抑制KDM2A基因对肝癌细胞增殖、侵袭和迁移的影响

Effects of inhibiting KDM2A gene on the proliferation,invasion,and migration of liver cancer cells

目的探讨抑制KDM2A基因对肝癌细胞增殖、侵袭和迁移的影响及其可能的调控机制。方法收集2014-2017年华中科技大学同济医学院附属同济医院收治的40例肝细胞癌(HCC)患者的肝癌组织及其癌旁组织,体外培养人肝癌细胞株HepG2、Huh7、HCCLM3、MHCC-97H及正常肝细胞LO2,采用q RT-PCR及Western blotting检测肝癌组织及细胞中KDM2A mRNA及蛋白的表达情况。取Huh7细胞,设置:(1)si-NC组(转染si-NC)与si-KDM2A组(转染si-KDM2A);(2)mimic-NC组(转染mimic-NC)、miRNA-29a-3p mimic组(转染miRNA-29a-3p mimic)、inhibitor-NC组(转染inhibitor-NC)与miRNA-29a-3p inhibitor组(转染miRNA-29a-3p inhibitor)。采用q RT-PCR和Western blotting检测KDM2A mRNA及蛋白的表达情况,CCK-8法检测细胞增殖能力,Transwell实验检测细胞侵袭和迁移能力。采用在线数据库TargetScan、miRDIP、miRWalk、Starbase及miRDB预测KDM2A与miR-29a-3p的结合位点。将KDM2A 3'-UTR(WT)或KDM2A 3'-UTR(MUT)报告质粒分别与NC-miRNA或miR-29a-3p mimic共转染293T细胞48 h后,采用荧光素酶报告实验检测荧光素酶活性。结果与癌旁组织相比,肝癌组织中KDM2A mRNA和蛋白相对表达水平明显升高(P<0.05);与正常肝细胞LO2相比,肝癌细胞HepG2、Huh7、HCCLM3、MHCC-97H中KDM2A mRNA和蛋白相对表达水平明显升高(P<0.05)。与si-NC组比较,si-KDM2A组Huh7细胞增殖、侵袭和迁移能力明显降低(P<0.05或P<0.01)。在线数据库TargetScan、miRDIP、miRWalk、Starbase及miRDB分析结果显示,miR-29a-3p与KDM2A存在结合位点。双荧光素酶报告实验结果显示,miR-29a-3p mimic可明显降低KDM2A-MUT荧光素酶活性(P<0.01)。过表达miRNA-29a-3p后,Huh7细胞中KDM2A mRNA和蛋白相对表达水平降低(P<0.01),细胞增殖、侵袭和迁移能力下降(P<0.05);抑制miRNA-29a-3p的表达后,Huh7细胞中KDM2A mRNA和蛋白相对表达水平升高(P<0.05),细胞增殖、侵袭和迁移能力增强(P<0.05)。结论抑制KDM2A的表达可减弱人肝癌细胞Huh7的增殖和转移能力,而miR-29a-3p可能是KDM2A的上游调节因子,可参与肝癌的发生发展。

Objective To investigate the effects of inhibiting KDM2A gene on the proliferation,invasion and migration of liver cancer cells and its possible regulatory mechanism.Methods Forty pairs of HCC tissues and their adjacent normal counterparts were collected from 2014 to 2017 in Tongji Hospital,Tongji Medical College Affiliated to Huazhong University of Science and Technology.Human liver cancer cell lines HepG2,Huh7,HCCLM3,MHCC-97H and normal liver cells LO2 were cultured in vitro.The mRNA and protein expression levels of KDM2A in HCC tissues and cells were detected by qRT-PCR and Western blotting.Huh7 cells were taken and set up as follows:(1)si-NC group(transfected with si-NC)and si-KDM2A group(transfected with si-KDM2A);(2)mimic-NC group(transfected with mimic-NC),miRNA-29a-3p mimic group(transfected with miRNA-29a-3p mimic),inhibitor-NC group(transfected with inhibitor-NC)and miRNA-29a-3p inhibitor group(transfected with miRNA-29a-3p inhibitor).The mRNA and protein expression levels of KDM2A were detected by qRT-PCR and Western blotting.The invasion and migration of cells were detected by Transwell,the proliferation of cells was detected by CCK-8 methods.The online databases TargetScan,miRDIP,miRWalk,Starbase and miRDB were used to predict the binding sites of KDM2A and miR-29a-3p.The KDM2A 3'-UTR(WT)or KDM2A 3'-UTR(MUT)report plasmid was co-transfected with NC-miRNA or miR-29a-3p mimics respectively for 48 h in 293T cells,and the luciferase activity was detected by the luciferase reporter gene detection system.Results Compared with adjacent normal counterparts,the relative mRNA and protein expression levels of KDM2A in HCC tissues increased significantly(P<0.05).Compared with LO2,the relative mRNA and protein expression levels of KDM2A in HepG2,Huh7,HCCLM3 and MHCC-97H increased significantly(P<0.05).Compared with si-NC group,the proliferation,invasion and migration of Huh7 cells in si-KDM2A group decreased significantly(P<0.05 or P<0.01).The analysis results of TargetScan,miRDIP,miRWalk,Starbase and miRDB showed that there were binding sites between KDM2A and miR-29a-3p.The results of the dual luciferase reporter assay showed that miR-29a-3p mimic significantly reduced KDM2A-MUT luciferase activity(P<0.01).After overexpression of miRNA-29a-3p,the relative mRNA and protein expression levels of KDM2A were decreased(P<0.01),the proliferation,invasion and migration abilities were decreased(P<0.05)in Huh7 cells.After inhibiting the expression of miRNA-29a-3p,the relative mRNA and protein expression levels of KDM2A were increased(P<0.05),the proliferation,invasion and migration abilities were enhanced(P<0.05)in Huh7 cells.Conclusion Inhibiting the expression of KDM2A can reduce the proliferation and migration ability of Huh7 cells.miR-29a-3p may be the upstream regulator of KDM2A and participate in the occurrence and development of hepatocellular carcinoma.