MiR-106b对子宫肌瘤细胞增殖、侵袭和凋亡的影响及与SULT2A1的靶向关系

Effect of miR-106b on the Proliferation,Invasion and Apoptosis of Uterine Leiomyoma Cells and Its Targeting Relationship with SULT2A1

目的:探讨微小RNA(miR)-106b在子宫肌瘤细胞增殖、侵袭和凋亡中的作用与潜在机制。方法:将体外培养的人子宫肌瘤细胞分为阴性对照(NC)组(转染miR-106b模拟物阴性对照)、miR-106b组(转染miR-106b模拟物)、反义(anti)-NC组(转染miR-106b抑制剂阴性对照)和anti-miR-106b组(转染miR-106b抑制剂),采用逆转录-聚合酶链反应(RT-PCR)检测各组细胞中miR-106b的表达,噻唑蓝(MTT)法检测细胞存活率,平板克隆实验检测细胞的克隆形成能力,Transwell小室检测细胞侵袭,流式细胞仪检测细胞凋亡;生物信息学软件预测、双荧光素酶报告基因实验检测miR-106b和磺基转移酶2A1(SULT2A1)的靶向关系,免疫印迹(Western blotting)法检测miR-106b对SULT2A1蛋白的调控作用。结果:与NC组比较,miR-106b组细胞中miR-106b的表达水平、细胞存活率、克隆形成率、侵袭细胞数均明显升高,细胞凋亡率和SULT2A1蛋白的表达水平均明显降低(P<0.05);与anti-NC组比较,anti-miR-106b组细胞中miR-106b的表达水平、细胞存活率、克隆形成率、侵袭细胞数均明显降低,细胞凋亡率和SULT2A1蛋白的表达水平均明显升高(P<0.05)。生物信息学软件Target Scan预测到miR-106b与SULT2A1 3’UTR存在互补的结合位点;双荧光素酶报告基因实验证实SULT2A1是miR-106b的靶基因。结论:miR-106b可促进子宫肌瘤细胞增殖、侵袭并抑制其凋亡,其作用机制可能与靶向调控SULT2A1表达有关。

Objective: To investigate the effect of microRNA(miR)-106b on the proliferation,invasion and apoptosis of uterine leiomyoma cells and the possible mechanisms. Methods: Human uterine leiomyoma cells cultured in vitro were divided into negative control(NC) group(transfected miR-106b mimic negative control),miR-106b group(transfected miR-106b mimic),antisense(anti)-NC group(transfected miR-106b inhibitor negative control) and anti-miR-106b group(transfected miR-106b inhibitor). The expression of miR-106b in each group was detected by reverse transcription-polymerase chain reaction(RT-PCR),the cell viability was tested by methylthiazolyldiphenyl-tetrazolium bromide(MTT) assay,and plate cloning assay was used to detect the cell colony forming efficiency,transwell chamber detection was applied to detect the cell invasion,and flow cytometry detection was used for the apoptosis detection;bioinformatics software prediction and dual luciferase reporter gene assay were used to detect the targeting relationship between miR-106b and sulfate transferase 2 A1(SULT2A1),and Western blotting was used to detect the regulation of miR-106b on SULT2A1 protein. Results: Compared with those in NC group,the miR-106b expression level,cell survival rate,colony forming efficiency and invasive cell number in miR-106b group were significantly increased,while the apoptosis rate and SULT2A1 protein expression level were significantly decreased(P< 0.05);compared with anti-NC group,the miR-106b expression level,cell survival rate,colony forming efficiency and invasive cell number in anti-miR-106b group were decreased,while the apoptosis rate and expression level of SULT2A1 protein were significantly increased(P<0.05). Bioinformatics software predicted that there was a complementary binding site between miR-106b and SULT2A1 3 ’UTR. Double luciferase reporter gene experiment confirmed that SULT2A1 was the target gene of miR-106b.Conclusion: miR-106b can promote the proliferation and invasion,and inhibit the apoptosis of uterine leiomyoma cells,and its mechanism may be related to the targeted regulation of SULT2A1 expression.