cullen.Sequence requirements for microRNA processing and function in human cells[J].RNA,2003,9:112-123.
26 Schmittgen TD,Jiang J,Liu Q,et al.A highthroughput method to monitor the expression of microRNA precursors[J].Nucleic Acids Res,2004,32(4):e43.
27 Jiang J,Lee EJ,Gusev Y,et al.Real-time expression profiling of microRNA Pecrursors in human cancer celllines[J].Nucleic Acids Res,2005,33(17):5394-5403.
28 Lee EJ,Gusev Y,Jiang J,et al.Expression profiling identifies microRNA signature in pancreatic cancer.Int J Cancer,2007,120(5):1046-1054.
29 Zhang HH,Wang XJ,Li GX,et al.Detection of let-Ta microRNA by realtime PCR ingastric Carcinoma.World Gastroenterol,2007,13(20):2883-2888.
30 Chen C,Ridzon DA,Broomer AJ,et al.Real-time quantification of microRNAs by stem-loop RT-PCR.Nucleic Acids Research,2005,33(20):e179.
31 Shi R,Chiang VL.Facile means for quantifying microRNA expression by realtime PCR[J].Biotechniques,2005,39(4):519-525.
32 张旗,何湘君,潘秀英.RNA加尾和引物延伸RT-PCR法实时定量检测microRNA[J].北京大学学报,2007,39(1):87-91.
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26 Ramaswamy S,Tamayo p Rifkin R,et al.Multiclass cancer diagnosis using tumor gene expression signature.Proc Natl Acad Sci USA,2001,98: 15149-15154.
27 Sempere LF,Christensen M,Silahtaroglu A,et al.Altered microRNA expression confined to specific epithelial cell subpopulations in breast cancer[J].Caner Res,2007,67:11612-11620.
28 Slaby O,Svoboda M,Fabian P,et al.Alter expression of miR-21,miR-31,miR-143 and miR-145 is related to clinicopathologic features of colorectal cancer[J].Oncology,2007,72:397-402.
29 Jiang J,Gusev Y,Aderca L,et al.Association of microRNA expression in hepatocellular carcinomas with hepatitis infection,cirrhosis,and patient survival[J].Clin Cancer Res,2008,14:419-427.
30 Guo Y,Chen Z,Zhang L,et al.Distinctive microRNA profiles relating to patient survival in esophageal squamous cell carcinoma[J].Cancer Res,2008,68:26-33.
31 Chan JA,Krichevsky AM and Kosik KS:MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells[J].Cancer Res,2005,65:6025-6029.
32 Meng F,Henson F,Lang M,et al.Involvement of human microRNA in growth and response to chemotherapy in human cholangiocarcinoma cell lines[J].Gastroemerology,2006,130:2113-2129.
33 Chen C,Ridzon DA,BroomerAJ.Real-time quantification of microRNAs by stem-loop RT-PCR.[J].Nucleic Acids Res,2005,33:791.
34 Raymond CK,Roberts BS,Garrett2Engele P,et al.Simple,quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs[J].RNA,2005,11:1737-1744.
35 Shi R,Chiang VL.Facile means for quantifying microRNA expression by real-time PCR[J].Biotechniques,2005,39:519 -525.
36 Liang P,Zhu W,Zhang X,et al.Differential display using onebase anchored olig-dT primers[J].Nucleic Acids Res,1994,22:563-5764.
37 Shi R,Chiang VL.Facile means for quantifying microRNA expression by real-ti-me PCR[J].Biotechniques,2005,39(4):519-525.
38 张旗,何湘君,潘秀英.RNA加尾和引物延伸RT-PCR法实时定量检测microRNA[J].北京大学学报,2007,39(1):87-91.
39 Shih-hsuan C,Chew-wun wu,Anna F-Y Li,et al.miR-21 microRNA expression in hunman gastric carcinoma and its clinical association[J].Anticancer Research,2008,28: 907-912.
40 Zhiyu Z,Zejuan Li,Caiping G,et al.miRNA plays a pivotal role in gastric cancer pathogenesis and progression [J],Laboratory Investigation,2008,12:1-9.
1 Rodriguez A,Griffiths-Jones S,Ashurst JL,et al.Identification of mammalian microRNA Host genes and transcription units [J].Genome Res,2004; 14:1902-10.
2 Suh M R,Lee Y,Kim J Y,et al.Human embryonic stem cells express a unique set of microRNAs [J].Dev Biol,2004,270,488-498.
3 Ambros,V.MicroRNA pathways in flies and worms: growth,death,fat,stress,and timing [J].Cell,2003,113,673-676.
4 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: 843-854.
5 Reinhart BJ,Slack F J,Basson M,et al.The 21 nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans [J].Nature,2000,403: 901-906.
6 Pasquinelli A,Reinhart B,Slack F,et al.Conservation of the sequence and temporal expressionof let-7 heterochronic regulatory RNA [J].Nature,2000,4 08: 8689.
7 Yin JQ,Wang Y.siRNA-mediated gene regulation system: Now and the future [J].Int J Mol Med,2002,10: 355-365.
8 Tan FL,inJQ.Application of RNAi to cancer research and therapy [J].Front Biosci,2005,10:1946-1960.
9 Bentwich I,Avniel A,Karov Y,et al.Identification of hundreds of conserved and nonconserved human microRNAs [J].Nat Genet,2005,37: 766-770.
10 Zhang W,Dahiberg JE,Tam W,et al.MicroRNAs in Tumorigenesis,A primer [J].Am J Pathol,2007,171(3 ): 728-738.
11 Sempere LF,Freemantle S,Pitha-Rowe I,et al.Expression profiling of mammalian microRNAs uncovers a subset of brain-expressed microRNAs with possible roles in murine and human neuronal differentiation [J].Genome Biol,2004,5(3): R13.
12 Lim LP,Lau NC,Weinstein EG,et al.The microRNAs of Caenorhabditis elegans[J].Genes Dev,2003,17(8): 991-1008.
13 Hayasbita 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.
14 Weinholds E,Kloosterman W.P,Miska,E,et al.MicroRNA expression in zebrafish embryonic development [J].Science,2005,309,310-311.
15 Kloosterman W.P,Weinholds E,Debruun,E,et al.In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes [J].Nat Methods,2006,3(l):27-29.
16 Krichevsky AM,King K S,Donahue CP,et al.A microRNA array reveals extensive regulation of miRNA during brain development [J].RNA,2003,9(10): 1274-1281.
17 Liu CG,Calin GA,Meloon B,et al.An oligonucleotide micro-chip for genomewide microRNA profiling in human and mouse tissues [J].Proc Natl Acad Sci USA,2004,101(26): 9740-9744.
18 Calin GA,Liu CG,Sevignani C,et al.MicroRNA profiling reveals Distinct signatures in B cell chronic lymphocytic leukemias [J].Proc Natl Acad Sci USA,2004,101(32): 11755-11760.
19 Murakami Y,Yasuda T,Saigo K,et al.Comprehensivea nalysis of microRNA expression pattenrs in hepatocellularc arcinoma and nontumorous tissues [J].Oncogene,2006,25(17): 2537-2545
20 Nelson PT,Baldwin DA,Scearce LM,et al.Microarray-based,high-Throughput gene expression profiling of microRNAs [J].Nat Methods,2004,1(2): 155-161.
21 Castoldi M,Schmidt S,Benes V,et al.A sensitive array for microRNA expression profiling (miChip) based on locked nucleic acids (LNA).RNA,2006,12: 913-920.
22 Akao Y,Nakagawa Y,Naoe T.MicroRNA-143 and-145 in colon cancer [J].DNA and Cell Biology,2007,26(5): 311-320.
23 Akao Y,Nakagawa Y,Naoe T.let-7 MicroRNA Functions as a potential growth Suppressor in Human Colon Cancer Cells [J].Biol Pharm Bull,2006,29(5): 903-906.
24 Fu HJ,Zhu J,Yang M,et al.A novel method to monitor the expression of microRNAs [J].Mol Biotechnol,2006,32(3): 197-204.
25 Yan Z,Bryan R,Cullen.Sequence requirements for microRNA processing and function in human cells[J].RNA,2003,9:112-123.
26 Schmittgen TD,Jiang J,Liu Q,et al.A highthroughput method to monitor the expression of microRNA precursors[J].Nucleic Acids Res,2004,32(4):e43.
27 Jiang J,Lee EJ,Gusev Y,et al.Real-time expression profiling of microRNA Pecrursors in human cancer celllines[J].Nucleic Acids Res,2005,33(17):5394-5403.
28 Lee EJ,Gusev Y,Jiang J,et al.Expression profiling identifies microRNA signature in pancreatic cancer.Int J Cancer,2007,120(5):1046-1054.
29 Zhang HH,Wang XJ,Li GX,et al.Detection of let-Ta microRNA by realtime PCR ingastric Carcinoma.World Gastroenterol,2007,13(20):2883-2888.
30 Chen C,Ridzon DA,Broomer AJ,et al.Real-time quantification of microRNAs by stem-loop RT-PCR.Nucleic Acids Research,2005,33(20):e179.
31 Shi R,Chiang VL.Facile means for quantifying microRNA expression by realtime PCR[J].Biotechniques,2005,39(4):519-525.
32 张旗,何湘君,潘秀英.RNA加尾和引物延伸RT-PCR法实时定量检测microRNA[J].北京大学学报,2007,39(1):87-91.