microRNA-935在胰腺癌组织及细胞中表达及对细胞增殖、迁移、凋亡的影响及相关机制研究

发布时间：2018-04-30 19:40

本文选题：miR-935 + 靶基因　；参考：《山东大学》2017年博士论文

【摘要】：研究目的检测miR-935在胰腺癌组织及细胞中的表达情况,并研究其对胰腺癌细胞增殖、迁移、凋亡的影响。预测miR-935作用的直接靶基因,并探讨其影响胰腺癌细胞恶性生物学行为的作用机制。研究方法(1)收集新鲜胰腺癌组织及癌旁正常组织标本,选取正常胰腺导管HPDE6-C7细胞及胰腺癌PANC-1细胞进行培养;(2)以实时定量PCR(Real-time PCR)方法测定胰腺癌组织及胰腺癌PANC-1细胞中miR-935表达情况;(3)利用生物学信息学数据库预测miR-935直接靶基因,并应用荧光素酶基因报告实验验证靶基因的合理性;(4)分别应用miR-935类似物,mir-935抑制物和miR-935拮抗物对胰腺癌PANC-1细胞进行转染,MTT法及克隆形成法检测转染细胞增殖情况、Transwell法检测转染细胞迁移能力、流式细胞仪检测转染细胞凋亡情况、通过qRT-PCR及Western blot检测P21、P27、EMT相关分子及凋亡相关分子在转染细胞中的表达情况、沉默靶基因表达以探讨miR-935与靶基因之间的调控方式。结果(1)与正常胰腺组织相比,miR-935在胰腺癌组织中表达显著上调,而INPP4A的表达显著下调;(2)与正常胰腺导管HPDE6-C7细胞相比,miR-935在胰腺癌PANC-1细胞中表达显著上调,而INPP4A的表达显著下调;(3)胰腺癌PANC-1细胞转染miR-935抑制物后,miR-935表达被明显抑制,表达下调的miR-935显著抑制细胞的增殖、迁移,并促进诱导细胞的凋亡;(4)miR-935表达抑制导致P27表达水平显著上调;导致EMT相关分子表达水平显著变化:E-cadherin的表达水平明显上调,N-cadherin、Snail和Vimentin的表达水平均明显下调;导致凋亡相关分析表达水平显著变化:Bax的表达水平明显上调,Bcl-2、pro-caspase-3及active-caspase-3的表达水平明显下调;(5)胰腺癌PANC-1细胞转染miR-935类似物后,miR-935表达被明显上调,表达上调的miR-935显著促进细胞的增殖、迁移,并抑制诱导细胞的凋亡;(6)miR-935表达上调导致P27表达水平显著下调;导致EMT相关分子表达水平显著变化:E-cadherin的表达水平明显下调,N-cadherin、Snail和Vimentin的表达水平均明显上调;导致凋亡相关分析表达水平显著变化:Bax的表达水平明显下调,Bcl-2、pro-caspase-3及active-caspase-3的表达水平明显上调;(7)INPP4A是miR-935直接作用靶基因。沉默INPP4A表达显著抵消了miR-935表达抑制对胰腺癌PANC-1细胞迁移及凋亡的抑制作用;也显著抵消了miR-935表达抑制对EMT及凋亡相关分子表达水平的影响。结论miR-935在胰腺癌组织及胰腺癌PANC-1中均有表达,上调miR-935表达水平可以促进胰腺癌细胞增殖及迁移能力,并抑制细胞凋亡。miR-935可能通过直接作用于靶基因INPP4A调控胰腺癌细胞恶性生物学行为。miR-935及INPP4A可能能成为胰腺癌基因治疗的潜在靶点。
[Abstract]:Objective to investigate the expression of miR-935 in pancreatic carcinoma and its effect on proliferation, migration and apoptosis of pancreatic cancer cells. To predict the direct target gene of miR-935 and to explore the mechanism of its influence on malignant biological behavior of pancreatic cancer cells. Methods 1) fresh pancreatic cancer tissues and adjacent normal tissues were collected. Normal pancreatic duct HPDE6-C7 cells and pancreatic cancer PANC-1 cells were selected for culture. The expression of miR-935 in pancreatic carcinoma tissues and pancreatic cancer PANC-1 cells was detected by real-time quantitative PCR(Real-time PCR. The direct target gene of miR-935 was predicted by bioinformatics database. Using luciferase gene report experiment to verify the rationality of target gene. (4) using miR-935 analogues (miR-935 analogs) and miR-935 antagonists to detect the proliferation of pancreatic cancer PANC-1 cells by MTT assay and clone formation method respectively. The ability of transfection cells to migrate was detected by Elisa. Apoptosis of transfected cells was detected by flow cytometry. The expression of P21 P27EMT related molecules and apoptosis-related molecules in transfected cells was detected by qRT-PCR and Western blot. The expression of target genes was silenced to explore the regulation between miR-935 and target genes. Results 1) compared with normal pancreatic tissues, the expression of mmiR-935 was significantly up-regulated in pancreatic carcinoma tissues, while the expression of INPP4A was significantly down-regulated in pancreatic carcinoma tissues. Compared with normal pancreatic ductal HPDE6-C7 cells, the expression of mmiR-935 was significantly up-regulated in pancreatic cancer PANC-1 cells. However, the expression of INPP4A was significantly down-regulated in PANC-1 cells of pancreatic cancer. After transfection of miR-935 inhibitor, the expression of miR-935 was significantly inhibited, and the down-regulated miR-935 significantly inhibited the proliferation and migration of the cells. The expression level of P27 and the expression of EMT related molecules were significantly up-regulated and down-regulated by up-regulation of N-cadherin Snail and down-regulation of Vimentin expression, and the expression of P27 was significantly up-regulated by promoting the expression inhibition of apoptosis-inducing cell line 4miR-935, leading to a significant change in the expression level of EMT related molecules, and a marked down-regulation of the expression levels of N-cadherin and Snail. The apoptosis-related analysis showed that the expression level of Bcl-2P pro-caspase-3 and active-caspase-3 were up-regulated significantly (P < 0.05). The expression of miR-935 was upregulated after transfection of miR-935 analogues in PANC-1 cells of pancreatic carcinoma, and the up-regulated miR-935 significantly promoted cell proliferation. Migration and inhibition of apoptosis-induced cell apoptosis resulted in a significant down-regulation of P27 expression, a significant change in the expression level of EMT related molecules, and a marked down-regulation of the expression level of N-cadherin Snail and Vimentin. As a result, the expression level of Bcl-2 pro-caspase-3 and active-caspase-3 were significantly down-regulated in the expression level of Bcl-2P -caspase-3 and active-caspase-3, and the expression level of Bcl-2P -caspase-3 and active-caspase-3 were up-regulated significantly in apoptosis-related analysis, which was the target gene of direct action of miR-935. The silencing of INPP4A significantly counteracted the inhibition of miR-935 expression on the migration and apoptosis of pancreatic cancer PANC-1 cells and the effect of miR-935 inhibition on the expression of EMT and apoptosis-related molecules. Conclusion miR-935 is expressed in pancreatic cancer tissues and PANC-1. Upregulation of miR-935 expression can promote the proliferation and migration of pancreatic cancer cells. Inhibition of apoptosis. MiR-935 may be a potential target for gene therapy of pancreatic cancer by directly acting on target gene INPP4A to regulate malignant biological behavior of pancreatic cancer cells. MiR-935 and INPP4A may be potential targets for gene therapy of pancreatic cancer.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.9

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