芦荟大黄素调控miR-30b促进子宫内膜癌细胞自噬的研究

Aloe emodin regulates miR-30b to promote autophagy in endometrial cancer cells

目的探讨芦荟大黄素(AE)调控子宫内膜癌细胞HEC-1-B中miR-30b表达促进细胞自噬的作用机制。方法实时荧光定量聚合酶链式反应(q RT-PCR)检测miR-30b在正常子宫内膜细胞和不同子宫内膜癌细胞株HEC-1-A、HEC-1-B、RL95-2中的表达水平。取miR-30b表达水平最高的HEC-1-B癌细胞分为HEC-1-B癌细胞组、miR-30b inhibitor NC组(转染miR-30b inhibitor NC)、miR-30b inhibitor组(转染miR-30b inhibitor)以及AE组(30μmol/L AE,未转染)和AE+转染组:AE+miR-30b mimics NC组(30μmol/L AE,转染miR-30b mimics NC)、AE+miR-30b mimics组(30μmol/L AE,转染miR-30b mimics)。q RT-PCR法检测各组细胞miR-30b表达水平;CCK-8法检测各组细胞增殖活性;流式细胞术检测各组细胞凋亡率;单丹磺酰尸胺(MDC)荧光染色法检测各组细胞自噬率;蛋白免疫印迹法(Western blot)法检测各组细胞自噬相关蛋白Beclin1、LC3-I、LC3-II和p62表达水平。结果与人正常子宫内膜上皮细胞相比,HEC-1-A、HEC-1-B和RL95-2癌细胞中miR-30b表达水平显著升高(P<0.05)。其中HEC-1-B癌细胞中miR-30b表达水平最高,所以后续将选择HEC-1-B癌细胞进行转染实验。与HEC-1-B癌细胞组和miR-30b inhibitor NC组相比,miR-30b inhibitor组癌细胞miR-30b表达水平、细胞增殖率以及p62蛋白表达水平显著降低(P<0.05),细胞凋亡率、自噬率以及Beclin1蛋白表达水平和LC3-II/I比值显著升高(P<0.05)。与HEC-1-B癌细胞组相比,AE组和AE+miR-30b mimics NC组癌细胞miR-30b表达水平、细胞增殖率以及p62蛋白表达水平显著降低(P<0.05),细胞凋亡率、自噬率以及Beclin1蛋白表达水平和LC3-II/I比值显著升高(P<0.05)。与AE+miR-30b mimics NC组相比,AE+miR-30b mimics组癌细胞miR-30b表达水平、细胞增殖率以及p62蛋白表达水平显著升高(P<0.05),细胞凋亡率、自噬率以及Beclin1蛋白表达水平和LC3-II/I比值显著降低(P<0.05)。结论子宫内膜癌细胞中miR-30b表达较高,AE可能通过抑制miR-30b表达,促进癌细胞自噬和凋亡,抑制癌细胞增殖,而过表达miR-30b可逆转AE发挥的作用。

Objective To investigate the mechanism of aloe emodin(AE)in promoting autophagy by regulating miR-30 b expression in endometrial carcinoma cell line HEC-1-B.Methods The expression level of miR-30 b in normal endometrial cells and endometrial cancer cell lines HEC-1-A,HEC-1-B,and RL95-2 was measured by real-time fluorescence quantitative polymerase chain reaction(q RT-PCR).HEC-1-B cancer cells with the highest expression level of miR-30 b were divided into a HEC-1-B cancer cell group,miR-30 b inhibitor NC group(transfected with miR-30 b inhibitor NC),miR-30 b inhibitor group(transfected with miR-30 b inhibitor),AE group(30μmol/L AE,untransfected),AE+transfection group:AE+miR-30 b mimics NC group(30μmol/L AE,transfected with miR-30 b mimics NC),and AE+miR-30 b mimics group(30μmol/L AE,transfected with miR-30 b mimics).The expression level of miR-30 b,cell proliferation,apoptosis rate,and autophagy rate were determined by q RT-PCR,CCK-8 assays,flow cytometry,and monodansulfonyl cadaverine fluorescence staining respectively.Western blot was used to measure the protein expression levels of Beclin 1,LC3-I,LC3-II,and p62.Results Compared with normal human endometrial epithelial cells,expression levels of miR-30 b were significantly higher in HEC-1-A,HEC-1-B,and RL95-2 cancer cells(P<0.05).The expression level of miR-30 b was the highest in HEC-1-B cancer cells.Thus,HEC-1-B cancer cells were selected for transfection experiments.Compared with HEC-1-B cancer cell and miR-30 b inhibitor NC groups,the miR-30 b inhibitor group had a significantly lower miR-30 b expression level,cell proliferation rate,and p62 protein expression level(P<0.05),and significantly higher apoptosis rate,autophagy rate,Beclin 1 protein expression level,and LC3-II/I ratio(P<0.05).Compared with the HEC-1-B group,AE and AE+miR-30 b mimics NC groups had significantly lower miR-30 b expression levels,cell proliferation rates and p62 protein expression levels(P<0.05),and a significantly higher apoptosis rate,autophagy rate,Beclin 1 protein expression level,and LC3-II/I ratio(P<0.05).Compared with the AE+miR-30 b mimics NC group,the AE+miR-30 b mimics group had a significantly higher miR-30 b expression level,cell proliferation rate,and p62 protein expression level(P<0.05),and significantly lower apoptosis rate,autophagy rate,Beclin 1 protein expression level,and LC3-II/I ratio(P<0.05).Conclusions Expression of miR-30 b is high in endometrial carcinoma cells.AE may inhibit the expression of miR-30 b,promote autophagy and apoptosis of cancer cells,and inhibit the proliferation of cancer cells.Overexpression of miR-30 b reverses the effect of AE.