Overexpression Of HBx Protein Induces ER Stress In Liver Cancer Cells And Effects Of MANF On Hepatoma Cells Biological Behavior

HBV (hepatitis B virus, HBV) infection causes chronic hepatitis, part of themfinally converted to liver cancer. Recent studies have shown that endoplasmic reticulum(ER) stress involved in the occurrence and development of tumorigenesis. However, themechanism remains unclear. The genome of HBV contains four open reading frames(open reading frame, ORF), where X region coding product (hepatitis B virus X protein,HBx) is a kind of multi-functional regulatory proteins, which including the intracellularsignal transduction, viral replication, transcription, cell proliferation and apoptosis.Therefore, HBx protein plays an important role in the development of chronic liverdisease. ER stress is one of the eukaryotic cell protective reactions. To reduce theabnormal proteins in the cell for cell protection effect, it responses to unfolded ormalfolded proteins by activating unfolded protein response (UPR) pathway. MANF(mesencephalic astrocyte derived neurotrophic factor, MANF) is a secretory protein ERstress induces its expression. We hypothesize that MANF probobaly is involved in HBx-mediated hepatocellular carcinoma.Objective:To establish stable expression of hepatitis B virus X protein (HBx) cell line bytransfection of HBx to HepG2. To investigate whether HBx causes ER stress and clarifythe effects of MANF on the biology activity of HepG2cell, with or without HBxexpression.Methods:We cloned HBx gene encoding fragment from HepG2.2.15cells by RT-PCRmethod to build pcDNA3.1-HBx eukaryotic expression plasmid and obtained the stableexpresses HBx cell line of HepG2, after G418screening. Cell proliferation activity wasdetermined by MTT method. The expressions of UPR genes were detected by PCRmethod. The effects of MANF, a inducible protein of ER stress, on the biologicalbehavior of hepatoma cells were determined by MTT, and transwell migration test,transwell invation test.Results:1. Establishment of cell line stably expressing HBx proteinThe gene fragment encoding HBx protein was cloned from HepG2.2.15cells by PT-PCR method. The PCR product was inserted into the pcDNA3.1vector after enzymedigestion with EcoR I and Hind Ⅲ. The construct was identified by enzyme digestionand plasmid sequencing. The result showed that we successfully constructed theplasmid pcDNA3.1–HBx Then we transfected the plasmid pcDNA3.1–HBx to HepG2cells and screened with G418(0.5u/ml) for2months. The expression of HBx in thestable cells was detected by immunoblot tests with anti-HBx antibody. 2. HBx promotes proliferation of HepG2cellsThe viability of HepG2stably expressing HBx was determined by MTT method.We found that the proliferation of the cells expressing HBx was increased comparedwith the cells transfected with blank vector. However, there is no difference in theproliferation between HepG2cells and HepG2.2.15cells expressing the whole genomeDNA of HBV, which may be related to comprehensive results in HBV genome.3. HBx protein differentially regulates UPR genesTo investigate the effect of HBx on the UPR genes, we detected PERK, BiP, MANF,ATF6, XBP1, IRE1, CHOP, and EIF2using RT-PCR. To our surprise, HBxdifferentially regulated these genes. For example, HBx up-regulated PERK, BiP,MANF, ATF6, and XBP1, while down-regulated IRE1, CHOP and EIF2expression inHepG2cells. These results suggest that HBx protein can induce ER stress via diverseUPR pathway.4. MANF inhibits liver cancer cell growth，migration and invasion4.1Effect of MANF overexpression on the proliferation of hepatoma cellsIn our previous studies, we found that hepatitis B and hepatocellular carcinoma(HCC) up-reuglated MANF expression. In this study, we also found HBx promotedMANF transcription. Therefore, we wondered that whether MANF involves in thetumorigenesis caused by HBx. To test it, we firstly overexpressed MANF in the livercancer cells. The results showed that MANF exerted different influences on differenttypes of liver cancer cell lines. MANF inhibited the proliferation of SMMC-7721,HepG2, HepG2.2.15, and HepG2cells stably expressing HBx.4.2Knockdown endogenous MANF promotes the proliferation，migration andinvasion of hepatoma cellsThe MANF-siRNA was transfected into HepG2cells. The expression of MANF was detected by RT-PCR. The result showed that the MANF-siRNA effectively inhibitedthe expression of endogenous MANF. MTT assay showed that MANF-siRNAtransfection promoted the proliferation of SMMC-7721cells, HepG2.2.15, HepG2,HepG2-HBx cellines, compared with the cells with blank vector transfection,suggesting MANF may be an inhibitor of HCC. We also found transfection withMANF-siRNA promotes the migration and invasion of HepG2cells.Conclusion:We successfully established the cell line stably expressing HBx; HBx differentiallyregulates ER (or UPR) related genes; MANF has inhibitory effect on liver cancer cellgrowth, migration and invasion.