miR-205-5p靶向CDKN1C调节滋养层细胞生物学行为的研究

The miR-205-5p regulates the biological behavior of trophoblast cells by targeting CDKN1C

目的探究miR-205-5p靶向细胞周期蛋白依赖激酶抑制因子1C(cyclin-dependent kinase inhibitor 1C,CDKN1C)在调节滋养层细胞生物学行为中的作用。方法双荧光素酶报告检测miR-205-5p与CDKN1C的靶向关系。滋养层细胞系HTR-8/SVneo细胞分为NC组、miR-205-5p mimic组、miR-205-5p inhibitor组、CDKN1C组和miR-205-5p mimic+CDKN1C组。qRT-PCR及Western blot检测Bax、Bcl-2及TGF-β1的表达。Western blot检测上皮间质转化相关因子Vimentin、N-cadherin及E-cadherin的表达,MTT检测细胞增殖情况,Transwell检测细胞侵袭能力,流式细胞术检测细胞的凋亡情况。结果CDKN1C被证实为miR-205-5p的靶基因。qRT-PCR及Western blot检测发现,与NC组比较,miR-205-5p inhibitor组和CDKN1C组Bcl-2和TGF-β1表达上升,Bax表达下降,Vimentin和N-cadherin的蛋白水平表达上升,E-cadherin表达下降(均P<0.05),细胞的增殖与侵袭能力上升,凋亡率下降(均P<0.05)。与NC组比较,miR-205-5p mimic组中Vimentin和N-cadherin的蛋白水平表达下调,E-cadherin表达上调,细胞的增殖、侵袭能力削弱,细胞凋亡增加(均P<0.05)。相对于miR-205-5p mimic组,miR-205-5p mimic+CDKN1C组细胞增殖和侵袭增加,凋亡减少,提示过表达miR-205-5p的作用能够被CDKN1C过表达逆转。结论抑制miR-205-5p表达能够促进HTR-8/SVneo细胞系中CDKN1C的表达,促进HTR-8/SVneo细胞的增殖和侵袭能力,miR-205-5p/CDKN1C可能是子痫前期治疗的潜在靶点。

Objective To explore the effect of miR-205-5p on biological behavior of trophoblast cells via targeting cyclin-dependent kinase inhibitor 1(CDKN1C).Methods Dual luciferase reporter assay was adopted to test the targeting regulatory relationship between miR-205-5p and CDKN1C.Trophoblast cell line HTR-8/Svneo were divided into five groups:NC group,miR-205-5p mimic group,miR-205-5p inhibitor group,CDKN1C group,miR-205-5p mimic+CDKN1C group.QRT-PCR and Western blot were used to quantify the expression of Bax,Bcl-2 and TGF-β1.At the same time,the protein expression of EMT related factors(vimentin,N-cadherin,E-cadherin)was detected by Western blot.MTT was performed to detect the proliferation of cells.The invasion ability of cells was tested using Transwell assay.Flow cytometry was performed to detect the apoptosis of cells.Results CDKN1C was confirmed as a target gene of miR-205-5p.QRT-PCR and Western blot results showed that,compared with NC group,Bcl-2 and TGF-β1 expression was increased while Bax expression was decreased in miR-205-5p inhibitor group and CDKN1C group(P<0.05).Compared with NC group,the expression of Vimentin and N-cadherin was increased,while the expression of E-cadherin was decreased in miR-205-5p inhibitor group and CDKN1C group(all P<0.05),meanwhile the proliferation and the invasion of cells were increased,the apoptosis of cells was decreased(all P<0.05).Compared with NC group,the expression of Vimentin and N-cadherin was downregulated,the expression of E-cadherin was upregulated,the proliferation and invasion abilities of cells were weakened,the apoptosis of cells was increased in miR-205-5p mimic group(all P<0.05).Compared with miR-205-5p mimic group,the cell proliferation and invasion abilities were increased while the apoptosis was decreased in miR-205-5p mimic+CDKN1C group,indicating that the effect of miR-205-5p overexpression was reversed by overexpression of CDKN1C.Conclusion Inhibition of miR-205-5p expression can promote the expression of CDKN1C in HTR-8/SVneo cell line,further accelerate the proliferation and the invasion of HTR-8/SVneo cells.The miR-205-5p/CDKN1C may be potential targets for preeclampsia treatment.