MDM2促进人乳腺癌细胞上皮间质转化及其分子机制

发布时间：2018-05-18 13:43

本文选题：MDM2 + 乳腺癌　；参考：《南京医科大学》2016年博士论文

【摘要】：研究背景：乳腺癌是世界上女性最常见的恶性肿瘤,其死亡率在女性恶性肿瘤中占第二位。上皮间质转化(epithelial-mesenchymal transitions,EMT)是启动肿瘤侵袭和转移的一个公认的机制,上皮间质转化发生时,乳腺癌上皮细胞获得一个能动的间质表型。MDM2基因在人类多种恶性肿瘤,如软组织肉瘤、乳腺癌、卵巢癌、宫颈癌、脑癌、肺癌、前列腺癌、结肠癌和肾癌中均高表达。在我们以前的研究中,我们发现MDM2能够通过上调MMP9的表达促进乳腺癌的侵袭和转移。MDM2是否还通过其他途径促进乳腺癌的侵袭和转移,值得我们进一步深入研究。研究目的：研究MDM2在人乳腺癌细胞上皮间质转化中的作用及其可能的分子机制。研究方法：我们首先通过定量PCR和western blotting法检测了三种乳腺癌细胞系MCF-7、MDA-MB-231、MDA-MB-435和一种正常乳腺细胞系HBL-100中MDM2的mRNA和蛋白水平的表达情况。然后,我们以慢病毒为载体,构建了过表达MDM2的乳腺癌稳转细胞株MCF-7-MDM2-a,MCF-7-MDM2-d及对照细胞株MCF-7-pCMV,通过光镜观察三种细胞株的形态,定量PCR及western blot检测上皮标记物E-钙粘蛋白的表达,转录因子Snail的表达以及间质标记物N-钙粘蛋白和波形蛋白的表达。然后,我们通过干扰RNA在MDA-MB-231细胞中敲低MDM2的表达,通过光镜观察两种细胞株的形态,定量PCR及western blotting检测上皮标记物E-钙粘蛋白的表达,间质标记物N-钙粘蛋白和波形蛋白的表达以及转录因子Snail的表达。接着我们通过干扰RNA在MCF-7-MDM2-a细胞中敲低Snail的表达,通过光镜观察两种细胞株的形态,定量PCR及western blotting检测上皮标记物E-钙粘蛋白的表达及间质标记物N-钙粘蛋白和波形蛋白的表达。然后,我们在MCF-7细胞中敲低Snail后再瞬时转染入MDM2质粒,定量PCR及western blotting检测EMT标记物E-cadherin,N-cadherin及Vimentin的表达情况。接着,我们进一步在MCF-7细胞中敲低NF-kappaB/P65亚基后再瞬时转染入MDM2质粒,定量PCR及western blotting检测转录因子Snail的表达。然后,我们用构建的乳腺癌稳转细胞株MCF-7-MDM2-a及对照细胞株MCF-7-pCMV接种雌性裸鼠腋下成瘤,六周后处死裸鼠,测量肿瘤的大小及重量,并通过定量PCR、western blotting及免疫组化法检测瘤体中上皮标记物E-钙粘蛋白的表达,转录因子Snail的表达以及间质标记物N-钙粘蛋白和波形蛋白的表达。最后,我们通过人乳腺癌芯片研究上皮标记物E-钙粘蛋白,间质标记物N-钙粘蛋白和波形蛋白以及转录因子Snail在人乳腺癌组织中的表达,并研究其相关性。研究结果：MDM2在侵袭性乳腺癌细胞系MDA-MB-231和MDA-MB-435中均高表达,在MCF-7细胞中,MDM2过表达通过NF-kappa/B P65亚基上调Snail的表达,上调的Snail可以促进细胞发生上皮间质转化。而在MDA-MB-231细胞中,敲低MDM2促进间质上皮转化。在MCF-7-MDM2-a细胞中,通过干扰RNA敲低Snail可导致MCF-7-MDM2-a细胞发生间质上皮转化,细胞恢复上皮性状。在动物水平,我们发现MDM2可以促进肿瘤生长,诱导乳腺癌细胞发生上皮间质转化及赋予人乳腺癌细胞转移潜能。最后,在乳腺癌芯片中,我们发现MDM2的表达与上皮标记物E-cadherin的表达呈负相关,而与间质标记物N-cadherin、Vimentin以及转录因子Snail的表达呈正相关。Snail的表达与上皮标记物E-cadherin的表达呈负相关。研究结论：MDM2通过NF-kappa/B P65亚基上调Snail的表达,上调的Snail促使人乳腺癌细胞发生上皮间质转化,MDM2有可能成为乳腺癌转移治疗和预防的一个潜在靶点。
[Abstract]:Background: breast cancer is the most common malignant tumor in women in the world. The mortality rate is second in female malignant tumors. Epithelial-mesenchymal transitions (EMT) is a recognized mechanism for the initiation of tumor invasion and metastasis. Interstitial phenotypic.MDM2 gene is highly expressed in a variety of human malignant tumors, such as soft tissue sarcoma, breast cancer, ovarian, cervical, brain, lung, prostate, colon and renal cancer. In our previous study, we found that MDM2 can promote the invasion of breast cancer and transfer.MDM2 through other routes through the up regulation of MMP9. The path to promote the invasion and metastasis of breast cancer is worthy of further study. Objective: To study the role of MDM2 in the epithelial mesenchymal transition of human breast cancer cells and its possible molecular mechanism. Research methods: we first detected three kinds of breast cancer cell lines, MCF-7, MDA-MB-231, MDA-MB-435 by quantitative PCR and Western blotting method. And the expression of mRNA and protein levels of MDM2 in a normal mammary cell line HBL-100. Then, we use lentivirus as the carrier to construct a breast cancer cell line that overexpresses MDM2, MCF-7-MDM2-a, MCF-7-MDM2-d and the control cell line MCF-7-pCMV, and observe the morphology of three cell lines by light microscopy, quantitative PCR and Western blot detection of epithelium. Expression of marker E- cadherin, expression of transcription factor Snail and expression of stromal marker N- cadherin and vimentin. Then, we observe the morphology of two cell lines by interfering with RNA in MDA-MB-231 cells and observe the morphology of the cell lines by light microscopy, quantitative PCR and Western blotting detection of the epithelial markers E- cadherin. Expression, expression of stromal marker N- and vimentin and expression of transcription factor Snail. Then we observe the morphology of two cell lines by interfering with RNA in MCF-7-MDM2-a cells and observe the morphology of the two cell lines by light microscopy, quantitative PCR and Western blotting to detect the expression of the epithelial markers E- cadherin and the interstitial marker N. The expression of cadherin and vimentin. Then, we transfected into MDM2 plasmids after knocking down Snail in MCF-7 cells, and quantified PCR and Western blotting to detect the EMT marker E-cadherin, N-cadherin and Vimentin expression. Then, we further transfected into the substance after knocking down the NF-kappaB/P65 subunit in the MCF-7 cell. The expression of transcription factor Snail was detected by grain, quantitative PCR and Western blotting. Then, we used the constructed breast cancer cell line MCF-7-MDM2-a and the control cell strain MCF-7-pCMV to be inoculated into the axillary tumor of female nude mice. After six weeks, the nude mice were killed and the size and weight of the tumor were measured, and the quantitative PCR, Western blotting and immunohistochemistry were detected. Expression of E- cadherin, expression of transcription factor Snail and expression of stromal marker N- cadherin and vimentin in the tumor. Finally, we study the epithelial markers E- cadherin by human breast cancer microarray, interstitial marker N- cadherin, wave white egg white and transcription factor Snail in human breast cancer tissue The expression of MDM2 is highly expressed in invasive breast cancer cell lines MDA-MB-231 and MDA-MB-435. In MCF-7 cells, MDM2 overexpression increases the expression of Snail through the NF-kappa/B P65 subunit, and up-regulated Snail can promote epithelial mesenchymal transition. In MDA-MB-231 cells, low MDM2 promotes low MDM2. Mesenchymal epithelial transformation. In MCF-7-MDM2-a cells, RNA knockdown Snail can lead to interstitial epithelial transformation in MCF-7-MDM2-a cells and cells restore epithelial traits. At animal levels, we found that MDM2 can promote tumor growth, induce epithelial stroma transformation of breast cancer cells and give human breast cancer cell metastasis potential. Finally, In the breast cancer chip, we found that the expression of MDM2 was negatively correlated with the expression of the epithelial marker E-cadherin, but the expression of the interstitial marker N-cadherin, Vimentin and the transcription factor Snail was positively correlated with the expression of.Snail and the expression of the epithelial marker E-cadherin. The conclusion: MDM2 is up Snail by the NF-kappa/B P65 subunit. The expression of upregulation of Snail promotes epithelial mesenchymal transition in human breast cancer cells. MDM2 may be a potential target for breast cancer metastasis therapy and prevention.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9

【相似文献】