miR-575在稽留流产的表达及作用机制的研究

发布时间：2018-01-05 15:33

本文关键词：miR-575在稽留流产的表达及作用机制的研究　出处：《南方医科大学》2017年博士论文　论文类型：学位论文

【摘要】：目的:本研究检测miR-575在稽留流产患者中的表达及临床意义,分析其对人绒毛膜细胞凋亡的影响及作用机制,为临床上预防和治疗稽留流产提供可靠依据。方法:(1)本研究选取2015年3月～2015年10月在我院妇产科门诊就诊的孕7周稽留流产的妇女30例为流产组,同期健康妊娠的女性30例为对照组,采集上述两组妇女的绒毛组织,荧光定量PCR法检测miR-575的表达,TUNEL法检测细胞的凋亡率,分析miR-575与组织细胞凋亡的相关性。(2)给JEG-3(人绒毛滋养细胞)转染 miR-575 scramble、inhibitor 和 mimic,将 JEG-3 分为 control 组、miR-575 scramble 组、miR-575inhibitor 组和 miR-575 mimic组。检测上述各组细胞miR-575的表达;MTT法检测增殖活性;流式细胞术检测细胞凋亡率;荧光定量PCR法和westernblot法检测Bcl-2、Bax、p53、VEGF、Ang-2mRNA和蛋白的表达;(3)生物信息学软件预测miR-575的靶基因,构建含有荧光素酶报告基因的SOD2 3' UTR WT和SOD2 3' UTR Mut质粒,并与miR-575共转染JEG-3细胞,检测荧光素酶的表达量。荧光定量PCR和westernblot法检测 SOD2mmRNA 和蛋白在 control 组、miR-575scramble 组、miR-575 inhibitor组和miR-575 mimic组的表达。将pc-SOD2质粒转染JEG-3细胞,检测control组和pc-SOD2组SOD2mRNA和蛋白的表达;将JEG-3细胞分为control组,inhibitor 组和 miR-575inhibitor+pc-SOD2 组,检测各组 Ang-2、VEGF mRNA 和蛋白的表达,流式细胞术检测凋亡率。结果:(1)稽留流产组绒毛组织中miR-575的表达量高于正常对照组(P=0.033);绒毛细胞的凋亡率明显高于正常对照组(P=0.008)。稽留流产患者绒毛组织细胞的凋亡率与miR-575的表达呈正相关(r=0.419,P=0.001);(2)与 miR-575scramble 组相比,miR-575inhibitor 组miR-575表达量明显减少(t=3.217,P=0.044),细胞的凋亡率明显降低(t=3.566,P=0.042);而 miR-575mimic 组 miR-575 表达量显著增高(t=4.773,P=0.007),细胞凋亡率显著增高(t=3.859,P0.023)。与miR-575scramble组相比,miR-575inhibitor组细胞在48、72、96h时的增殖率明显增高(P0.05),而miR-575mimic组细胞的增值率明显降低(P0.05)。与miR-575scramble组相比,miR-575inhibitor 组 Bcl-2、Ang-2 和 VEGFmRNA 和蛋白的表达量明显增高,Bax、p53mRNA和蛋白的表达量明显减少;而miR-575mimic组Bcl-2、Ang-2和VEGF mRNA和蛋白的表达量明显减少,Bax、p53mRNA的表达量明显增高(P0.05)。(3)SOD2被预测为mmiR-575的靶基因。与正常对照组相比,稽留流产组患者绒毛组织中 SOD2mRNA(t=3.122,P=0.048)和蛋白(t=3.245,P=0.046)的表达量均明显减少。miR-575inhibitor+SOD2 3' UTR WT组荧光素酶的表达量明显低于miR-control+SOD2 3' UTR WT 组(t=4.618,P=0.005),miR-575 inhibitor+SOD23' UTR Mut组荧光素酶的表达量与mmiR-control+SOD2 3' UTR Mut组无统计学差异(t=0.245,T=0.911)。与 control 组相比,pc-SOD2 组 SOD2mRNA(t=0.349,P=0.044)和蛋白(t=0.388,P=0.040)的表达量明显增高。与Scramble组相比,mimic组SOD2 mRNA和蛋白的表达量明显降低(P0.01),inhibitor组则明显增高(P0.01)。与 control 组相比,inhibitor 组 Ang-2、VEGFmRNA 和蛋白的表达量均明显增高(P0.05),细胞凋亡率明显减少(P0.01);inhibitor+pc-SOD2 组 Ang-2、VEGFmRNA 和蛋白的表达量则明显减少(P0.05),细胞凋亡率则无统计学差异(P0.05)。结论:miR-575通过靶向抑制SOD2的表达,进而降低Ang-2的表达促进绒毛滋养细胞凋亡,通过抑制VEGF的表达来抑制血管新生,最终引起稽留流产的发生。
[Abstract]:Objective: To study the expression of miR-575 was detected in missed abortion patients and its clinical significance, analyzes its influence on the apoptosis of human chorionic cells and the mechanism of action for clinical prevention and treatment of missed abortion and provide a reliable basis. Methods: (1) this study selected women from March 2015 to October 2015 in our hospital obstetrics and gynecology clinic. 7 weeks of missed abortion and 30 cases of abortion group, healthy pregnant women as a control group of 30 cases, collected the two groups of women in the villous tissues, miR-575 expression was detected by fluorescence quantitative PCR method, the apoptosis rate was detectedby TUNEL. The correlation between miR-575 and apoptosis. (2) to JEG-3 (people trophoblast cells transfected with miR-575 scramble), inhibitor and mimic, JEG-3 were divided into control group, miR-575 scramble group, miR-575inhibitor group and miR-575 mimic group. The expression of miR-575 cells were detected by MTT; Detection of proliferation activity; cell apoptosis was detected by flow cytometry; fluorescence quantitative detection of Bcl-2, PCR and Westernblot Bax, p53, VEGF, Ang-2mRNA and protein expression; (3) the target genes were predicted by bioinformatics software miR-575, construct luciferase reporter SOD2 3'and UTR WT SOD2 3' UTR Mut plasmid miR-575, and JEG-3 cells were co transfected with luciferase expression. MiR-575scramble group and SOD2mmRNA protein was detected by fluorescence quantitative PCR and Westernblot method in the control group, miR-575 inhibitor group and miR-575 mimic group. The expression of pc-SOD2 plasmid was transfected into JEG-3 cells, the expression of control group and pc-SOD2 group SOD2mRNA and protein; JEG-3 cells divided into control group, inhibitor group and miR-575inhibitor+pc-SOD2 group were detected Ang-2, VEGF expression of mRNA and protein, the rate of apoptosis was detected by flow cytometry. Results: (1) missed abortion The expression of miR-575 in villi group is higher than the normal control group (P=0.033); apoptosis of villus cells was significantly higher than the normal control group (P=0.008). The expression of miR-575 was positively correlated with the apoptosis rate of missed abortion chorionic villi in patients with the cells (r=0.419, P=0.001); (2) compared with miR-575scramble group, miR-575inhibitor group, miR-575 expression decreased (t=3.217, P=0.044), the apoptosis rate was significantly lower (t=3.566, P=0.042); group miR-575mimic miR-575 expression increased significantly (t=4.773, P=0.007), cell apoptosis rate increased significantly (t=3.859, P0.023). Compared with the miR-575scramble group, significantly higher rates of proliferation of miR-575inhibitor cells in the 48,72,96h group (P0.05) but, the added value of miR-575mimic cells decreased significantly (P0.05). Compared with the miR-575scramble group, miR-575inhibitor group, Bcl-2, Ang-2 and VEGFmRNA and protein expression increased significantly, Bax, the expression of p53mRNA and protein significantly decreased; and the miR-575mimic group Bcl-2, expression of Ang-2 and VEGF mRNA and protein significantly decreased, Bax, p53mRNA expression was significantly increased (P0.05). (3) SOD2 was predicted to be the target gene of mmiR-575. Compared with the normal control group, SOD2mRNA group of patients with missed abortion villi in (t=3.122, P=0.048) and protein (t=3.245, P=0.046) expression significantly decreased expression of.MiR-575inhibitor+SOD2 3'UTR in WT group was significantly lower than that of miR-control+SOD2 3' UTR luciferase WT group (t=4.618, P=0.005), miR-575 inhibitor+SOD23'UTR group Mut luciferase expression and mmiR-control+SOD2 3' UTR Mut group showed no significant difference (t=0.245, T=0.911). Compared with the control group, pc-SOD2 group, SOD2mRNA (t=0.349, P=0.044) and protein (t=0.388, P=0.040) expression was significantly increased. Compared with Scramble group, mimic group and SOD2 mRNA protein The expression was lower (P0.01), inhibitor group was significantly increased (P0.01). Compared with control group, inhibitor group Ang-2, the expression of VEGFmRNA and protein were significantly increased (P0.05), the apoptosis rate was significantly reduced (P0.01); group inhibitor+pc-SOD2, Ang-2, VEGFmRNA and protein expression were significantly reduced (P0.05), the apoptosis rate were not statistically significant (P0.05). Conclusion: miR-575 can inhibit the expression of SOD2, thereby reducing the expression of Ang-2 promotes apoptosis of trophoblast cells by inhibiting the expression of VEGF to inhibit angiogenesis, resulting in missed abortion.

【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R714.21

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