YTHDF1对HBV蛋白表达和HBsAg和HBeAg抗原分泌的作用

目的:探讨YTH N6-甲基腺苷RNA结合蛋白1（YTHDF1）对乙型肝炎病毒（HBV）蛋白表达和抗原分泌的影响。方法:分别以pCD3/Flag-KBE和pSilencer2.1-U6 neo为载体，构建过表达和敲降YTHDF1质粒。在Huh7细胞中过表达HBV蛋白质粒的同时表达pshR-YTHDF1和对照质粒，通过Western印迹检测YTHDF1对HBV蛋白表达的影响。在HepG2.2.15细胞中过表达和敲降YTHDF1, 在Huh7细胞中同时过表达pHBV1.3质粒，通过ELISA检测YTHDF1对HBsAg、HBeAg抗原分泌的影响。结果:与正常肝细胞相比，YTHDF1在肝癌细胞Huh7（t=17.4）、HepG2.2.15细胞中（t=39.6）高表达。成功构建过表达和敲降YTHDF1质粒并验证其有效。敲降YTHDF1抑制HBV基因组编码的HBc蛋白（t=9.5）、HBs蛋白（t=5.2）、preS1蛋白（t=25.7）、preS2蛋白（t=9.0）、HBx蛋白（t=19.7）、HBV DNA Pol蛋白（t=23.0）表达，差异均有统计学意义（均P＜0.05）。过表达YTHDF1促进HBsAg（Huh7细胞t48 h=5.5，t72 h=5.0；HepG2.2.15细胞 t48 h=5.3，t72 h=27.4）、HBeAg抗原（Huh7细胞t48 h=5.7，t72 h=6.3；HepG2.2.15细胞t48 h=29.0，t72 h=6.6）的分泌，而敲降YTHDF1抑制HBsAg（Huh7细胞t48 h=16.0，t72 h=7.9；HepG2.2.15细胞t48 h=22.3，t72 h=7.0）、HBeAg抗原（Huh7细胞t48 h=9.0，t72 h=14.3；HepG2.2.15细胞t48 h=8.1，t72 h=6.6）的分泌（均P＜0.05）。结论:YTHDF1可能具有增加HBV蛋白表达及促进HBsAg和HBeAg抗原分泌的作用。

Role of YTHDF1 on HBV protein expression and secretion of HBsAg and HBeAg antigens

Objective: To investigate the effect of YTH N6-methyladenosine RNA-binding protein 1（YTHDF1） on hepatitis B virus （HBV） protein expression and antigen secretion. Methods: Overexpression and knockdown YTHDF1 plasmids were constructed using pCD3/Flag-KBE and pSilencer2.1-U6 neo as vectors，respectively. HBV protein plasmid was overexpressed in Huh7 cells along with pshR-YTHDF1 and control plasmid，and the effect of YTHDF1 on HBV protein expression was examined by Western blotting assay. YTHDF1 was overexpressed and knocked down in HepG2.2.15 cells，simultaneous pHBV1.3 plasmid was overexpressed in Huh7 cells，and the effect of YTHDF1 on HBsAg and HBeAg antigen secretion were detected by ELISA. Results: YTHDF1 was highly expressed in hepatocellular carcinoma cells Huh7（t=17.4），HepG2.2.15 cells（t=39.6） compared to normal hepatocytes. Overexpression and knockdown YTHDF1 plasmids were successfully constructed and verified to be effective. Knockdown of YTHDF1 inhibited the expression of HBc protein（t=9.5），HBs protein（t=5.2），preS1 protein（t=25.7），preS2 protein（t=9.0），HBx protein（t=19.7），and HBV DNA Pol protein（t=23.0） encoded by HBV genome，and the differences were statistically significant（all P<0.05）. Over expression of YTHDF1 promoted the secretion of HBsAg （Huh7 cells t48 h=5.5，t72 h=5.0；HepG2.2.15 cells t48 h=5.3，t72 h=27.4），HBeAg antigen（Huh7 cells t48 h=5.7，t72 h=6.3； HepG2.2.15 cells t48 h=29.0，t72 h=6.6） and knockdown of YTHDF1 inhibited the secretion of HBsAg（Huh7 cells t48 h=16.0，t72 h=7.9，HepG2.2.15 cells t48 h=22.3，t72 h =7.0），HBeAg antigen（Huh7 cells t48 h=9.0，t72 h=14.3，HepG2.2.15 cells t48 h=8.1，t72 h=6.6），all P<0.05. Conclusion: YTHDF1 may have a role in increasing HBV protein expression and promoting the secretion of HBsAg and HBeAg antigens.