鼻咽癌相关基因ID2与EMT关系的初步探讨
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摘要
研究背景和目的
鼻咽癌(NasoPharyngeal Carcinoma,NPC)是一种主要发生在我国南方地区的鼻咽部恶性肿瘤,由于NPC发生的部位比较隐蔽,早期症状常不明显而容易被忽略,因此确诊的患者60%以上都是中、晚期病例,常伴有转移发生。寻找鼻咽癌发生发展及转移的机制,是我们当前研究的主要方向。
肿瘤的形成和侵袭转移是一个多步骤、多阶段、多因子参与的复杂而又连续的过程,涉及了一些关键的瘤基因和抑瘤基因。在本研究中,我们从公共基因芯片数据库GEO(Gene expression omnibus)中寻找并下载鼻咽癌的相关基因芯片数据,并使用Genclip软件对其进行分析,找到鼻咽癌公共基因芯片数据中与上皮间充质转化(Epithelial——mesenchymal transition, EMT)相关的基因35个,其中包括本课题所研究的ID2,它在鼻咽癌组织和细胞中表达明显上调,提示该基因可能正向参与促进了鼻咽癌的发病过程。
ID蛋白家族成员共有四个ID1, ID2, ID3, ID4,它们最初被发现在负性调节细胞分化方面起了重要作用,后来越来越多的研究证明它们在调节谱系定型,基因表达,细胞命运,细胞增殖,以及在神经发生,淋巴组织形成,新生血管形成中细胞的分化时间也起了关键作用。ID蛋白家族成员被发现在很多肿瘤中过表达,比如大肠癌,胶质母细胞瘤,髓母细胞瘤,神经细胞瘤,胰腺癌,甲状腺癌,鳞状细胞癌,前列腺癌,乳腺癌,子宫内膜癌,宫颈癌和黑色素瘤等。
为了阐明ID2基因在鼻咽癌发病中的作用,我们利用RNAi技术高效、特异的靶向沉默机制干扰鼻咽癌细胞SUNE1中ID2表达,建立稳定靶向干扰ID2表达的siRNA鼻咽癌细胞,以观察干扰ID2后鼻咽癌细胞生物学功能改变,并探寻其与EMT的关系,为鼻咽癌的发病机制和治疗提供新的思路。
方法
1.鼻咽癌EMT相关基因的筛选
从公共基因芯片数据库GEO (Gene expression omnibus)中寻找并下载鼻咽癌的相关基因芯片数据,并使用Genclip软件对下载的数据进行分析,寻找鼻咽癌公共基因芯片数据中与上皮间充质转化(Epithelial——mesenchymal transition, EMT)相关的基因；并用生物信息学方法对筛选出来的基因进一步分析。
2. ID2在鼻咽癌中的表达特性鉴定
免疫组化SP法从蛋白水平检测ID2在非癌鼻咽组织和鼻咽癌中的表达水平,初步探讨ID2在组织水平与鼻咽癌发生发展的关系。
荧光定量PCR法从mRNA水平检测ID2在永生化鼻咽上皮细胞NP69和鼻咽癌细胞SUNE1、5-8F、6-10B、CNE2、HNE1、CNE1、C666-1、HONE1中的表达水平,初步探讨ID2在永生化鼻咽上皮细胞和鼻咽癌细胞中的表达差异,并找到高表达ID2的鼻咽癌细胞作为下一步干扰对象。
3. ID2表达沉默对鼻咽癌细胞生物学影响及部分与EMT相关基因表达水平的检测
构建ID2慢病毒干扰载体,包装成成熟的慢病毒颗粒感染高表达ID2的SUNE1细胞,有限稀释法筛选出稳定的阳性和空载克隆,荧光定量PCR检测各干扰克隆细胞中ID2干扰效率,筛选干扰效率最高的细胞克隆并将该细胞克隆作为实验对象被进一步研究。MTT法、平板克隆形成实验检测ID2沉默后对细胞体外增殖的影响；Transwell检测ID2沉默后细胞迁移运动能力的改变。
荧光定量PCR法检测与ID2相关的TGF-beta以及与EMT相关的转录因子Slug, SIP1, Twist, Snail,上皮标志物E-cadherin,间质标志物Vimentin在四株细胞株SUNE1/plv-ID2-1, SUNE1/plv-ID2-2, SUNE1/plv, SUNE1中mRNA的表达水平。
4.统计学方法
采用SPSS 13.0统计软件包进行统计学处理,第二章中因免疫组化各指标均为等级资料,故其组间的比较采用非参数秩和检验,ID2的表达在非癌鼻咽组织和鼻咽癌比较采用Mann-Whitney U检验,ID2的表达与鼻咽癌临床分期的关系检验用Kruskal Wallis Test,检验水准均取双侧α=0.05；荧光定量PCR法检测鼻咽癌细胞株ID2表达特性结果比较采用单因素方差分析,多重比较采用SNK检验。第三章中荧光定量PCR、运动、平板克隆形成实验采用单因素方差分析；MTT采用析因设计的方差分析；多重比较采用SNK或LSD检验。
结果
1.鼻咽癌EMT相关基因的筛选
在公共的鼻咽癌芯片数据中找到35个与EMT相关的差异表达基因,而且这些基因功能大致与细胞组分、细胞粘附、通信、信号转导、分化、运动、迁移以及细胞表面受体相关的信号转导等有关,这些功能往往都被认为与肿瘤的侵袭和转移有关。
2.ID2在鼻咽癌中的表达特性鉴定
1、共收集鼻咽炎组18例、鼻咽癌组52例临床标本。SP免疫组化结果表明,ID2表达主要定位在细胞核和细胞浆。ID2在鼻咽癌和非癌鼻咽组织中的表达具有统计学差异(Z=-5.553, P=0.000), ID2的表达情况与临床分期没有统计学差异(χ2=0.963,P=0.810)
2、荧光定量PCR法检测ID2在永生化鼻咽上皮细胞NP69和鼻咽癌细胞SUNE1、5-8F、6-10B、CNE2、HNE1、CNE1、C666-1、HONE1 mRNA水平表达具有统计学差异(F=2.980, P=0.002), SUNE1表达最高,表达量为NP69 ID2表达量的78倍,作为下一步干扰对象。
3.ID2表达沉默对鼻咽癌细胞生物学影响及部分与EMT相关基因表达水平的检测
1、构建ID2慢病毒干扰载体,包装慢病毒后感染SUNE1细胞,有限稀释法筛选GFP+单克隆,荧光定量PCR检测各克隆的ID2表达情况,筛选出干扰效率最高的2个单克隆为下一步实验的研究对象。阳性克隆的干扰效率与SUNE1和阴性对照细胞表达具有统计学差异(F=48.693,P=0.000),同时检测ID2在阴性对照中的表达相对于SUNE1强度较一致(0.933±0.006)。阳性干扰克隆命名为SUNE1/plv-ID2-1和SUNE1/plv-ID2-2,阴性对照克隆为SUNE1/plv。
2、MTT法检测细胞增殖情况,与阴性对照SUNE1/Plv和SUNE1细胞相比,干扰克隆细胞SUNE1/plv-ID2-1和SUNE1/plv-ID2-2的增殖速度明显减慢并且呈时间依赖关系,具有统计学差异(F=3.301,P=0.000)；平板克隆形成实验显示四组细胞的克隆形成率具有统计学差异(F=253.434,P=0.000), SUNE1/plv-ID2细胞克隆形成减少,SUNE1和阴性对照组不具有统计学差异(P=0.866)。综合表明ID2表达干扰后,SUNE1细胞体外生长被显著抑制。
3、Transwell小室检测结果显示,与SUNE1和SUNE1/plv细胞相比,SUNE1 /plv-ID2细胞穿过聚碳酸酯膜的细胞数没有明显变化,差异不具有显著性(F=0.624,P=0.610)。
4、荧光定量PCR法检测结果显示TGF-beta, E-cadherin, Slug, SIP1, Vimentin在4组细胞间的mRNA表达不具有统计学差异(F=0.405,P=0.754；F=0.571,P=0.650; F=0.541,P=0.668; F=0.456,P=0.721; F=1.058,P=0.419),而Twist, Snail在4组细胞间的表达具有统计学差异(F=94.627,P=0.000;F=155.295,P=0.000)。
结论
1、生物信息学方法筛选出35个与鼻咽癌EMT相关的基因
2、免疫组化SP法证实ID2的表达与鼻咽癌的发生发展有关系。
3、荧光定量PCR法检测ID2在永生化鼻咽上皮细胞NP69和鼻咽癌细胞SUNE1、5-8F、6-10B、CNE2、HNE1、CNE1、C666-1、HONE1中的表达具有统计学差异,SUNE1表达最高。
4、ID2干扰后部分细胞体外生物学功能发生改变。
5、ID2干扰后部分与EMT相关的基因1nRNA表达水平出现改变。
BACKGROUND & OBJECTIVE
Nasopharyngeal carcinoma (NPC) is a common malignant epithelial tumor in Southern China. Because the primary anatomical site of tumor growth is located in a cryptic area, with slightly early symptoms, the NPC patients tend to present a more advanced stage of disease in clinic with higher metastatic potential. So finding the mechanism of the NPC tumorigenicity and metastasis is still our main research direction.
Tumor formation and metastasis is a complex and continuous process that needs multi-step, multi-stage, multi-factor involving in a number of key oncogenes and tumor suppressors. In this study, we obtained the gene chip data of nasopharyngeal carcinoma from GEO database and used the GenClip software to identify the genes related to EMT in nasopharyngeal carcinoma by bioinformatics analysis, and found 35 EMT-related genes include ID2. Markedly upregulated expression of inhibitor of differentiation protein ID2 was showed in NPC tissues and NPC cells,which suggested that this gene might promote the pathogenesis of NPC.
There are four members in the ID protein family, ID1, ID2, ID3, and ID4. These proteins were initially discovered as proteins involved in the negative control of cell differentiation. Id proteins may play a central role in coordinating gene expression, cell proliferation, tumorigenesis, and angiogenesis. Id proteins have been found to be over-expressed in many types, including Glioblastoma, Medulloblastoma, Neuroblastoma, Pancreatic Cancer, Thyroid Cancer, Squamous Cell Carcinoma, Breast Carcinoma, Endometrial Cancer, Cervical Cancer, Melanoma, and Retinob-lastoma.
In order to clarify the role of ID2 in the pathogenesis of NPC, we used RNAi technology to specifically interfere ID2 expression in NPC cells, and observed the alteration of the biological function of NPC cells retrieval the relationship between ID2 and EMT. This investigation may provide new ideas for searching the pathogenesis and the treatment of NPC.
METHODS
1. Screening for Nasopharyngeal carcinoma EMT-related genes
The gene chip data of nasopharyngeal carcinoma were obtained from GEO database and analyzed by GenClip software to identify the genes related to EMT in nasopharyngeal carcinoma with bioinformatics analysis.
2. Expression characterization of ID2 in NPC
Immunohistochemisty (IHC) method was used to detect the ID2 expression in non-cancerous nasopharyngeal tissues and NPC tissues for preliminary study the relationship between ID2 and the occurrence of NPC.
Fluorescence quantitative PCR methods was used to detect the ID2 expression in immortalized nasopharyngeal epithelial cell NP69 and NPC cells SUNE1、5-8F、6-10B、CNE2、HNE1、CNE1、C666-1、HONE1 in the mRNA level. Compared with NP69 cells, NPC SUNE1 cells with the high ablility of tumorigenesis and metastasis showed the highest mRNA expression level of ID2.
3. The influences on cell biology of NPC by ID2 silence and mRNA expression of some EMT-related genes
Constructing ID2 interference and blank lentivirus expression vectors to infect SUNE1 cells.The cell clones with stably expressing anti-ID2 shRNA and blank vectors were picked out. Fluorescence quantitative PCR was used to detect the interfere efficiency of each cell clone. The cell clone with highest interference efficiency would be selected as the next experiment object. MTT and colony forming experiments were used to detect cells proliferation in vitro;Transwell chamber was employed to detect cells migration in vitro; Fluorescence quantitative PCR was used to detect the mRNA expression of some EMT-related genes in the four cell lines SUNE1/plv-ID2-1, SUNE1/plv-ID2-2, SUNE1/plv, SUNE1.
4. Statistical methods
We use SPSS 13.0 statistical software package for statistical treatment, In ChapterⅠ, Mann-Whitney U test was used to analyze the immunohistochemistry result of protein expression between NPC and non-cancerous nasopharyngeal tissue. Kruskal Wallis Test was utilized to evaluate the relationship between ID2 expression and clinical stage of NPC. QRT-PCR detection results was analyzed using one-way ANOVA, and multiple comparison test using SNK. In ChapterⅡ, QRT-PCR, colony forming experiments and migrate assay were tested by one-way ANOVA, MTT results using factorial design analysis of variance, multiple comparisons test using SNK or LSD test.
RESULTS
1. Screening for Nasopharyngeal carcinoma EMT-related genes
35 differentially expressed EMT-related genes were identified in the public gene chip database of nasopharyngeal carcinoma. These genes were associated with the cellular component, adhesion, communication, signal transduction, differentia- tion, motion, migration and cell surface receptor linked signal transduction which attribute to the tumor invasion and metastasis.
2. Expression of ID2 and its clinical significance in NPC
1、18 cases of non-cancerous nasopharyngeal tissue and 52 cases of NPC specimens were collected. The results of IHC showed that ID2 mainly located in nuclear and cytoplasm. There is significantly differentiation between NPC and non-cancerous nasopharyngeal tissues (Z=—5.553, P=0.000). No correlation was found between ID2 expression and cliniccal stage (χ2= 0.963, P=0.810)
2、The results of fluorescence quantitative PCR showed ID2 expression with statistical significance (F=2.980, P=0.002)in immortalized nasopharyngeal epithelial NP69 cells and NPC cells SUNE1、5-8F、6-10B、CNE2、HNE1、CNE1、C666-1、HONE1. Compared with NP69 cells, NPC SUNE1 cells showed the highest expression of ID2(78 fold) in all 8 NPC cell lines. So we chosed SUNEl cells as the interference object for further experiment study.
3. The influences on cell biology of NPC by ID2 silence and mRNA expression of some EMT-related genes
1、The lentiviral vectors containing specific anti-ID2 shRNA and negative control were transfected into 293FT cell line to produce lentivirus particles, followed by transduced into SUNE1 cell line. After separated by limiting dilution assay,7 resistant cell clones were picked out, fluorescence quantitative PCR was used to detect the interfere efficiency for each clone. Clones with the highest efficiency was chosen for the following study. There is markedly differentiation between positive clone and negative control clone, SUNE1 in ID2 expression (F=48.693,P =0.000).Interference clone was named SUNE1/plv-ID2 and SUNE1/plv-ID2, negative control clone named SUNE1/plv.
2、A significantly time-dependent inhibitory proliferation was found in SUNE1/ plv-ID2 cells compared with SUNE1/plv and SUNE1 by in vitro MTT assay (F=3.301,P=0.000). In addition, SUNE1/plv-ID2-1 and SUNE1/plv-ID2-2 cells had a significant impaired ability to form colonies in plates, as compared with SUNE1/plv and SUNE1 cells (F=253.434,P=0.000).000).The results indicate that ID2 silence suppressed proliferation of SUNE1 cells.
3、SUNE1/plv-ID2 cells did not have migrate difference as compared with SUNE1/plv and SUNE1 cells detected by Transwell in vitro (F=0.624,P=0.61)
4、There are no significant difference of mRNA expression of TGF-beta, E-cadherin, Slug, SIP1, Vimentin between four cell lines SUNE1/plv-ID2-1, SUNE1/plv-ID2-2, SUNE1/plv and SUNE1 (F=0.405,P=0.754; F=0.571, P=0.650; F=0.541,P=0.668;F=0.456,P=0.721;F=1.058,P=0.419), but except for Twist and Snail (F=94.627,P=0.000;F=155.295,P=0.000)
CONCLUSION
1. 35 nasopharyngeal carcinoma EMT-related genes have been found by bioinformatics
2. Overexpression was showed in NPC tissuse compared with non-cancerous nasopharyngeal tissues by IHC detection.
3. The results of fluorescence quantitative PCR showed ID2 expression with statistical significance in NP69 cells and NPC cells SUNE1、5-8F、6-10B、CNE2、HNE1、CNE1、C666-1、HONE1. Compared with NP69 cells, NPC SUNE1 cells showed the highest expression of ID2 in all 8 NPC cell lines.
4. Some of the biological function in vitro were changed after ID2 silence.
5. The mRNA expression of some EMT-related genes were changed after ID2 silence
鼻咽癌(NasoPharyngeal Carcinoma,NPC)是一种主要发生在我国南方地区的鼻咽部恶性肿瘤,由于NPC发生的部位比较隐蔽,早期症状常不明显而容易被忽略,因此确诊的患者60%以上都是中、晚期病例,常伴有转移发生。寻找鼻咽癌发生发展及转移的机制,是我们当前研究的主要方向。
肿瘤的形成和侵袭转移是一个多步骤、多阶段、多因子参与的复杂而又连续的过程,涉及了一些关键的瘤基因和抑瘤基因。在本研究中,我们从公共基因芯片数据库GEO(Gene expression omnibus)中寻找并下载鼻咽癌的相关基因芯片数据,并使用Genclip软件对其进行分析,找到鼻咽癌公共基因芯片数据中与上皮间充质转化(Epithelial——mesenchymal transition, EMT)相关的基因35个,其中包括本课题所研究的ID2,它在鼻咽癌组织和细胞中表达明显上调,提示该基因可能正向参与促进了鼻咽癌的发病过程。
ID蛋白家族成员共有四个ID1, ID2, ID3, ID4,它们最初被发现在负性调节细胞分化方面起了重要作用,后来越来越多的研究证明它们在调节谱系定型,基因表达,细胞命运,细胞增殖,以及在神经发生,淋巴组织形成,新生血管形成中细胞的分化时间也起了关键作用。ID蛋白家族成员被发现在很多肿瘤中过表达,比如大肠癌,胶质母细胞瘤,髓母细胞瘤,神经细胞瘤,胰腺癌,甲状腺癌,鳞状细胞癌,前列腺癌,乳腺癌,子宫内膜癌,宫颈癌和黑色素瘤等。
为了阐明ID2基因在鼻咽癌发病中的作用,我们利用RNAi技术高效、特异的靶向沉默机制干扰鼻咽癌细胞SUNE1中ID2表达,建立稳定靶向干扰ID2表达的siRNA鼻咽癌细胞,以观察干扰ID2后鼻咽癌细胞生物学功能改变,并探寻其与EMT的关系,为鼻咽癌的发病机制和治疗提供新的思路。
方法
1.鼻咽癌EMT相关基因的筛选
从公共基因芯片数据库GEO (Gene expression omnibus)中寻找并下载鼻咽癌的相关基因芯片数据,并使用Genclip软件对下载的数据进行分析,寻找鼻咽癌公共基因芯片数据中与上皮间充质转化(Epithelial——mesenchymal transition, EMT)相关的基因；并用生物信息学方法对筛选出来的基因进一步分析。
2. ID2在鼻咽癌中的表达特性鉴定
免疫组化SP法从蛋白水平检测ID2在非癌鼻咽组织和鼻咽癌中的表达水平,初步探讨ID2在组织水平与鼻咽癌发生发展的关系。
荧光定量PCR法从mRNA水平检测ID2在永生化鼻咽上皮细胞NP69和鼻咽癌细胞SUNE1、5-8F、6-10B、CNE2、HNE1、CNE1、C666-1、HONE1中的表达水平,初步探讨ID2在永生化鼻咽上皮细胞和鼻咽癌细胞中的表达差异,并找到高表达ID2的鼻咽癌细胞作为下一步干扰对象。
3. ID2表达沉默对鼻咽癌细胞生物学影响及部分与EMT相关基因表达水平的检测
构建ID2慢病毒干扰载体,包装成成熟的慢病毒颗粒感染高表达ID2的SUNE1细胞,有限稀释法筛选出稳定的阳性和空载克隆,荧光定量PCR检测各干扰克隆细胞中ID2干扰效率,筛选干扰效率最高的细胞克隆并将该细胞克隆作为实验对象被进一步研究。MTT法、平板克隆形成实验检测ID2沉默后对细胞体外增殖的影响；Transwell检测ID2沉默后细胞迁移运动能力的改变。
荧光定量PCR法检测与ID2相关的TGF-beta以及与EMT相关的转录因子Slug, SIP1, Twist, Snail,上皮标志物E-cadherin,间质标志物Vimentin在四株细胞株SUNE1/plv-ID2-1, SUNE1/plv-ID2-2, SUNE1/plv, SUNE1中mRNA的表达水平。
4.统计学方法
采用SPSS 13.0统计软件包进行统计学处理,第二章中因免疫组化各指标均为等级资料,故其组间的比较采用非参数秩和检验,ID2的表达在非癌鼻咽组织和鼻咽癌比较采用Mann-Whitney U检验,ID2的表达与鼻咽癌临床分期的关系检验用Kruskal Wallis Test,检验水准均取双侧α=0.05；荧光定量PCR法检测鼻咽癌细胞株ID2表达特性结果比较采用单因素方差分析,多重比较采用SNK检验。第三章中荧光定量PCR、运动、平板克隆形成实验采用单因素方差分析；MTT采用析因设计的方差分析；多重比较采用SNK或LSD检验。
结果
1.鼻咽癌EMT相关基因的筛选
在公共的鼻咽癌芯片数据中找到35个与EMT相关的差异表达基因,而且这些基因功能大致与细胞组分、细胞粘附、通信、信号转导、分化、运动、迁移以及细胞表面受体相关的信号转导等有关,这些功能往往都被认为与肿瘤的侵袭和转移有关。
2.ID2在鼻咽癌中的表达特性鉴定
1、共收集鼻咽炎组18例、鼻咽癌组52例临床标本。SP免疫组化结果表明,ID2表达主要定位在细胞核和细胞浆。ID2在鼻咽癌和非癌鼻咽组织中的表达具有统计学差异(Z=-5.553, P=0.000), ID2的表达情况与临床分期没有统计学差异(χ2=0.963,P=0.810)
2、荧光定量PCR法检测ID2在永生化鼻咽上皮细胞NP69和鼻咽癌细胞SUNE1、5-8F、6-10B、CNE2、HNE1、CNE1、C666-1、HONE1 mRNA水平表达具有统计学差异(F=2.980, P=0.002), SUNE1表达最高,表达量为NP69 ID2表达量的78倍,作为下一步干扰对象。
3.ID2表达沉默对鼻咽癌细胞生物学影响及部分与EMT相关基因表达水平的检测
1、构建ID2慢病毒干扰载体,包装慢病毒后感染SUNE1细胞,有限稀释法筛选GFP+单克隆,荧光定量PCR检测各克隆的ID2表达情况,筛选出干扰效率最高的2个单克隆为下一步实验的研究对象。阳性克隆的干扰效率与SUNE1和阴性对照细胞表达具有统计学差异(F=48.693,P=0.000),同时检测ID2在阴性对照中的表达相对于SUNE1强度较一致(0.933±0.006)。阳性干扰克隆命名为SUNE1/plv-ID2-1和SUNE1/plv-ID2-2,阴性对照克隆为SUNE1/plv。
2、MTT法检测细胞增殖情况,与阴性对照SUNE1/Plv和SUNE1细胞相比,干扰克隆细胞SUNE1/plv-ID2-1和SUNE1/plv-ID2-2的增殖速度明显减慢并且呈时间依赖关系,具有统计学差异(F=3.301,P=0.000)；平板克隆形成实验显示四组细胞的克隆形成率具有统计学差异(F=253.434,P=0.000), SUNE1/plv-ID2细胞克隆形成减少,SUNE1和阴性对照组不具有统计学差异(P=0.866)。综合表明ID2表达干扰后,SUNE1细胞体外生长被显著抑制。
3、Transwell小室检测结果显示,与SUNE1和SUNE1/plv细胞相比,SUNE1 /plv-ID2细胞穿过聚碳酸酯膜的细胞数没有明显变化,差异不具有显著性(F=0.624,P=0.610)。
4、荧光定量PCR法检测结果显示TGF-beta, E-cadherin, Slug, SIP1, Vimentin在4组细胞间的mRNA表达不具有统计学差异(F=0.405,P=0.754；F=0.571,P=0.650; F=0.541,P=0.668; F=0.456,P=0.721; F=1.058,P=0.419),而Twist, Snail在4组细胞间的表达具有统计学差异(F=94.627,P=0.000;F=155.295,P=0.000)。
结论
1、生物信息学方法筛选出35个与鼻咽癌EMT相关的基因
2、免疫组化SP法证实ID2的表达与鼻咽癌的发生发展有关系。
3、荧光定量PCR法检测ID2在永生化鼻咽上皮细胞NP69和鼻咽癌细胞SUNE1、5-8F、6-10B、CNE2、HNE1、CNE1、C666-1、HONE1中的表达具有统计学差异,SUNE1表达最高。
4、ID2干扰后部分细胞体外生物学功能发生改变。
5、ID2干扰后部分与EMT相关的基因1nRNA表达水平出现改变。
BACKGROUND & OBJECTIVE
Nasopharyngeal carcinoma (NPC) is a common malignant epithelial tumor in Southern China. Because the primary anatomical site of tumor growth is located in a cryptic area, with slightly early symptoms, the NPC patients tend to present a more advanced stage of disease in clinic with higher metastatic potential. So finding the mechanism of the NPC tumorigenicity and metastasis is still our main research direction.
Tumor formation and metastasis is a complex and continuous process that needs multi-step, multi-stage, multi-factor involving in a number of key oncogenes and tumor suppressors. In this study, we obtained the gene chip data of nasopharyngeal carcinoma from GEO database and used the GenClip software to identify the genes related to EMT in nasopharyngeal carcinoma by bioinformatics analysis, and found 35 EMT-related genes include ID2. Markedly upregulated expression of inhibitor of differentiation protein ID2 was showed in NPC tissues and NPC cells,which suggested that this gene might promote the pathogenesis of NPC.
There are four members in the ID protein family, ID1, ID2, ID3, and ID4. These proteins were initially discovered as proteins involved in the negative control of cell differentiation. Id proteins may play a central role in coordinating gene expression, cell proliferation, tumorigenesis, and angiogenesis. Id proteins have been found to be over-expressed in many types, including Glioblastoma, Medulloblastoma, Neuroblastoma, Pancreatic Cancer, Thyroid Cancer, Squamous Cell Carcinoma, Breast Carcinoma, Endometrial Cancer, Cervical Cancer, Melanoma, and Retinob-lastoma.
In order to clarify the role of ID2 in the pathogenesis of NPC, we used RNAi technology to specifically interfere ID2 expression in NPC cells, and observed the alteration of the biological function of NPC cells retrieval the relationship between ID2 and EMT. This investigation may provide new ideas for searching the pathogenesis and the treatment of NPC.
METHODS
1. Screening for Nasopharyngeal carcinoma EMT-related genes
The gene chip data of nasopharyngeal carcinoma were obtained from GEO database and analyzed by GenClip software to identify the genes related to EMT in nasopharyngeal carcinoma with bioinformatics analysis.
2. Expression characterization of ID2 in NPC
Immunohistochemisty (IHC) method was used to detect the ID2 expression in non-cancerous nasopharyngeal tissues and NPC tissues for preliminary study the relationship between ID2 and the occurrence of NPC.
Fluorescence quantitative PCR methods was used to detect the ID2 expression in immortalized nasopharyngeal epithelial cell NP69 and NPC cells SUNE1、5-8F、6-10B、CNE2、HNE1、CNE1、C666-1、HONE1 in the mRNA level. Compared with NP69 cells, NPC SUNE1 cells with the high ablility of tumorigenesis and metastasis showed the highest mRNA expression level of ID2.
3. The influences on cell biology of NPC by ID2 silence and mRNA expression of some EMT-related genes
Constructing ID2 interference and blank lentivirus expression vectors to infect SUNE1 cells.The cell clones with stably expressing anti-ID2 shRNA and blank vectors were picked out. Fluorescence quantitative PCR was used to detect the interfere efficiency of each cell clone. The cell clone with highest interference efficiency would be selected as the next experiment object. MTT and colony forming experiments were used to detect cells proliferation in vitro;Transwell chamber was employed to detect cells migration in vitro; Fluorescence quantitative PCR was used to detect the mRNA expression of some EMT-related genes in the four cell lines SUNE1/plv-ID2-1, SUNE1/plv-ID2-2, SUNE1/plv, SUNE1.
4. Statistical methods
We use SPSS 13.0 statistical software package for statistical treatment, In ChapterⅠ, Mann-Whitney U test was used to analyze the immunohistochemistry result of protein expression between NPC and non-cancerous nasopharyngeal tissue. Kruskal Wallis Test was utilized to evaluate the relationship between ID2 expression and clinical stage of NPC. QRT-PCR detection results was analyzed using one-way ANOVA, and multiple comparison test using SNK. In ChapterⅡ, QRT-PCR, colony forming experiments and migrate assay were tested by one-way ANOVA, MTT results using factorial design analysis of variance, multiple comparisons test using SNK or LSD test.
RESULTS
1. Screening for Nasopharyngeal carcinoma EMT-related genes
35 differentially expressed EMT-related genes were identified in the public gene chip database of nasopharyngeal carcinoma. These genes were associated with the cellular component, adhesion, communication, signal transduction, differentia- tion, motion, migration and cell surface receptor linked signal transduction which attribute to the tumor invasion and metastasis.
2. Expression of ID2 and its clinical significance in NPC
1、18 cases of non-cancerous nasopharyngeal tissue and 52 cases of NPC specimens were collected. The results of IHC showed that ID2 mainly located in nuclear and cytoplasm. There is significantly differentiation between NPC and non-cancerous nasopharyngeal tissues (Z=—5.553, P=0.000). No correlation was found between ID2 expression and cliniccal stage (χ2= 0.963, P=0.810)
2、The results of fluorescence quantitative PCR showed ID2 expression with statistical significance (F=2.980, P=0.002)in immortalized nasopharyngeal epithelial NP69 cells and NPC cells SUNE1、5-8F、6-10B、CNE2、HNE1、CNE1、C666-1、HONE1. Compared with NP69 cells, NPC SUNE1 cells showed the highest expression of ID2(78 fold) in all 8 NPC cell lines. So we chosed SUNEl cells as the interference object for further experiment study.
3. The influences on cell biology of NPC by ID2 silence and mRNA expression of some EMT-related genes
1、The lentiviral vectors containing specific anti-ID2 shRNA and negative control were transfected into 293FT cell line to produce lentivirus particles, followed by transduced into SUNE1 cell line. After separated by limiting dilution assay,7 resistant cell clones were picked out, fluorescence quantitative PCR was used to detect the interfere efficiency for each clone. Clones with the highest efficiency was chosen for the following study. There is markedly differentiation between positive clone and negative control clone, SUNE1 in ID2 expression (F=48.693,P =0.000).Interference clone was named SUNE1/plv-ID2 and SUNE1/plv-ID2, negative control clone named SUNE1/plv.
2、A significantly time-dependent inhibitory proliferation was found in SUNE1/ plv-ID2 cells compared with SUNE1/plv and SUNE1 by in vitro MTT assay (F=3.301,P=0.000). In addition, SUNE1/plv-ID2-1 and SUNE1/plv-ID2-2 cells had a significant impaired ability to form colonies in plates, as compared with SUNE1/plv and SUNE1 cells (F=253.434,P=0.000).000).The results indicate that ID2 silence suppressed proliferation of SUNE1 cells.
3、SUNE1/plv-ID2 cells did not have migrate difference as compared with SUNE1/plv and SUNE1 cells detected by Transwell in vitro (F=0.624,P=0.61)
4、There are no significant difference of mRNA expression of TGF-beta, E-cadherin, Slug, SIP1, Vimentin between four cell lines SUNE1/plv-ID2-1, SUNE1/plv-ID2-2, SUNE1/plv and SUNE1 (F=0.405,P=0.754; F=0.571, P=0.650; F=0.541,P=0.668;F=0.456,P=0.721;F=1.058,P=0.419), but except for Twist and Snail (F=94.627,P=0.000;F=155.295,P=0.000)
CONCLUSION
1. 35 nasopharyngeal carcinoma EMT-related genes have been found by bioinformatics
2. Overexpression was showed in NPC tissuse compared with non-cancerous nasopharyngeal tissues by IHC detection.
3. The results of fluorescence quantitative PCR showed ID2 expression with statistical significance in NP69 cells and NPC cells SUNE1、5-8F、6-10B、CNE2、HNE1、CNE1、C666-1、HONE1. Compared with NP69 cells, NPC SUNE1 cells showed the highest expression of ID2 in all 8 NPC cell lines.
4. Some of the biological function in vitro were changed after ID2 silence.
5. The mRNA expression of some EMT-related genes were changed after ID2 silence
引文
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