Effect Of Hepatitis B Virus X Protein (HBx) On ST2 Expression In Hepatocytes And Its Molecular Mechanism

Objective: Interleukin-33(IL-33),a new member of the IL-1 family,exerts biological functions by binding to its receptor ST2(also known as IL-1 receptor-like 1,IL1RL1).When IL-33 forms a complex with ST2,it recruits and binds IL-1 receptor accessory protein(IL-1Rac P),binds to form a trimer,which conducts signal transduction with NF-κB by activating mitogen-activated protein kinase(MAPK).The IL-33/ST2 axis is involved in a variety of diseases and plays an important role in the body’s immune response.Studies have indicated higher expression of s ST2 in serum of chronic hepatitis B patients comparing to healthy controls,however the molecular mechanism remains unknown.As the X gene expression product X protein(HBx)in hepatitis B virus(HBV)plays a multi-functional role in the progression of hepatitis,this study investigated the relationship between HBx and IL-33/ST2 axis as well as the impact of HBx on the expression of ST2 in hepatocytes under related regulatory mechanisms.Methods: Serum samples were collected from the patients with chronic hepatitis B(CHB),the patients with HBV-related hepatocellular carcinoma(HBV-HCC)and the healthy human,and then the content of soluble ST2(s ST2)in different groups were detected using an ELISA kit.Next,the supernatant of cultured HL7702-HBx cells at different culture time were collected and then detected by s ST2 ELISA.Moreover,the ST2 promoter was constructed to determine whether HBx affects the transcriptional activity of the ST2 promoter via GATA-2.The Ch IP assay was used to confirm the interaction between HBx and GATA-2.The effects of GATA-2 on ST2 promoter transcription were also investigated by using GATA-2 overexpression and si RNA interference experiments.Results:(1)Serum s ST2 levels in CHB group and HBV-HCC group were significantly higher than those in healthy control group(P<0.05).Serum s ST2 levels in HBV-HCC group were significantly higher than those in CHB group(P<0.05);(2)By ROC curve analysis,the optimal cut-off points of CHB and HBV-HCC were 23.255 ng/ml and 33.740 ng/ml,respectively.The optimal cut-off point of s ST2 is valuable for monitoring the progress of HBV infection-related diseases.(3)The verification experiment of cultured cells indicated that the level of s ST2 in HL7702-HBx cells was significantly higher than that in HL7702-WT and HL7702-NC cells(P<0.05).There was no difference in s ST2 level between HL7702-WT and HL7702-NC cells.The s ST2 level of the three cells increased gradually with the prolongation of culture time(P<0.05);(4)HBx affected the transcriptional activity of ST2 promoter via GATA-2;The protein and m RNA expression of ST2 could be affected by up-regulation and interference the expressing of GATA-2(P<0.05).Conclusion:(1)HBV infection can promote the expression of ST2 in hepatocytes,and it shows an obvious upward trend with the development of the disease.(2)spearman correlation analysis and ROC curve analysis show that the detection of s ST2 in peripheral blood has reference value for the monitoring the progression of HBV infection-related disease;(3)GATA-2 is important for HBx to up-regulate the transcriptional activity of ST2 promoter and promote ST2 expression.