miR-425通过靶向PTPRN2调节肝癌细胞的增殖、迁移和侵袭

MiR-425 regulates proliferation,migration and invasion of hepatocellular carcinoma cells by targeting PTPRN2

目的探讨miR-425对肝癌细胞增殖、迁移和侵袭的影响及其分子机制。方法RT-qPCR检测肝癌细胞系(HepG2、SMMC-7721、MHCC-97H)和正常肝细胞系HL-7702中miR-425的表达水平;利用生物信息学预测miR-425作用的靶基因;双荧光素酶实验确认miR-425和PTPRN2的直接靶向关系;蛋白质印迹检测PTPRN2蛋白表达水平;MTT实验检测miR-425和PTPRN2对肝癌细胞增殖能力的影响;Transwell迁移和侵袭实验检测miR-425和PTPRN2对肝癌细胞迁移和侵袭能力的影响。结果miR-425在肝癌细胞系HepG2、SMMC-7721、MHCC-97H相对表达量分别为:2.01±0.13、3.97±0.24、9.14±0.26,高于正常肝细胞系的1.00±0.03(P<0.05);封闭miR-425的表达(miR-425 inhibitor)可明显抑制肝癌细胞的增殖、迁移和侵袭能力;荧光素酶实验结果显示:与对照组相比,野生型质粒组荧光素酶活性明显降低(0.99±0.09比0.40±0.03,P<0.05),突变型质粒组荧光素酶活性无明显变化(1.00±0.05比0.93±0.07,P>0.05);沉默PTPRN2蛋白表达后可以逆转miR-425 inhibitor对肝癌细胞增殖、迁移和侵袭能力的抑制作用。结论miR-425通过靶向下调PTPRN2可促进肝癌细胞的增殖、迁移和侵袭过程。

Objective To investigate the effect of microRNA(miR)-425 on the proliferation,migration and invasion of hepatocellular carcinoma cells,and the molecular mechanism.Methods The expression level of miR-425 in hepatocellular carcinoma cell lines(HepG2,SMMC-7721 and MHCC-97H)and normal hepatic cell line(HL-7702)was detected using reverse transcription-quantitative polymerase chain reaction.We predicted the target genes of miR-425 by bioinformatics analysis,and confirmed that protein tyrosine phosphatase,receptor type,N polypeptide 2(PTPRN2)was directly targeted by miR-425 through dual luciferase reporter assay.The expression level of PTPRN2 protein was examined by western blotting.The effect of miR-425 and PTPRN2 on the proliferation,migration and invasion of hepatocellular carcinoma cells was tested by MTT assay and Transwell assay,respectively.Results The relative expressions of miR-425 in hepatocellular carcinoma cell lines were 2.01±0.13,3.97±0.24 and 9.14±0.26,which were higher than 1.00±0.03 in normal hepatic cell line(P<0.005).Inhibiting the expression of miR-425 by transfecting miR-425 inhibitor could significantly suppress the proliferation,migration and invasion of hepatocellular carcinoma cells.The luciferase reporter assay showed that miR-425 could reduce the activity of luciferase in wild-type vectors(0.99±0.09 vs 0.40±0.03,P<0.05).But there was no statistically significant difference in mutant vectors(1.00±0.05 vs 0.93±0.07,P>0.05).Knockdown of PTPRN2 could reverse the inhibitory effect of miR-425 inhibitor on the proliferation,migration and invasion of hepatocellular carcinoma cells.Conclusion MiR-425 promotes the proliferation,migration and invasion of hepatocellular carcinoma cells by targeting PTPRN2.