miR-575在稽留流產(chǎn)的表達(dá)及作用機(jī)制的研究
本文關(guān)鍵詞:miR-575在稽留流產(chǎn)的表達(dá)及作用機(jī)制的研究 出處:《南方醫(yī)科大學(xué)》2017年博士論文 論文類(lèi)型:學(xué)位論文
更多相關(guān)文章: 稽留流產(chǎn) miR-575 絨毛滋養(yǎng)細(xì)胞 細(xì)胞凋亡 SOD2
【摘要】:目的:本研究檢測(cè)miR-575在稽留流產(chǎn)患者中的表達(dá)及臨床意義,分析其對(duì)人絨毛膜細(xì)胞凋亡的影響及作用機(jī)制,為臨床上預(yù)防和治療稽留流產(chǎn)提供可靠依據(jù)。方法:(1)本研究選取2015年3月~2015年10月在我院婦產(chǎn)科門(mén)診就診的孕7周稽留流產(chǎn)的婦女30例為流產(chǎn)組,同期健康妊娠的女性30例為對(duì)照組,采集上述兩組婦女的絨毛組織,熒光定量PCR法檢測(cè)miR-575的表達(dá),TUNEL法檢測(cè)細(xì)胞的凋亡率,分析miR-575與組織細(xì)胞凋亡的相關(guān)性。(2)給JEG-3(人絨毛滋養(yǎng)細(xì)胞)轉(zhuǎn)染 miR-575 scramble、inhibitor 和 mimic,將 JEG-3 分為 control 組、miR-575 scramble 組、miR-575inhibitor 組和 miR-575 mimic組。檢測(cè)上述各組細(xì)胞miR-575的表達(dá);MTT法檢測(cè)增殖活性;流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡率;熒光定量PCR法和westernblot法檢測(cè)Bcl-2、Bax、p53、VEGF、Ang-2mRNA和蛋白的表達(dá);(3)生物信息學(xué)軟件預(yù)測(cè)miR-575的靶基因,構(gòu)建含有熒光素酶報(bào)告基因的SOD2 3' UTR WT和SOD2 3' UTR Mut質(zhì)粒,并與miR-575共轉(zhuǎn)染JEG-3細(xì)胞,檢測(cè)熒光素酶的表達(dá)量。熒光定量PCR和westernblot法檢測(cè) SOD2mmRNA 和蛋白在 control 組、miR-575scramble 組、miR-575 inhibitor組和miR-575 mimic組的表達(dá)。將pc-SOD2質(zhì)粒轉(zhuǎn)染JEG-3細(xì)胞,檢測(cè)control組和pc-SOD2組SOD2mRNA和蛋白的表達(dá);將JEG-3細(xì)胞分為control組,inhibitor 組和 miR-575inhibitor+pc-SOD2 組,檢測(cè)各組 Ang-2、VEGF mRNA 和蛋白的表達(dá),流式細(xì)胞術(shù)檢測(cè)凋亡率。結(jié)果:(1)稽留流產(chǎn)組絨毛組織中miR-575的表達(dá)量高于正常對(duì)照組(P=0.033);絨毛細(xì)胞的凋亡率明顯高于正常對(duì)照組(P=0.008);袅鳟a(chǎn)患者絨毛組織細(xì)胞的凋亡率與miR-575的表達(dá)呈正相關(guān)(r=0.419,P=0.001);(2)與 miR-575scramble 組相比,miR-575inhibitor 組miR-575表達(dá)量明顯減少(t=3.217,P=0.044),細(xì)胞的凋亡率明顯降低(t=3.566,P=0.042);而 miR-575mimic 組 miR-575 表達(dá)量顯著增高(t=4.773,P=0.007),細(xì)胞凋亡率顯著增高(t=3.859,P0.023)。與miR-575scramble組相比,miR-575inhibitor組細(xì)胞在48、72、96h時(shí)的增殖率明顯增高(P0.05),而miR-575mimic組細(xì)胞的增值率明顯降低(P0.05)。與miR-575scramble組相比,miR-575inhibitor 組 Bcl-2、Ang-2 和 VEGFmRNA 和蛋白的表達(dá)量明顯增高,Bax、p53mRNA和蛋白的表達(dá)量明顯減少;而miR-575mimic組Bcl-2、Ang-2和VEGF mRNA和蛋白的表達(dá)量明顯減少,Bax、p53mRNA的表達(dá)量明顯增高(P0.05)。(3)SOD2被預(yù)測(cè)為mmiR-575的靶基因。與正常對(duì)照組相比,稽留流產(chǎn)組患者絨毛組織中 SOD2mRNA(t=3.122,P=0.048)和蛋白(t=3.245,P=0.046)的表達(dá)量均明顯減少。miR-575inhibitor+SOD2 3' UTR WT組熒光素酶的表達(dá)量明顯低于miR-control+SOD2 3' UTR WT 組(t=4.618,P=0.005),miR-575 inhibitor+SOD23' UTR Mut組熒光素酶的表達(dá)量與mmiR-control+SOD2 3' UTR Mut組無(wú)統(tǒng)計(jì)學(xué)差異(t=0.245,T=0.911)。與 control 組相比,pc-SOD2 組 SOD2mRNA(t=0.349,P=0.044)和蛋白(t=0.388,P=0.040)的表達(dá)量明顯增高。與Scramble組相比,mimic組SOD2 mRNA和蛋白的表達(dá)量明顯降低(P0.01),inhibitor組則明顯增高(P0.01)。與 control 組相比,inhibitor 組 Ang-2、VEGFmRNA 和蛋白的表達(dá)量均明顯增高(P0.05),細(xì)胞凋亡率明顯減少(P0.01);inhibitor+pc-SOD2 組 Ang-2、VEGFmRNA 和蛋白的表達(dá)量則明顯減少(P0.05),細(xì)胞凋亡率則無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。結(jié)論:miR-575通過(guò)靶向抑制SOD2的表達(dá),進(jìn)而降低Ang-2的表達(dá)促進(jìn)絨毛滋養(yǎng)細(xì)胞凋亡,通過(guò)抑制VEGF的表達(dá)來(lái)抑制血管新生,最終引起稽留流產(chǎn)的發(fā)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.
【學(xué)位授予單位】:南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】:博士
【學(xué)位授予年份】:2017
【分類(lèi)號(hào)】:R714.21
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