微RNA-182-5p激动剂和抑制剂对肝癌细胞增殖和迁移的影响

Effects of miR-182-5p agomir or antagomir treatment on proliferation and migration of hepatocellular carcinoma

目的研究微RNA(miR)-182-5p激动剂和抑制剂对肝癌细胞增殖和迁移的影响。方法采用实时荧光定量-聚合酶链反应(RT-qPCR)和蛋白质印迹法检测非肝细胞癌(HCC)肝组织,HCC组织及邻近组织标本中miR-182-5p的表达水平以及RND3的mRNA/蛋白表达水平。建立患者来源的HCC细胞培养物,并进行miR-182-5p激动剂和抑制剂转染处理,以分别模拟miRNA的过表达和敲减。通过Transwell细胞侵袭实验监测体外HCC细胞的迁移。通过细胞活力和增殖评价体外HCC细胞的生长。结果与非HCC或邻近的HCC组织相比,HCC组织中的miR-182-5p明显上调,与RND3 mRNA表达呈负相关。使用miR-182-5p激动剂可显著降低HCC细胞中RND3 mRNA/蛋白质表达水平。通过荧光素酶试验和顺乌头酸酶2(AGO2)-RNA免疫沉淀分析,验证了miR-182-5p对RND3 mRNA具有靶向作用。MiR-182-5p激动剂可显著降低RND3表达,促进HCC细胞体外迁移和侵袭,可能是通过Rho相关卷曲螺旋蛋白激酶1(ROCK1)和ROCK2的抑制来实现。结论miR-182-5p可以通过靶向RND3来提高体外HCC细胞的迁移和增殖。使用miR-182-5p抑制剂,可以抑制体外肝癌细胞的迁移和增殖。

Objective To investigate the effects of miR-182-5p agomir or antagomir treatment on proliferation and migration of hepatocellular carcinoma(HCC).Methods The expression level of miR-182-5p and mRNA/protein expression levels of RND3 in non-HCC liver tissue,HCC tissue and adjacent tissue specimens were evaluated by RT-qPCR and western blotting.Patient-derived HCC cell culture was established,and transfected with miR-182-5p agomir or antagomir to mimic the overexpression or knockdown of the miRNAs,respectively.HCC cell migration in vitro was assessed by Transwell migration and invasion assay,while the HCC cell growth in vitro was evaluated by cell viability,proliferation and apoptosis assay.Results miR-182-5p were significantly upregulated in HCC tissue specimens comparing to non-HCC or adjacent ones,and negatively correlated with RND3 mRNA expression level.Treatment with miR-182-5p agomir significantly reduced RND3 mRNA/protein expression level in HCC cells.MiR-182-5p targeting of RND3 mRNA was verified by luciferase reporter assay and AGO2-RNA immunoprecipitation assay.MiR-182-5p agomir treatment significantly suppressed RND3 expression and promoted HCC cell migration and invasion in vitro,possibly via inhibition of Rho-related coiled-coil containing protein kinase 1(ROCK1)and ROCK2.Conclusion MiR-182-5p may increase HCC cell migration and proliferation in vitro via targeting RND3.Treatment with miR-182-5p antagomir may result in suppression of HCC cell migration and proliferation in vitro.