Study On MicroRNA Expression And Function In Peripheral Blood Mononuclear Cells From Patients With Chronic Hepatitis B Virus Infection

Backgroud Hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF) is a serious liver disease associated with significant morbidity and mortality. The pathogenesis of HBV infection is closely associated with HBV replication and host immune response. HBV is a typically noncytopathic virus that dose not directly damage infected cells. The pathogenesis is largely mediated by the immune response following HBV infection. microRNA (miRNA), a new category of non-coding RNA, has been found to be involved in immune reponse and cell differentiation and development. miRNA have been shown to modulate innate immune responses through Toll-like receptors (TLRs) and cytokine signaling pathway. In addition to regulating innate immune responses, miRNAs participate in adaptive immune responses by influencing antigen presentation and modulating T cell receptors (TCRs) signaling. The relationship between miRNA and HBV increased our attention. Results from recent researches have suggested that miRNAs are associated with the viral replication, disease progression following HBV infection. Studies have also demonstrated that miRNA expression profiles in Peripheral blood mononuclear cells (PBMC) contributes to predicting the clinical outcome of interferon (IFN) therapy in HBV-and hepatitis C virus (HCV)-infected patients. Despite recent advances in relationship between miRNA and HBV, the miRNA involved in ACLF Is not understood.Objective (1) To investigate the miRNA expression profiles in PBMC from HBV-infected patients with different clinical manifestations.(2) To identify the significant miRNAs which are potentially associated with HBV symptom severity.(3) To predict the target genes of the significant miRNAs and investigate the target genes expression profiles in PBMC from HBV-infected patients with different clinical manifestations.(4) To identify the target genes of miR-197and miR-328and explore the biological function of miR-197and miR-328.Methods (1) PBMC isolated from4asymptomatic carrier (ASC) and4ACLF patients were used for the miRNA microarray profiles.(2) The possible target genes for miR-197and miR-328were identified using TargetScan5.2, DIANA-microT3.0, and Pictar target prediction program.(3) The expression levels of miR-150, miR-197, miR-223, miR-328, miR-30a, miR-574-3p were validated in PBMC from51healthy control (HC),70ASC,107chronic hepatitis B (CHB) patients, and76ACLF patients by real-time polymerase chain reaction (real-time PCR). The expression levels of interleukin18(IL18), tumor necrosis factor ligand superfamily member10(TNFSF10), and tumor necrosis factor receptor superfamily member9(TNFRSF9) were detected by real-time PCR.(4) Gene transfection was used to introduce the miR-197mimic/inhibitor and miR-328mimic/inhibitor into THP-1cell lines, respectively. Cell counting kit-8(CCK-8) was used to detect the cell increment rate of cells in each group. The IL18expression of cell culture supernatant was investigated by enzyme-linked immunosorbent assay (ELISA). The real-time PCR was employed to analyze the expression of miR-197, miR328, TNFRSF9, TNFSF10and IL18, and the western blot was employed to analyze the protein expression of TNFRSF9and TNFSFIO in each group.Results (1) The microarray profiles suggested that a total of17PBMC miRNAs were found to be differentially expressed between ASC and ACLF patients, including5up-regulated (miR-1246, miR-30a, miR-1305, miR-193a-3p and miR-196b) and12down-regulated (miR-223, miR-574-3p, miR-150and so on).(2) The validation experiment showed that miR-197, miR-574-3p and miR-30a profiles were consistent with the initial miRNA microarray profiles, but miR-150, miR-223and miR-328profiles were inconsistent with the initial miRNA microarray profiles. The PBMC miR-197levels regularly decreased as the severity of liver disease symptoms aggravated. Meanwhile, the PBMC miR-328level was lower in HC than in CHB and ACLF patients. Furthermore, miR-150, miR-30a, and miR-574-3p showed differential expression in the PBMC of HBV-induced liver disease patients presenting with various symptoms. But the miR-223expression did not differ significantly.(3) Using target prediction programs, such as TargetScan5.2, we found that miR-197can interact with the3’untranslated region (UTR) of TNFRSF9, TNFSF10, and IL18, and miR-328can interact with the3’UTR of TNFRSF9. The PBMC TNFRSF9expression regularly decreased as the severity of liver disease symptoms aggravated, that was contrast to miR-328results. The PBMC TNFSF10and IL18expression regularly increased as the severity of liver disease symptoms aggravated, that was contrast to miR-197results.(4) The mRNA and protein expression of IL18, TNFRSF9and TNFSF10were significantly lower in miR-197mimic group than in blank control group and mimic control group. Compared with blank control group and inhibitor control group, the mRNA and protein expression of IL18in miR-197inhibitor group were dramatically higher. Although the mRNA expression of TNFRSF9was significantly increased in miR-197inhibitor group, the protein expression was similar. The mRNA expression of TNFSF10was significantly increased in miR-197inhibitor group, but the protein expression was similar. The mRNA and protein expression of TNFRSF9was significantly lower in miR-328mimic group than in blank control group and mimic control group, and that was noticeably higher in miR-328inhibitor group than in blank control group and inhibitor control group.Conclusions (1) The microarray has low repeatability, so the miRNA profiles was required to combine microarray and real-time PCR.(2) Multiple miRNAs in the PBMC of HBV-infected patients were highly correlated with the severity of clinical manifestations, especially miR-197and miR-328, which may be a potential biomarker for HBV-induced liver disease.(3) TNFRSF9, TNFSF10, and IL18showed differential expressions in the PBMC of the HBV-infected patients presenting with various symptoms.(4) miR-197negative regulated the mRNA and protein expression of EL18, and miR-328negative regulated the mRNA and protein expression of TNFRSF9.(5) miR-197negative regulated the mRNA expression of TNFRSF9.(6) miR-197negative regulated the mRNA expression of TNFSF10.(7) miR-197and miR-328were involved in the pathogenesis of HBV infection through IL18and TNFRSF9pathway, respectively.