敲低miR-375对子痫前期模型滋养细胞增殖、凋亡的影响及机制

Effects of miR-375 knockdown on the proliferation and apoptosis of trophoblast cells in a preeclampsia model and its mechanism

目的探讨敲低miR-375对子痫前期(PE)滋养细胞模型增殖、凋亡的影响及作用机制。方法取人绒毛膜滋养细胞株HTR-8/Svneo,随机分为常氧对照组、PE模型组、miR-NC组和miR-375 inhibitor组。其中,miR-NC组和miR-375 inhibitor组细胞分别转染miR-NC和miR-375 inhibitor;常氧对照组细胞在常氧环境下培养,其余3组细胞均在缺氧条件下培养以建立PE滋养细胞模型。采用RT-PCR检测miR-375的表达,EdU荧光染色、CCK-8实验检测细胞增殖情况,流式细胞术检测细胞凋亡情况,Western blot检测YAP1蛋白及凋亡相关蛋白(Bcl-2、caspase-3及Bax蛋白)的表达。双荧光素酶基因实验检测miR-375与YAP1的靶向调控关系。结果与常氧对照组比较,PE模型组细胞miR-375表达升高(P<0.05),EdU阳性细胞百分比下降(P<0.05),细胞增殖能力降低(P<0.05),细胞凋亡率升高(P<0.05),Bax、caspase-3蛋白表达水平升高(P<0.05),YAP1、Bcl-2蛋白表达水平降低(P<0.05)。PE模型组和miR-NC组细胞上述各项指标比较差异均无统计学意义(P>0.05)。与miR-NC组比较,miR-375 inhibitor组细胞miR-375表达降低(P<0.05),EdU阳性细胞百分比升高(P<0.05),细胞增殖能力增强(P<0.05),细胞凋亡率降低(P<0.05),Bax、caspase-3蛋白表达降低(P<0.05),YAP1、Bcl-2蛋白表达升高(P<0.05)。双荧光素酶报告基因实验显示,miR-375对YAP1具有靶向调控作用(P<0.05)。结论敲低miR-375可促进PE滋养细胞增殖并抑制细胞凋亡,其作用机制可能与其对YAP1基因的靶向调控有关。

Objective To investigate the effects of miR-375 knockdown on the proliferation and apoptosis of trophoblast cells in preeclampsia(PE)model and its mechanism.Methods Human chorionic trophoblast cell line HTR-8/Svneo was randomly divided into the normoxic control group,the PE model group,the miR-NC group,and the miR-375 inhibitor group.Among them,cells in the miR-NC group and miR-375 inhibitor group were transfected with miR-NC and miR-375 inhibitor,respectively;cells in the normoxic control group were cultured in a normoxic enviroment,and cells in the remaining three groups were cultured under hypoxic conditions to establish the PE trophoblast cell model.The expression of miR-375 was detected by RT-PCR,the proliferation of cells was detected by EdU fluorescence staining and CCK-8 assay,the apoptosis of cells was detected by flow cytometry,and the expression of YAP1 protein and apoptosis-related proteins(Bcl-2,caspase-3,and Bax proteins)were detected by Western blot.The target regulation relationship between miR-375 and YAP1 was detected by dual luciferase reporter gene assay.Results Compared with the normoxic control group,the PE model group showed an increase in the expression of miR-375(P<0.05),a decrease in the proportion of EdU-positive cells(P<0.05),a decline in the cell proliferation ability(P<0.05),increases in the apoptosis rate(P<0.05)and the expression levels of Bax and caspase-3 proteins(P<0.05),and a decrease in the expression levels of YAP1 and Bcl-2 proteins(P<0.05).There was no significant difference in the above indicators between the PE model group and the miR-NC group(P>0.05).Compared with the miR-NC group,the miR-375 inhibitor group showed a lower expression of miR-375(P<0.05),an increased percentage of EdU-positive cells(P<0.05),an enhanced cell proliferation ability(P<0.05),a lower apoptosis rate(P<0.05),and a lower expression of Bax and caspase-3 proteins(P<0.05),while with a higher expression of YAP1 and Bcl-2 proteins(P<0.05).The dual luciferase reporter gene assay showed that miR-375 had a targeted regulatory effect on YAP1(P<0.05).Conclusion Knocking down miR-375 can promote the proliferation and inhibit the apoptosis of PE trophoblast cells,and its mechanism may be related to its targeted regulation of YAP1 gene.