TGFBI Promoter Methylation Resulted From HBV RtA181T Mutation

Objective:This study aims to clarify whether TGFBI promoter methylation involved in the oncogenic activity of the HBV rtA181 T truncation mutant and investigate the molecular mechanism of the oncogenic potential of HBV rtA181 T surface truncation mutant via constructing HBsAg and its rtA181 T mutant HBsAg?rtA181T? expression vectors in LO2 cells. Methods:1.cDNA of HBsAg was amplified by RT-PCR with total DNA extracted and cloned into eukaryotic expression vector p IRES2. The recombinant plasmid p IRES2-HBsAg was identified by colony PCR and DNA sequencing;2. The mutant plasmid pIRES2-HBsAg?rtA181T? was obtained by site directed mutagenesis by using the recombinant plasmid p IRES2-HBsAg as the template. The recombinant plasmid p IRES2-HBsAg?rtA181T? was identified by colony PCR and DNA sequencing;3. LO2 cells were transiently transfected with p-HBsAg?rtA181T. Chromatin immunoprecipitation sequence?Ch IP-seq? assay was performed for the binding of rtA181 T surface truncation mutant to TGFBI genes;4. The recombinant plasmid of TGFBI?NM₀00358? promoter was constructed according to the results of CHIP-seq, and identified by colony PCR and DNA sequencing. The pGL3-TGFBI-transfected LO2 cells were transiently transfected with p-HBsAg, rtA181 T or the control vector for 24 h and then subjected to 0.5 nM TGF-? for 24 h. Luciferase reporter gene assay was perform for the gene expression.5. The LO2 cells were transiently transfected with p-HBsAg, rtA181 T or the control vector for 24 h and then subjected to 0.5 nM TGF-? for 24 h. The expression level of mRNA TGFBI was detected by RT-qPCR. The expression level of protein TGFBI was detected by western blot.6.The LO2 cells were transiently transfected with p-HBsAg, rtA181 T or the control vector. The methylation status of TGFBI promoter was detected by MSP method. The LO2 cells were transiently transfected with p-HBsAg, rtA181 T or the control vector for 24 h and then subjected to 5-Aza for 24 h. The expression level of mRNA TGFBI was detected by RT-qPCR. The expression level of protein Cyclin D1 and pCREB were detected by western blot. Results:1. DNA sequencing confirmed that the recombinant eukaryotic expression plasmid pIRES2-HBsAg and p IRES2-HBsAg?rtA181T? were constructed successfully;2. LO2 cells were transiently transfected with p-HBsAg, rtA181 T or the control vector. Chromatin immunoprecipitation sequence?ChIP-seq? results show that TGFBI gene may be responsible for rtA181 T truncated HBV surface protein;3.The recombinant plasmid of TGFBI?NM₀00358? promoter was constructed according to the results of CHIP, and identified by colony PCR and DNA sequencing. The pGL3-TGFBI-transfected LO2 cells were transiently transfected with p IRES2-HBsAg, rtA181 T or the control vector for 24 h and then subjected to 0.5 nM TGF-? for 24 h. Luciferase reporter gene assay was perform for the gene expression, the activity of rtA181 T group obviously on the low side.4. TGF-? treatment can significantly increase TGFBI mRNA and protein expression in p-HBsAg-transfected LO2 cells, however, in rtA181T-transfected LO2 cells, the rtA181 T mutation completely abrogated the TGF-?-induced TGFBI mRNA and protein expression.5. In rtA181T-transfected LO2 cells, the rtA181 T mutation notably increased the methylation status but not in p-HBsAg-transfected LO2 cells.However, the 5-Aza pretreatment abolished the effect. Meanwhile, the protein expression of Cyclin D1 and pCREB were significantly up-regulation. Conclusion: The inhibitory expression of TGFBI and the promoter methylation of TGFBI gene is involved in the oncogenic potential of HBV rtA181 T surface truncation mutant.