ING5基因过表达对乳腺癌细胞恶性表型的影响

发布时间：2018-03-14 16:19

本文选题：乳腺癌　切入点：Bcap-37细胞　出处：《锦州医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的研究生长抑制因子5(the protein of the inhibitor of growth 5,ING5)基因过表达对人乳腺癌细胞Bcap-37细胞增殖,细胞周期阻滞和细胞凋亡、侵袭与迁移和上皮间质转化(epithelial-mesenchymal transition,EMT)的影响,以及可能通过PI3K/AKT信号通路影响EMT。方法人乳腺癌细胞Bcap-37稳转p EGFP-N1-ING5真核表达载体和p EGFP-N1空载体,荧光显微镜检查绿色荧光蛋白的表达,实时荧光定量聚合酶链反应PCR和蛋白质印迹方法筛选ING5过表达细胞株。Bcap-37-ING5细胞为实验组,Bcap-37-GFP细胞作为空载体组,Bcap-37细胞作为空白对照组。采用MTT法检测ING5对Bcap-37细胞增殖的影响,采取流式细胞仪检测细胞凋亡及周期的影响,采取细胞划痕实验和Transwell实验来检测Bcap-37细胞的迁移和侵袭情况,实时荧光定量PCR和蛋白质印迹检测ING5对乳腺癌细胞EMT相关基因E-cadherin和N-cadherin表达水平的影响。构建裸鼠成瘤模型,采用免疫组化法检测ING5过表达对人乳腺癌细胞在裸鼠体内成瘤的EMT相关蛋白Ecadherin和N-cadherin的影响。采用蛋白质印迹检测ING5基因过表达对PI3K/AKT磷酸化水平的改变。结果人乳腺癌细胞Bcap-37经稳转并筛选得到稳定高表达绿色荧光蛋白(green fliorescent protein,GFP)标记的ING5蛋白的Bcap-37细胞株。相比空白对照组和空载体组,ING5过表达可以降低人乳腺癌细胞Bcap-37细胞增殖率,并导致G2期阻滞,诱导人乳腺癌细胞Bcap-37发生细胞凋亡率增加,抑制人乳腺癌细胞Bcap-37的侵袭与迁移能力,并逆转EMT表型(P0.05),这可能是通过抑制PI3K/AKT信号通路的磷酸化水平从而逆转EMT进程。结论ING5基因表达与乳腺癌发生、转移和分化紧密相关,ING5过表达对人乳腺癌细胞Bcap-37控制增殖、发生G2期阻滞、诱导凋亡、抑制侵袭与迁移,并可能通过抑制PI3K/AKT途径抑制乳腺癌细胞中EMT进程,逆转肿瘤的恶性表型,可作为乳腺癌的基因治疗和预后的分子靶标。
[Abstract]:Objective to investigate the effects of overexpression of the protein of the inhibitor of growth 5 gene on proliferation, cell cycle arrest and apoptosis, invasion and migration and epithelial-mesenchymal transition of human breast cancer cell line Bcap-37. Methods Human breast cancer cell line Bcap-37 was stably transformed into p EGFP-N1-ING5 eukaryotic expression vector and p EGFP-N1 empty vector, and the expression of green fluorescent protein was detected by fluorescence microscope. Real-time fluorescence quantitative polymerase chain reaction (PCR) and Western blot were used to screen ING5 overexpression cell line .Bcap-37-ING5 cells as blank control group. MTT assay was used to detect the effect of ING5 on the proliferation of Bcap-37 cells. Flow cytometry was used to detect cell apoptosis and cell cycle, cell scratch test and Transwell assay were used to detect the migration and invasion of Bcap-37 cells. The effect of ING5 on the expression of EMT related genes E-cadherin and N-cadherin in breast cancer cells was detected by real-time fluorescence quantitative PCR and Western blot. The effect of ING5 overexpression on EMT associated protein Ecadherin and N-cadherin in human breast cancer cells in nude mice was detected by immunohistochemical method. Western blotting was used to detect the changes of PI3K/AKT phosphorylation level in human breast cancer cells. Bcap-37 was stably transformed into Bcap-37 cell line which was stably expressed green fliorescent protein (GFP). Overexpression of ING5 could reduce the proliferation rate of human breast cancer Bcap-37 cells compared with blank control group and empty vector group. It also resulted in G2 arrest, increased apoptosis rate of human breast cancer cell Bcap-37, and inhibited the invasion and migration of human breast cancer cell Bcap-37. And reverse the EMT phenotype P0.05, which may reverse the process of EMT by inhibiting the phosphorylation level of PI3K/AKT signaling pathway. Conclusion the expression of ING5 gene is closely related to the occurrence, metastasis and differentiation of breast cancer, and the overexpression of ING5 can control the proliferation of human breast cancer cell line Bcap-37. G _ 2 arrest induces apoptosis inhibits invasion and migration and inhibits EMT progression in breast cancer cells by inhibiting PI3K/AKT pathway and reversing the malignant phenotype of breast cancer. It can be used as a molecular target for gene therapy and prognosis of breast cancer.
【学位授予单位】：锦州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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