The Regulation Mechanism Of NFAT5 On The Development Of HBV-associated HCC

Objects:Infection with the hepatitis B virus(HBV)is closely associated with the development of hepatocellular carcinoma(HCC).In China,HBV infection ranks the first among all causes for HCC development.With its low survival rate and high recurrence rate and mortality,HCC is one of the most difficult cancer disease for diagnosis and treatment.Nuclear factor of activated T-cells 5(NFAT5)is documented that mediated by osmolality and proved to be an oncogene in some tumors.Methods:We applied RT-qPCR for estimating NFAT5 mRNA expression in tumor and non-tumor tissue and evaluated NFAT5 protein expression in tumor and non-tumor tissue by immunohistochemistry.Then we verified NFAT5 expression in different cell lines with RT-qPCR and Western Blot.We also studied NFAT5 protein expression in different osmotic solution by WB.Afterwards we estimated the effect of NFAT5 on hepatoma cell apoptosis by Flow Cytometry(FCM),also we adopted WB for apoptotic biomarker.Then we analysis regulation of NFAT5 for HCC invasion through applying Transwell invasion assay and Scratch assay.We also tested biomarkers for invasion with Western Blot.Then we used FCM again for analyzing role of NFAT5 in cell cycle progression.After that,we predicted regulator of NFAT5 through bio-information analysis(KEGG,Targetscan).Then we proceeded Western Blot,RT-qPCR and electrochemiluminescence immunoassay for studying interaction between HBV and miR-30e-5p.We applied WB for variation of protein in MAPK signaling pathway.Via ChIP-PCR method,we testified the correlation between c-Myc and NFAT5 promoter.Afterwards,we proceeded ChIP-Seq for prognosis of target genes of NFAT5.We then knocked down and overexpressed NFAT5 in order to learn the correlation between NFAT5 and DARS2.Then we analyzed DARS2 expression in HCC cell lines with WB and RT-qPCR.We applied FCM for studying the function of DARS2 in apoptosis and cell cycle progression,meanwhile we evaluated the biomarkers of cell cycle and apoptosis.Then we collected the clinical data for studying correlation between DARS2 expression and clinical characteristics by statistical analysis.Moreover,we applied SPSS 21 for analysis of ROC curve,Kaplan-Meier survival analysis and COX regression analysis.Result:In our study,we considered NFAT5 as a tumor suppressor of HCC,regulated by osmolality.Besides,we discovered NFAT5 inhibited cell cycle progression and tumor invasion,and induced HCC cell apoptosis.On the other hand,hyperosmolality upregulated NFAT5,which is considered as protective factor for HCC.Furthermore we investigated the regulatory mechanisms in the upstream of NFAT5 in HBV-associated HCC,the results showed that HBV suppressed NFAT5 expression via inhibiting miR-30e-5p in hepatoma cells,and miR-30e-5p inhibited HBV replication in turn.MiR-30e-5p upregulated NFAT5 expression was not binding to the 3’UTR of the NFAT5,but inactivated the MAPK signaling pathway by directly targeting MAP4K4.Finally,we used CHIP-seq to find a novel NFAT5 target:DARS2,which is unknown in HCC.We discovered NFAT5 suppressed DARS2 by binding to its promoter,as determined by a ChIP-seq assay.DARS2 was identified as an HCC oncogene that promotes HCC cell cycle progression and inhibits HCC cell apoptosis.In conclusion,HBV promotes HCC tumorigenesis by inducing DARS2,via miR-30e-5p/MAPK/NFAT5 pathway.These findings should provide new insights into our understanding how the molecular mechanisms underline the effects of HBV infection on the expression of NFAT5 and the development of HCC.