| Patients suffering from severe hepatitis B have a high mortality and poor prognosis. Searching for molecular biomarkers, which can be used for early warming and diagnosis of severe hepatitis, has become a hot topic of viral hepatitis. Increasing evidence indicates that circulating microRNAs (cir-miRNAs) may serve as potential diagnostic biomarkers for human diseases. The aims of the present study are to investigate the change of expression pattern of serum miRNAs during severe exacerbation of chronic hepatitis B in order to screen differentialy expressed microRNA-210(miR-210), to evaluate the correlation of serum miR-210level with severe exacerbation progression of chronic hepatitis B, to explore the potential function and mechanism of miR-210in the exacerbation of chronic hepatitis B and to provide novel target for predicting, preventing and treating severe hepatitis B.Methods1miRNA microarray experiment(1) miRNA expression profile analysisTaqMan(?) Human MicroRNA Arrays A were employed to profile miRNAs in mixed serum from10mild chronic hepatitis B (CHB-M) patients,10HBV-associated acute-on-chronic liver failure (ACLF) patients and10healthy controls, respectively.(2) Screening differentially expressed miRNAs miRNA with cycle threshold (Ct) value below35was considered as miRNA detectable in serum. Differentially expressed miRNAs between each group was preliminarily screened based on the criteria:|△△Ct|≥2.(3) Selecting candidate miR-210miRNA with cycle threshold (Ct) value below35was considered as miRNA detectable in serum. Candidate miR-210, whose expression level increased with disease severity, was selected for further RT-qPCR identification.2Investigation of patients with hepatitis B(1) One hundred and twenty subjects consisted of30cases with mild chronic hepatitis B (CHB-M),30cases with severe chronic hepatitis B (CHB-S),30cases with HBV-associated acute-on-chronic liver failure (ACLF) and30healthy controls.(2) RT-qPCR was performed to further identify differentially expressed miR-210screened by miRNA microarray.(3) The hepatic function (ALT, AST, TBIL, PTA), serum HBV markers and the titer of HBV DNA were measured.3Animal experimentLiver failure in mice was established by intravenously injection with Con A. The mice were divided into the exposure group exposed to Con A (20mg/kg) and control group exposed to normal saline for6h,12h and24h, respectively. Serum and hepatic tissues were collected from normal or Con A-treated mice.(1) Hypoxic hepatocytes were detected by using the HypoxyprobeTM-1Plus Kit.(2) RT-qPCR was performed to detect the levels of hepatic and serum miR-210.(3) RT-qPCR was performed to detect the levels of Iscu, Ndufa4, Sdhd, Gpdll andCox10mRNA in hepatic tissues.4Experiment in vitro The HepG2.2.15cells were transfected with blank control (DEPC-H2O), mimics negative control RNA and miR-210mimics, respectively. Transfected cells were cultured under hypoxic condition for24h or48h.(1) HepG2.2.15cell dehydrogenase activity was determined by using the CCK8assay.(2) The concentrations of HBsAg and HBeAg in the supernatants were determined by enzyme immunoassay kits. HBV DNA in the supernatants was quantified using a commercial PCR diagnostic kit. RT-qPCR was performed to detect the levels of HBsAg mRNA in HepG2.2.15cells.Results1The results of miRNA microarray(1) The number and expression levels of miRNAs detectable in serum increased markedly with disease severity.(2) The expression levels of20miRNAs detectable in serum of hepatitis B patients were significantly higher than those in healthy control group.(3) The expression levels of miR-210, miR-324-5p and miR-423-5p detectable in serum increased markedly with disease severity.2The results of patient investigation(1) The levels of serum miR-210in CHB-M, CHB-S and HBV-ACLF patients were significantly higher than those in control group (P<0.05). Serum miR-210levels increased with the severity of patients with hepatitis B.(2) The elevated levels of serum miR-210were positively correlated with the increased levels of serum ALT/AST and total bilirubin in CHB (P<0.05), and the levels of serum miR-210were negatively correlated with the prothrombin activity (P<0.05).3The results of animal experiment (1) Dynamic hypoxia existed in Con A-induced hepatitis model. The hypoxic rates of hepatocytes in6and12h groups significantly enhanced as compared with0h group (P<0.05), the hypoxic rate of hepatocytes in24h group was higher than that in0h group but lower than those in6and12h groups (P<0.05).(2) The levels of hepatic and serum miR-210increased obviously with the development of liver failure.(3)The levels of Iscu, Ndufa4, Sdhd, Gpd11and Cox10mRNA were significantly decreased during liver injury (P<0.05).4The results of experiment in vitro(1) miR-210overexpression inhibited HepG2.2.15cell dehydrogenase activity under hypoxic condition.(2) miR-210overexpression inhibited HBV DNA replication and HBsAg expression under hypoxic condition.Conclusions(1) The results of miRNA microarray indicated that the number and expression levels of microRNAs detectable in serum were correlated with disease severity of hepatitis B.(2) The expression levels of miR-210increased with disease severity, miR-210may serve as a novel marker for early diagnosis of severe hepatitis B.(3)Liver inflammation causing liver hypoxia may be the possible mechanism that resulted in miR-210levels elevation during hepatitis progression.(4) miR-210may play a protective role during hepatitis progression through regulation of mitochondrial metabolism. |
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