miRNA-34a在人胃癌中的表达及对胃癌细胞增殖、凋亡的影响

发布时间：2018-06-20 00:08

本文选题：胃癌 + SGC-7901细胞　；参考：《安徽医科大学》2015年硕士论文

【摘要】：目的：研究miR-34a在人胃腺癌及配对癌旁正常组织、人正常胃粘膜上皮细胞及多种胃癌细胞株中的表达水平；通过构建过表达miR-34a慢病毒载体并感染人胃癌SGC-7901细胞,观察其对细胞增殖、细胞周期和细胞凋亡的影响；预测miR-34a可能的肿瘤相关靶基因,探讨miR-34a在胃癌发生、发展中的作用及其调控机制。方法：1.通过Real-time PCR检测34例人胃腺癌及配对的癌旁正常组织,人正常胃黏膜上皮细胞GES及人胃癌细胞株AGS、SGC-7901、MKN-45、BGC-823中miR-34a的表达水平。2.构建过表达hsa-miR-34a慢病毒载体并体外感染人胃癌SGC-7901细胞,同时设对照组。荧光显微镜观察评估慢病毒细胞感染情况,MTT实验、流式细胞术检测感染后胃癌SGC-7901细胞的细胞增殖、周期及细胞凋亡情况。3.采用microRNA在线靶基因预测软件miRanda、TargetScan、PICTAR共同预测并筛选miR-34a可能肿瘤相关靶基因。4.应用SPSS 19.0软件对所有实验数据进行统计学分析。结果：1.胃癌组织的miR-34a相对表达量为0.65+0.69,明显低于配对癌旁组织的相对表达量1.02±0.04(t=3.085,P0.01),但miR-34a的表达水平与胃癌的分化程度、浸润深度、局部淋巴结转移无明显相关性(P0.05)。2.胃癌细胞AGS、SGC-7901、MKN-45、BGC-823中miR-34a的相对表达量分别为0.24±0.01,0.03±0.00,0.31±0.04和0.25±0.05,均分别明显低于miR-34a在正常人胃黏膜上皮细胞GES的相对表达量(0.98±0.07),差异均具有统计学意义(t=17.522、22.879、13.836、14.393,P0.05),其中miR-34a在胃癌SGC-7901细胞中的相对表达量降低最为明显(P0.01)。3. miR-34a慢病毒感染组在检测第3到5天的相对增殖倍数分别为1.39±0.19、1.82±0.16和2.37±0.04,均明显低于阴性对照慢病毒感染组第3到5天的相对增殖倍数(分别为1.844±0.10、2.46±0.11和3.144±0.38),差异有统计学意义(t=4.567、7.265、4.500,P0.01)。4. miR-34a慢病毒感染组G1/G0期细胞比例为58.72%,显著高于阴性对照组的56.36%(t=-4.804,P0.05)。5. miR-34a慢病毒感染组细胞凋亡率为6.44%,显著高于阴性对照组的3.06%(t=-13.226,P0.01)。6.通过对miRanda、TargetScain、PICTAR三个在线数据库检索交集的分析,筛选出与肿瘤增殖/凋亡/细胞周期/侵袭转移相关的7个靶基因：E2F3、MET、NOTCH1、 SIRT1、Bcl-2、YY1、CCNE2。结论：1.miR-34a在人胃癌组织及多种人胃癌细胞中呈低表达,提示miR-34a与胃癌的发病关系密切。2.miR-34a可以抑制胃癌SGC7901细胞的增殖,诱导细胞G1/G0期停滞及细胞凋亡,在胃癌SGC-7901细胞中可能发挥抑癌基因的作用；miR-34a可能通过抑制E2F3、MET、NOTCH1、SIRT1、Bcl-2、YY1、CCNE2等靶基因发挥促进胃癌细胞增值抑制、细胞周期停滞及细胞凋亡的抗肿瘤作用。
[Abstract]:Objective: to study the expression of miR-34a in human gastric adenocarcinoma and adjacent normal tissues, normal gastric mucosal epithelial cells and gastric cancer cell lines, and to construct and infect human gastric cancer SGC-7901 cells by overexpression of miR-34a lentivirus vector. The effects of miR-34a on cell proliferation, cell cycle and apoptosis were observed, the possible tumor-related target genes of miR-34a were predicted, and the role and regulatory mechanism of miR-34a in carcinogenesis and development of gastric cancer were discussed. Method 1: 1. Real-time PCR was used to detect the expression of miR-34a in 34 cases of human gastric adenocarcinoma and matched adjacent normal tissues, the expression level of miR-34a in human gastric epithelial cells (GES) and human gastric cancer cell line AGSN SGC-7901 (MKN-45) BGC-823. Expression of hsa-miR-34a lentivirus vector was constructed and transfected into human gastric cancer SGC-7901 cells in vitro. MTT assay and flow cytometry were used to detect the proliferation, cycle and apoptosis of SGC-7901 cells after infection. MicroRNA online target gene prediction software miRandaScant PICTAR was used to predict and screen miR-34a possible tumor-related target gene. 4. All experimental data were statistically analyzed by SPSS 19.0 software. The result is 1: 1. The relative expression of miR-34a in gastric cancer was 0.65 0.69, which was significantly lower than that in matched paracancerous tissues (1.02 卤0.04). However, the expression level of miR-34a was not correlated with the differentiation, depth of invasion and local lymph node metastasis of gastric carcinoma. The relative expression of miR-34a was 0.24 卤0.01 卤0.03 卤0.001 卤0.31 卤0.04 and 0.25 卤0.05 in gastric cancer cell line AGSC-7901MKN-45-BGC-823, respectively, which was significantly lower than that of miR-34a in normal gastric epithelial cells (0.98 卤0.07). The difference was statistically significant (P 0.05), and the miR-34a expression in SGC-7901 cells was significantly lower than that in normal gastric cancer SGC-7901 cells. There were significant differences in the expression of miR-34a in gastric cancer SGC-7901 cells. There were significant differences in the expression of miR-34a in gastric cancer SGC-7901 cells. There were significant differences in the relative expression of miR-34a in gastric cancer SGC-7901 cells. There were significant differences in the expression of miR-34a in SGC-7901 cells. The relative proliferative times of lentivirus infection group were 1.39 卤0.19 卤1.82 卤0.16 and 2.37 卤0.04, respectively, which were significantly lower than those of the negative control group on the 3rd to 5th day (1.844 卤0.102.46 卤0.11, respectively). The difference was statistically significant between 3.144 卤0.38 and 4.5677.265A4.500P0.01n.4miR-34a lentivirus infection group, the cell ratio of G1 / G0 phase was 58.722.It was significantly higher than that of negative control group (56.36t-4.804P0.05.5.MiR-34a lentivirus infection group), which was significantly higher than that of negative control group (3.06tti-13.226P0.01p0.01. 6.) By analyzing the intersections of three online databases of MiRanda TargetScainTar, seven target genes: E2F3METTCH1, SIRT1 Bcl-2OY1 (YY1) and CCNE2 were screened out, which were related to tumor proliferation / apoptosis / cell cycle / invasion and metastasis. Conclusion the low expression of miR-34a in human gastric cancer tissues and various human gastric cancer cells suggests that miR-34a is closely related to gastric cancer. 2.miR-34a can inhibit the proliferation of gastric cancer SGC7901 cells and induce G1 / G0 phase arrest and apoptosis of SGC7901 cells. The inhibitory effect of miR-34a on the proliferation, cell cycle arrest and apoptosis of gastric cancer cells may be promoted by inhibiting the target genes, such as E2F3METNTCH1, SIRT1, Bcl-2Y1 and CCNE2, in SGC-7901 cells.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.2

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