MiRNA-199a-3p通过mTOR通路与阿霉素联合抑制子宫内膜癌细胞的增殖和促凋亡作用

MiRNA-199a-3p combined with doxorubin inhibits endometrial cancer cell proliferation and promotes apoptosis through the mTOR pathway

目的:探讨阿霉素对转染miRNA-199a-3p的子宫内膜癌(EC)的作用及机制。方法:构建pSIREN-miRNA-199a-3p过表达质粒并稳定转染内膜癌细胞系Ishikawa、SPEC-2细胞。应用逆转录PCR(RT-PCR)检测miRNA-199a-3p的表达;MTT、克隆形成实验检测细胞增殖;流式细胞术检测细胞凋亡;Western blot检测mTOR信号通路相关蛋白表达。结果:上调miRNA-199a-3p可显著抑制子宫内膜癌细胞Ishikawa、SPEC-2的克隆形成能力(P<0.01,P<0.05);miRNA-199a-3p可增强阿霉素对Ishikawa、SPEC-2细胞的生长抑制(P均<0.05)和凋亡诱导作用(P均<0.05);Western blot证实,miRNA-199a-3p可显著下调mTOR及其效应蛋白p70S6K的磷酸化水平。结论:miRNA-199a-3p可能通过mTOR通路与阿霉素联合抑制子宫内膜癌细胞的增殖并促进其凋亡,为联合应用miRNA和阿霉素治疗EC提供了实验依据。

Objective：To study the effect and mechanism of doxorubicin on miRNA- 199a-3p transfected endometrial carcinoma （EC）. Methods： The pSIREN-miRNA-199a-3p overexpression plasmids were stably tranfected into Ishikawa and SPEC-2 cells. RT-PCR was used to confirm the upregulation of miRNA-199a-3p. Cellular proliferation was determined by the MTT assay and colony formation assay. The apoptotic status was detected by flow cytometr. The alterations of proteins in the mTOR pathways were measured by Western blot. Results：Up- regulation of miRNA-199a-3p significantly suppressed the colon forming in Ishikawa and SPEC- 2 cells（P〈0.01 ,P〈0.05）,and miRNA-199a-3p sensitized these two cells to doxorubicin（P〈 0.05,P〈0.05）. Moreover, miRNA-199a-3p promoted the effects of doxorubicin on cellular ap- optosis（P〈0.05, P〈0.05）. Western blot showed that miRNA-199a-3p further enhanced the mTOR signal inhibition of doxorubicin. Conclusions： MiRNA-199a-3p may be combined with doxorubicin to inhibit proliferation of EC cells and promote the apoptosis through mTOR path- way. This study will provide experimental basis for the combination of miRNA and doxornbicin in the treatment of EC.