MTDH通过Wnt信号通路介导上皮间质转化对膀胱癌细胞功能学影响的机制研究

发布时间：2018-05-11 19:15

本文选题：MTDH + E-cadherin　；参考：《重庆医科大学》2016年博士论文

【摘要】：膀胱癌是泌尿系最常见的恶性肿瘤,临床上,膀胱癌多采用肿瘤切除,术后辅助化疗。然而,由于膀胱癌的高转移性,膀胱癌的复发率及死亡率仍然很高。故研究膀胱癌的转移机制是很有必要的。MTDH是近年来发现的癌基因,已有报道其在恶性肿瘤中表达较高。上皮间质转化(EMT)是上皮细胞获得间质表型,易于侵袭转移的特性,其经典上皮标志物为E-cadherin。有报道显示,在恶性肿瘤中,MTDH可促进EMT发生,而EMT是膀胱癌发生侵袭、转移的重要原因。目前,在膀胱癌中尚无MTDH与EMT的相关研究。本研究拟通过分析在膀胱癌组织及细胞中MTDH与E-cadherin的表达关系,探讨MTDH与EMT是否存在某种联系,进而采用基因沉默及过表达技术,深入研究MTDH对EMT的影响,及MTDH促进膀胱癌细胞侵袭、转移的机制。第一部分MTDH和E-cadherin在膀胱癌组织及细胞中的表达及临床意义目的检测MTDH、E-cadherin在膀胱癌组织及细胞中的表达,探讨MTDH、E-cadherin与临床病理特征的关系,以及MTDH与E-cadherin表达的相关性。方法1.应用免疫组化(immunohistochemistry,IHC)SP法检测膀胱癌组织中MTDH和EMT上皮标志物E-cadherin的表达水平。分析MTDH、E-cadherin蛋白表达与临床病理特征之间的关系,Spearman’s法分析MTDH与E-cadherin表达可能存在的相关性。2.Real-time PCR方法检测MTDH与E-cadherin在膀胱癌细胞株、尿路上皮细胞株中m RNA的表达水平。3.Western blot方法检测在膀胱癌细胞株、尿路上皮细胞株MTDH与E-cadherin蛋白的表达水平。结果1.与癌旁组织比较,癌组织MTDH的蛋白表达水平明显增高,E-cadherin的表达水平明显降低。MTDH、E-cadherin表达与患者年龄、性别、肿瘤大小、数量无关,与肿瘤远处转移、临床分期、病理分级有关。MTDH与E-cadherin的蛋白表达之间呈负相关(r=-0.722,P0.05)。2.膀胱癌细胞株5637、T24中MTDH m RNA表达水平相对于人尿路上皮细胞株SV-HUC-1中显著升高;E-cadherin m RNA表达水平在膀胱癌细胞株5637、T24中相对于人尿路上皮细胞株SV-HUC-1中显著降低。3.膀胱癌细胞株5637、T24中MTDH蛋白表达水平相对于人尿路上皮细胞株SV-HUC-1中显著升高;E-cadherin的蛋白表达水平在膀胱癌细胞株5637、T24中相对于其在人尿路上皮细胞株SV-HUC-1中显著降低。结论MTDH在膀胱癌组织及细胞中高表达,E-cadherin在癌旁组织及正常细胞中高表达。MTDH与肿瘤临床病理学特征密切相关,可作为判断肿瘤临床病理的分子指标。MTDH与E-cadherin呈负相关,相互之间存在负向调控。第二部分MTDH基因沉默对膀胱癌细胞功能学影响及其机制研究目的探讨抑制MTDH表达对膀胱癌细胞增殖、凋亡、迁移、侵袭的影响及MTDH沉默对上皮间质转化相关标志物的影响方法1.Real-time PCR检测si RNA-MTDH转染5637和T24细胞后MTDH m RNA变化情况,Western blot方法检测si RNA-MTDH转染5637和T24细胞后,MTDH蛋白表达情况,分析si RNA的抑制效率,最终筛选出沉默效率最高的si RNA。2.通过CCK8法检测MTDH基因沉默后5637和T24的增殖能力的变化,流式细胞术检测MTDH基因沉默对5637和T24细胞凋亡的影响,细胞划痕实验检测MTDH基因沉默对5637、T24细胞迁移能力的影响,Transwell实验检测MTDH基因沉默对5637、T24细胞的侵袭能力的影响。3.Real-time PCR方法检测si RNA2-MTDH转染5637、T24细胞后上皮间质转化相关标志物E-cadherin、N-cadherin、Vimentin m RNA的表达变化。Western blot检测si RNA2-MTDH转染5637、T24细胞后上皮间质转化相关标志物E-cadherin、N-cadherin、Vimentin蛋白的表达变化。细胞免疫荧光技术检测si RNA2-MTDH转染5637、T24细胞后上皮间质转化相关标志物E-cadherin、N-cadherin、Vimentin荧光表达变化。结果1.从基因及蛋白水平验证si RNA2-MTDH沉默效率大于70%,筛选确定si RNA2用于后续实验。2.CCK8法实验结果显示:与对照组相比,MTDH基因沉默后膀胱癌细胞5637、T24的增殖能力明显降低,流式细胞仪检测结果提示MTDH基因沉默后5637、T24凋亡率较对照组明显增加,细胞划痕实验、Transwell实验结果提示MTDH基因沉默后5637、T24细胞迁移及侵袭能力显著降低。3.Real-time PCR、Western blot实验提示si RNA2-MTDH转染5637、T24细胞后上皮相关标志物E-cadherin m RNA及蛋白表达显著增高,而间质标志物N-cadherin、Vimentin m RNA及蛋白表达显著降低。细胞免疫荧光技术检测提示si RNA2-MTDH转染5637、T24细胞后相关标志物E-cadherin荧光表达增强,阳性细胞数增多;而N-cadherin、Vimentin荧光表达减弱,阳性细胞数减少。结论MTDH基因沉默抑制膀胱癌细胞增殖,促进其凋亡,抑制膀胱癌细胞迁移及侵袭,促进上皮标志物表达,抑制间质标志物表达,抑制上皮间质转化。第三部分MTDH过表达对膀胱癌细胞功能学影响及其机制研究目的探讨MTDH过表达对膀胱癌细胞增殖、凋亡、迁移、侵袭的影响及MTDH过表达对上皮间质转化相关标志物的影响方法1.构建MTDH过表达慢病毒载体GV341-MTDH及GV341-NC,制备慢病毒浓缩液LV-MTDH,LV-NC,感染5637、T24细胞,建立过表达MTDH的LV-MTDH单克隆细胞株及阴性对照组LV-NC细胞株。Real-time PCR检测5637和T24细胞过表达MTDH后MTDH m RNA变化情况,Western blot方法检测5637和T24细胞过表达MTDH后,MTDH蛋白表达情况。2.通过CCK8法检测5637和T24细胞MTDH过表达后增殖能力的变化,流式细胞术检测MTDH基因过表达对5637和T24细胞凋亡的影响,细胞划痕实验检测MTDH基因过表达对5637、T24细胞迁移能力的影响,Transwell实验检测MTDH基因过表达对5637、T24细胞的侵袭能力的影响。3.Real-time PCR方法检测5637、T24细胞MTDH过表达后上皮间质转化相关标志物E-cadherin、N-cadherin、Vimentin m RNA的表达变化。Western blot检测5637、T24细胞MTDH过表达后上皮间质转化相关标志物E-cadherin、N-cadherin、Vimentin蛋白表达变化。细胞免疫荧光技术检测5637、T24细胞MTDH过表达后E-cadherin、N-cadherin、Vimentin荧光表达变化。结果1.MTDH过表达慢病毒载体构建成功,从基因及蛋白水平证实成功建立过表达MTDH的LV-MTDH单克隆细胞株及阴性对照组LV-NC细胞株。2.CCK8法实验结果显示:与对照组相比,MTDH过表达后膀胱癌细胞5637、T24的增殖能力明显增强,流式细胞仪检测结果提示MTDH过表达后5637、T24凋亡率较对照组明显降低,细胞划痕实验、Transwell实验结果提示MTDH基因过表达后5637、T24细胞迁移及侵袭能力显著增强。3.Real-time PCR、Western blot实验提示5637、T24细胞MTDH基因过表达后上皮间质转化相关标志物E-cadherin m RNA及蛋白表达明显减弱,而N-cadherin、Vimentin m RNA及蛋白表达显著增强。细胞免疫荧光技术检测提示5637、T24细胞MTDH基因过表达后E-cadherin荧光表达减弱,阳性细胞数减少;而N-cadherin、Vimentin荧光表达增强,阳性细胞数增多。结论MTDH基因过表达促进膀胱癌细胞增殖,抑制其凋亡,增强膀胱癌细胞迁移及侵袭能力,抑制上皮标志物表达,提高间质标志物表达,促进上皮间质转化。第四部分MTDH过表达对T24、5637细胞Wnt/β-catenin信号通路及其下游靶基因的调控目的探讨MTDH过表达对T24、5637细胞Wnt/β-catenin信号通路及其下游靶基因的调控方法1.Western blot检测MTDH过表达对5637、T24细胞Wnt/β-catenin信号通路、下游靶基因及基质金属蛋白酶MMP9蛋白表达的影响2.免疫荧光检测MTDH过表达5637和T24细胞对β-catenin荧光表达的影响结果1.Western blot实验提示5637、T24细胞MTDH基因过表达后Wnt通路被激活,细胞内β-catenin总蛋白表达增多,胞核β-catenin蛋白表达显著增多,而胞质β-catenin蛋白表达明显减低,c-Myc及MMP9蛋白表达显著增加。2.免疫荧光实验提示:5637、T24细胞MTDH基因过表达后,β-catenin蛋白荧光表达增强,阳性细胞数增多,且胞核内表达显著增强。结论慢病毒载体介导的MTDH基因过表达经Wnt/β-catenin信号通路,促进上皮间质转化,增强膀胱癌细胞侵袭能力。
[Abstract]:Bladder cancer is the most common malignant tumor of the urinary system. In clinic, tumor resection and adjuvant chemotherapy are mostly used in bladder cancer. However, the recurrence rate and death rate of bladder cancer are still high because of the high metastasis of bladder cancer. Therefore, it is necessary to study the metastasis mechanism of bladder cancer in recent years, and it has been reported that.MTDH is in the evil. The expression of epithelial mesenchymal transition (EMT) is the characteristic of epithelial cells obtaining interstitial phenotypes and prone to invasion and metastasis. The classical epithelial markers of E-cadherin. have reported that MTDH can promote the occurrence of EMT in malignant tumors, and EMT is an important reason for the invasion and metastasis of bladder cancer. At present, there is no MTDH and EMT in bladder cancer. The purpose of this study is to analyze the relationship between MTDH and E-cadherin in bladder cancer tissues and cells, to explore the relationship between MTDH and EMT, and then to use gene silencing and overexpression technology to study the effect of MTDH on EMT, and the mechanism of MTDH to promote the invasion and metastasis of bladder cancer cells. The first part MTDH and E-cadherin Expression and clinical significance in bladder cancer tissues and cells objective to detect the expression of MTDH, E-cadherin in bladder cancer tissues and cells, to explore the relationship between MTDH, E-cadherin and clinicopathological features, and the correlation between MTDH and E-cadherin expression. Method 1. the SP method of immunohistochemistry (IHC) was used to detect MTD in bladder cancer tissues. The expression level of H and EMT epithelial marker E-cadherin. Analysis of the relationship between MTDH, E-cadherin protein expression and clinicopathological features, Spearman 's method to analyze the possible correlation between MTDH and E-cadherin expression and.2.Real-time PCR method to detect the expression level of MTDH in bladder cancer cell lines and urinary tract epithelial cells. .Western blot method was used to detect the expression level of MTDH and E-cadherin protein in bladder cancer cell line and urinary tract epithelial cell line. Results 1. compared with para cancer tissue, the protein expression level of MTDH in cancer tissue was significantly higher, the expression level of E-cadherin decreased obviously, and the expression of E-cadherin was not related to age, sex, size and quantity of the patient, and the swelling of E-cadherin. Tumor distant metastasis, clinical staging, pathological grade related to the protein expression of.MTDH and E-cadherin was negatively correlated (r=-0.722, P0.05).2. bladder cancer cell line 5637, MTDH m RNA expression level in T24 was significantly higher in T24 than in human urinary tract epithelial cell strain SV-HUC-1; E-cadherin m (E-cadherin m) expression level in bladder cancer cell line 5637, relative to human The urinary tract epithelial cell line SV-HUC-1 significantly reduced the.3. bladder cancer cell line 5637, and the expression level of MTDH protein in T24 was significantly higher than that in human urinary tract epithelial cell strain SV-HUC-1; the protein expression level of E-cadherin was significantly lower in bladder cancer cell line 5637 and in T24 than in human urinary tract epithelial cell strain SV-HUC-1. Conclusion MTDH is in bladder. High expression of E-cadherin in cystocarcinoma tissue and cells, high expression of.MTDH in para cancer tissues and normal cells is closely related to the clinicopathological features of tumor. It can be a negative correlation between.MTDH and E-cadherin as a molecular marker of tumor clinicopathology, and there is a negative regulation between each other. Second part of MTDH gene silencing on the function of bladder cancer cells Effect and mechanism study to investigate the effect of inhibition of MTDH expression on the proliferation, apoptosis, migration, invasion of bladder cancer cells and the effect of MTDH silence on epithelial mesenchymal transition related markers. 1.Real-time PCR detection of MTDH m RNA changes after Si RNA-MTDH transfection of 5637 and T24 cells, Western blot method After 24 cells, the expression of MTDH protein was expressed and the inhibition efficiency of Si RNA was analyzed. Finally, the Si RNA.2. with the highest silence efficiency was selected to detect the proliferation of 5637 and T24 after MTDH gene silencing by CCK8 method. The effect of MTDH gene silencing on the apoptosis of 5637 and T24 cells was detected by flow cytometry, and the cell scratch test was used to detect the MTDH gene silencing of 563 7, the effect of T24 cell migration, Transwell test detected the influence of MTDH gene silencing on the invasiveness of 5637, T24 cells,.3.Real-time PCR method was used to detect Si RNA2-MTDH transfection 5637, T24 cell epithelial mesenchymal transition related markers E-cadherin, N-cadherin. The expression of epithelial mesenchymal transition related markers E-cadherin, N-cadherin, Vimentin protein expression after T24 cells. Cell immunofluorescence technique was used to detect Si RNA2-MTDH transfection 5637, E-cadherin, N-cadherin, Vimentin fluorescent expression of epithelial mesenchymal transition markers after T24 cells, and 1. from gene and protein level to verify Si RNA2-MTDH precipitation. The effect of Si RNA2 was more than 70%. The results of the screening and determination of Si RNA2 for subsequent experiment showed that compared with the control group, the proliferation ability of bladder cancer cells was 5637 and T24 was significantly reduced after the silence of the control group. The results of flow cytometry showed that the MTDH gene was 5637, and the apoptosis rate of T24 was significantly higher than that of the control group, and the cell scratch test and Transwell were real. The results suggested that after MTDH gene silencing, 5637, T24 cell migration and invasion ability significantly decreased.3.Real-time PCR, Western blot experiment suggested that Si RNA2-MTDH transfected 5637, E-cadherin m RNA and protein expression was significantly increased after T24 cell, and the interstitial marker and protein expression decreased significantly. The immunofluorescence technique showed that the transfection of Si RNA2-MTDH was 5637, the fluorescent expression of E-cadherin was enhanced and the number of positive cells increased, while N-cadherin, Vimentin fluorescent expression decreased, and the number of positive cells decreased. Conclusion MTDH gene silencing inhibits the proliferation of bladder cancer cells, promotes its apoptosis and inhibits the migration and invasion of bladder cancer cells. Promote the expression of epithelial markers, inhibit the expression of interstitial markers, inhibit epithelial mesenchymal transition. Third the effect of MTDH overexpression on the function of bladder cancer cells and its mechanism research to explore the effect of MTDH overexpression on the proliferation, apoptosis, migration, invasion of bladder cancer cells and the effect of MTDH overexpression on epithelial mesenchymal transition markers Methods 1. MTDH overexpressed lentivirus vectors, GV341-MTDH and GV341-NC, were constructed to prepare the lentivirus concentrate LV-MTDH, LV-NC, infection 5637, T24 cells, and the LV-MTDH McAb of MTDH was established and the LV-NC cell line of the negative control group was detected 5637 and the T24 cells were detected. After the overexpression of MTDH in 5637 and T24 cells, the expression of MTDH protein was detected by.2.. The proliferation ability of 5637 and T24 cells was detected by CCK8, and the effect of MTDH gene overexpression on the apoptosis of 5637 and T24 cells was detected by flow cytometry. The cell scratch test was used to detect the effect of MTDH gene over the migration ability of 5637 and T24 cells. The influence of the overexpression of MTDH gene on the invasiveness of 5637, T24 cells;.3.Real-time PCR method was used to detect the expression of E-cadherin, N-cadherin, Vimentin m RNA, after the overexpression of MTDH in 5637, T24 cell MTDH Cadherin, N-cadherin, Vimentin protein expression changes. Cell immunofluorescence technology detected 5637, T24 cell MTDH overexpression E-cadherin, N-cadherin, Vimentin fluorescence expression changes. Results 1.MTDH overexpression lentivirus vector construction successfully, from gene and protein level confirmed the successful establishment of LV-MTDH monoclonal cell lines and negative expression MTDH negative and negative The results of.2.CCK8 assay in the control group LV-NC showed that the proliferation ability of bladder cancer cells 5637 and T24 increased significantly after MTDH overexpression compared with the control group. The results of flow cytometry showed that after MTDH overexpression was 5637, the apoptosis rate of T24 was significantly lower than that of the control group, the cell scratch test, and the result of Transwell test suggested that the MTDH gene was overexpressed 56. 37, the migration and invasion ability of T24 cells significantly enhanced.3.Real-time PCR. Western blot experiments suggested that the expression of E-cadherin m RNA and protein expression of epithelial mesenchymal transition was significantly weakened after the overexpression of MTDH gene in T24 cells, while N-cadherin, Vimentin and protein expression was enhanced. Cell immunofluorescence technique detection suggested 5637. After the overexpression of MTDH gene, the fluorescence expression of E-cadherin decreased, the number of positive cells decreased, while the expression of N-cadherin, Vimentin increased and the number of positive cells increased. Conclusion MTDH gene overexpression promotes the proliferation of bladder cancer cells, inhibits its apoptosis, enhances the migration and invasion ability of bladder cancer cells, inhibits the expression of epithelial markers, and improves the standard of interstitial markers. Fourth part MTDH overexpression on T245637 cell Wnt/ beta -catenin signaling pathway and its downstream target genes regulation of MTDH overexpression on T245637 cells Wnt/ beta -catenin signaling pathway and its downstream target gene regulation, 1.Western blot detection MTDH overexpression of 5637, T24 cells The influence of tenin signaling pathway, downstream target gene and matrix metalloproteinase MMP9 protein expression 2. immunofluorescence detection of MTDH overexpression 5637 and T24 cells on the expression of beta -catenin fluorescent expression results 1.Western blot experiment 5637, T24 cell MTDH overexpression Wnt pathway is activated, intracellular beta -catenin total protein expression increased, the nucleus of the cell nucleus The expression of beta -catenin protein was significantly increased, while the expression of cytoplasmic beta -catenin protein was significantly reduced, and the expression of c-Myc and MMP9 protein was significantly increased by.2. immunofluorescence experiment: 5637, after the MTDH gene was overexpressed in T24 cells, the expression of beta -catenin protein increased, the number of positive cells increased, and the expression of the cell nucleus increased significantly. Conclusion lentivirus vector mediated M Over expression of TDH gene promotes epithelial mesenchymal transition and enhances invasion ability of bladder cancer cells through Wnt/ beta -catenin signaling pathway.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.14

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