Zhu X, Xie C, Li Y-M, Huang Z-L, Zhao Q-Y, Hu Z-X, Wang P-P, Gu Y-R, Gao Z-L, Peng L
Department of Infectious Diseases, Third Affiliated Hospital of Sun Yat-sen University, 600# Tianhe Road, Guangzhou, Guangdong Province, China.
Guangdong Provincial Key Laboratory of Liver Diseases, Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong Province, China.
Cell Death Dis. 2016 Jun 2;7(6):e2239. doi: 10.1038/cddis.2016.146.
We have previously observed the downregulation of TMEM2 in the liver tissue of patients with chronic hepatitis B virus (HBV) infection and in HepG2.2.15 cells with HBV genomic DNA. In the present study, we investigated the role and mechanism of TMEM2 in HepG2 and HepG2.2.15 during HBV infection HepG2 and HepG2.2.15. HepG2 shTMEM2 cells with stable TMEM2 knockdown and HepG2 TMEM2 and HepG2.2.15 TMEM2 cells with stable TMEM2 overexpression were established using lentivirus vectors. We observed reduced expression of TMEM2 in HBV-infected liver tissues and HepG2.2.15 cells. HBsAg, HBcAg, HBV DNA, and HBV cccDNA levels were significantly increased in HepG2 shTMEM2 cells but decreased in HepG2 TMEM2 and HepG2.2.15 TMEM2 cells compared with naive HepG2 cells. On the basis of the western blotting results, the JAK-STAT signaling pathway was inhibited in HepG2 shTMEM2 cells but activated in HepG2 TMEM2 and HepG2.2.15 TMEM2 cells. In addition, reduced and increased expression of the antiviral proteins MxA and OAS1 was observed in TMEM2-silenced cells (HepG2 shTMEM2 cells) and TMEM2-overexpressing cells (HepG2 TMEM2 and HepG2.2.15 TMEM2 cells), respectively. The expression of Interferon regulatory factor 9 (IRF9) was not affected by TMEM2. However, we found that overexpression and knockdown of TMEM2, respectively, promoted and inhibited importation of IRF9 into nuclei. The luciferase reporter assay showed that IRF9 nuclear translocation affected interferon-stimulated response element activities. In addition, the inhibitory effects of TMEM2 on HBV infection in HepG2 shTMEM2 cells was significantly enhanced by pre-treatment with interferon but significantly inhibited in HepG2.2.15 TMEM2 cells by pre-treatment with JAK1 inhibitor. TMEM2 inhibits HBV infection in HepG2 and HepG2.2.15 by activating the JAK-STAT signaling pathway.
我们之前观察到，在慢性乙型肝炎病毒（HBV）感染患者的肝组织以及含有HBV基因组DNA的HepG2.2.15细胞中，TMEM2表达下调。在本研究中，我们调查了TMEM2在HBV感染HepG2和HepG2.2.15过程中的作用及机制。使用慢病毒载体构建了稳定敲低TMEM2的HepG2 shTMEM2细胞，以及稳定过表达TMEM2的HepG2 TMEM2和HepG2.2.15 TMEM2细胞。我们观察到在HBV感染的肝组织和HepG2.2.15细胞中TMEM2表达降低。与未处理的HepG2细胞相比，HepG2 shTMEM2细胞中HBsAg、HBcAg、HBV DNA和HBV cccDNA水平显著升高，而在HepG2 TMEM2和HepG2.2.15 TMEM2细胞中则降低。基于蛋白质印迹结果，JAK-STAT信号通路在HepG2 shTMEM2细胞中受到抑制，但在HepG2 TMEM2和HepG2.2.15 TMEM2细胞中被激活。此外，在TMEM2沉默的细胞（HepG2 shTMEM2细胞）和TMEM2过表达的细胞（HepG2 TMEM2和HepG2.2.15 TMEM2细胞）中，分别观察到抗病毒蛋白MxA和OAS1表达降低和升高。干扰素调节因子9（IRF9）的表达不受TMEM2影响。然而，我们发现TMEM2的过表达和敲低分别促进和抑制了IRF9进入细胞核。荧光素酶报告基因检测表明IRF9核转位影响干扰素刺激反应元件活性。此外，干扰素预处理显著增强了TMEM2对HepG2 shTMEM2细胞中HBV感染的抑制作用，但JAK1抑制剂预处理则显著抑制了HepG2.2.15 TMEM2细胞中的这种抑制作用。TMEM2通过激活JAK-STAT信号通路抑制HepG2和HepG2.2.15中的HBV感染。
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