miR-223-3p通过靶向RAC1调控肝细胞癌SMMC-7721细胞的增殖和凋亡

目的:探讨miR-223-3p通过调控Ras相关C3肉毒素底物1(Ras-related C3 botulinum toxin substrate 1,RAC1)对肝细胞癌(hepatocellular carcinoma,HCC)细胞增殖和凋亡的影响及其可能的作用机制。方法:选用2016年8月至2018年8月吉林市中心医院手术切除的30例HCC组织及其癌旁组织标本和人HCC细胞系SMMC-7721、Bel-7402、HepG2及人正常肝细胞QSG-7701,用qPCR检测HCC组织和细胞系中miR-223-3p的表达水平。分别将miR-223-3p mimics、miR-223-3p inhibitor和siRAC1转染至SMMC-7721细胞,通过CCK-8、克隆形成实验和Annexin V-FITC/PI染色流式细胞术检测SMMC-7721细胞的增殖、克隆形成和凋亡水平。用双荧光素酶报告基因实验检测miR-223-3p与RAC1的靶向关系,Western blotting检测细胞中RAC1蛋白的表达水平。结果:miR-223-3p在HCC组织的表达水平显著低于癌旁组织(P<0.01),其表达水平与肿瘤大小、TNM分期及肿瘤分化病理特征相关(P<0.05或P<0.01);miR-223-3p在HCC细胞系表达水平显著低于QSG-7701细胞(均P<0.01),以在SMMC-7721细胞中表达水平最低。双荧光素酶报告基因实验证实RAC1是miR-223-3p靶基因,miR-223-3p靶向负调控RAC1的表达。转染miR-223-3p mimics显著抑制SMMC-7721细胞的增殖和克隆形成能力(P<0.05或P<0.01),并促进细胞凋亡(P<0.01);转染miR-223-3p inhibtor则逆转miR-223-3p mimics对细胞的抑制作用。结论:过表达miR-223-3p抑制HCC细胞增殖和克隆形成能力并促进细胞凋亡,其机制可能与靶向下调RAC1表达有关。

miR-223-3p regulates proliferation and apoptosis of hepatocellular carcinoma SMMC-7721 cells by targeting RAC1

Objective: To investigate the effects of miR-223-3p on the proliferation and apoptosis of hepatocellular carcinoma (HCC)cells by regulating Ras-related C3 botulinum toxin substrate 1 (RAC1) and its possible mechanism. Methods: Thirty pairs of HCC and corresponding para-cancer tissues resected in Jilin Central Hospital from August 2016 to August 2018 were collected for this study; in addition, human HCC cell lines SMMC-7721, BEL-7402, HepG2 and human normal hepatocyte QSG-7701 were also collected. The expression level of miR-223-3p in HCC tissue and cell lines was detected by qPCR. miR-223-3p mimics, miR-223-3p inhibitor and siRAC1 were transfected into SMMC-7221 cells, respectively. CCK-8 assay, Colony formation assay and Annexin V-FITC/PI staining Flow cytometry were used to detect the proliferation, clone formation and apoptosis of SMMC-7721 cells, respectively. The relationship between miR-223-3p and RAC1 was confirmed by Dual luciferase reporter gene assay. The protein level of RAC1 in SMMC-7721 cells was detected by Western blotting. Results: The expression of miR-223-3p in HCC tissues was significantly lower than that in paracaner tissues (P<0.01), and had significant correlation with pathological characteristics, such as tumor size, TNM stage, Edmondson-Steiner grade (all P<0.05 or P<0.01). miR-223-3p expression in HCC cell lines was significantly lower than that in QSG-7701 cells with the lowest expression in SMMC-7721 cells. Dual luciferase reporter gene assay confirmed that RAC1 was a target gene of miR-223-3p, and miR-223-3p negatively regulated RAC1 expression. Over-expression of miR-223-3p significantly inhibited the proliferation and colony formation (P<0.05 or P<0.01) of SMMC-7721 cells and promoted cell apoptosis (P<0.01). Contrarily, knockdown of miR-223-3p reversed the inhibitory effect of miR-223-3p mimics on cells. Conclusion: miR-223-3p over-expression inhibits proliferation and colony formation and promotes apoptosis of HCC cells, the mechanism of which may be related with its targeted down-regulation of RAC1.