多囊卵巢综合征外周血中miR-132和SMAD4的表达对颗粒细胞增殖和凋亡的影响

Effects of miR-132 and SMAD4 expression in peripheral blood of polycystic ovary syndrome on the proliferation and apoptosis of granulosa cells

目的:探讨多囊卵巢综合征(PCOS)外周血中miR-132和SMAD4的表达水平及对卵巢颗粒细胞增殖和凋亡的影响。方法:收集2019年5月—2021年3月接受治疗的PCOS患者(PCOS组,85例),同时期来院就诊的月经规律的健康女性40名设为对照组。采用全自动生化分析仪检测黄体生成素(LH)、睾酮(T)、雄烯二酮和卵泡刺激素(FSH)的水平,qRT-PCR检测miR-132、SMAD4 mRNA相对表达水平,分析以上指标在两组间的差异及PCOS组中miR-132和SMAD4的相关性。分别将si-NC质粒、si-miR-132质粒、miR-NC质粒、SMAD4 mimic质粒和SMAD4 mimic+miR-132 mimic质粒转至KGN细胞中,MTT和流式细胞术分别检测转染细胞的增殖和凋亡。双荧光素酶报告实验检测miR-132和SMAD4靶向关系。结果:PCOS组LH、T、雄烯二酮和miR-132相对表达水平高于对照组,FSH和SMAD4 mRNA相对表达水平低于对照组(t=3.618、4.253、15.412、30.921、5.207、24.194,均P<0.05)。PCOS组中miR-132和SMAD4 mRNA的相对表达水平呈负相关(r=-0.876,P<0.001)。si-miR-132组中miR-132相对表达水平为0.58±0.07,低于si-NC组(2.68±0.24,t=5.201,P<0.001)。SMAD4 mimic组SMAD4 mRNA水平为1.89±0.21,高于miR-NC组(0.82±0.10)和SMAD4 mimic+miR-132 mimic组(0.63±0.08,t=3.251、4.012,P<0.05)。si-miR-132组细胞增殖率(30.5±4.1)%低于si-NC组(65.3±4.8)%(t=8.512,P<0.001),细胞凋亡率(23.7±4.2)%高于si-NC组(5.7±1.8)%(t=7.625,P<0.001)。SMAD4 mimic组细胞增殖率(25.3±4.6)%低于miR-NC组(63.3±3.6)%和SMAD4 mimic+miR-132 mimic组(63.7±5.8)%(t=3.210、3.515,P<0.05),细胞凋亡率(25.6±3.7)%,高于miR-NC组(6.3±2.0)%和SMAD4 mimic+miR-132 mimic组(6.3±1.5)%(t=9.415、10.320,P<0.001)。miR-132与SMAD4存在靶向结合位点,miR-132可靶向调节SMAD4。结论:miR-132通过靶向调节SMAD4促进颗粒细胞的增殖并抑制其凋亡,可能参与PCOS的发病。

Objective:To detect the expression levels of miR-132 and SMAD4 in peripheral serum of polycystic ovary syndrome(PCOS),and analyze their effects on the proliferation and apoptosis of ovarian granulosa cells.Methods:A total of 85 patients with PCOS who received treatment in our hospital from May 2019 to March 2021 were collected as the PCOS group,and 40 healthy women with regular menstruation who came to the hospital during the same period were collected as the control group.An automatic biochemical analyzer was used to detect the levels of luteinizing hormone(LH),testosterone(T),and rostenedione and follicle stimulating hormone(FSH).qRT-PCR was used to detect the relative expression levels of miR-132 and SMAD4 mRNA,and the above indicators differences were analyzed between the two groups and analyzed correlation of miR-132 and SMAD4 in the PCOS group.The si-NC plasmid,si-miR-132 plasmid,miR-NC plasmid,SMAD4 mimic plasmid and SMAD4 mimic+miR-132 mimic plasmid were transferred to KGN cells respectively,and the proliferation and apoptosis rate of transfected cells were detected by MTT and flow cytometry.The dual luciferase reporter experiment detected the targeting relationship between miR-132 and SMAD4.Results:The relative expression levels of LH,T,androstenedione and miR-132 in the PCOS group were higher than those in the control group,and the relative expression levels of FSH and SMAD4 mRNA were lower than those in the control group(t=3.618,4.253,15.412,30.921,5.207,24.194,all P<0.05).The relative expression levels of miR-132 and SMAD4 mRNA in the PCOS group were negatively correlated(r=-0.876,P<0.001).The relative expression level of miR-132 in si-miR-132 group was 0.58±0.07,which was lower than that in si-NC group(2.68±0.24,t=5.201,P<0.001).The SMAD4 mRNA level in the SMAD4 mimic group was 1.89±0.21,which was higher than that in the miR-NC group(0.82±0.10)and the SMAD4 mimic+miR-132 mimic group(0.63±0.08)(t=3.251,4.012,P<0.05).The cell proliferation rate in the si-miR-132 group was(30.5±4.1)%,which was lower than that in the si-NC group(65.3±4.8)%(t=8.512,P<0.001),and the apoptosis rate was(23.7±4.2)%,which was high than that in si-NC group(5.7±1.8)%(t=7.625,P<0.001).The cell proliferation rate in the SMAD4 mimic group was(25.3±4.6)%,which was lower than that in the miR-NC group(63.3±3.6)%and the SMAD4 mimic+miR-132 mimic group(63.7±5.8)%(t=3.210,3.515,P<0.05),the apoptosis rate was(25.6±3.7)%higher than that in the miR-NC group(6.3±2.0)%and the SMAD4 mimic+miR-132 mimic group(6.3±1.5)%(t=9.415,10.320,P<0.001).Conclusion:miR-132 promotes granulosa cell proliferation and inhibits apoptosis by targeting SMAD4,which may be involved in the pathogenesis of polycystic ovary syndrome.