Characterisaton Of Novel Biomarkers In HBV-related Acute-on-chronic Liver Failure

Background:Hepatitis B-related Acute-on-chronic liver failure (HBV-ACLF) has a high mortality rate in the Asia-Pacific region, and the mechanisms of the development and progression of HBV-ACLF remains unclear. The establishment of sensitive and specific biomarkers to early diagnosis and predict the progression of HBV-ACLF is critical for identifying patients who require early treatment. Recently, blood microRNAs (miRNAs) and signaling proteins have been reported as potential biomarkers for the noninvasive diagnosis of various diseases, including liver diseases.Materials and Methods:In the first section, novel miRNA biomarkers of peripheral blood mononuclear cells (PBMC) in HBV-ACLF patients were characterized by high-throughput sequencing. Twelve subjects from groups of HBV-ACLF, chronic hepatitis B (CHB) and healthy control participated in this study for initial screening of dysregulated miRNAs and each group had four individuals. Total miRNA isolated from PBMC was sequenced with HiSeq 2000. The expression analysis of known miRNAs and identification of novel miRNAs were performed by miRDeep2. The expression of the dysregulated miRNAs and novel miRNAs were validated in another independent 10 individuals of each group using TaqMan probe-based quantitative real-time polymerase chain reaction (qRT-PCR).In the second section, novel serological biomarkers of HBV-ACLF were initially screened by a cytokine antibody array containing 274 known human proteins in 15 subjects and each group of HBV-ACLF, CHB and Healthy had five individuals. The analysis of significant proteins were performed by use of significance analysis of microarray (SAM) and predictive analysis of microarray (PAM). Significant biomarkers were then confirmed by enzyme-linked immunosorbent assay (ELISA) in 20 healthy individuals,116 paitents with CHB and 279 HBV-ACLF patients in the experimental group. Additional 136 HBV-ACLF patients were enrolled as an external validation group for validating the capacity to predict motality of HBV-ACLF of biomarkers.Results:In the first section,1167 miRNAs were expressed in at least one sample, as well as 107 novel miRNAs. Among these detected miRNAs,78 miRNAs were significantly differentially expressed in HBV-ACLF patients compared to CHB patients and healthy controls. Seven miRNAs increased and one miRNA decreased more than 4-fold in HBV-ACLF patients with read counts greater than median in HBV-ACLF group. The expression patterns of foresaid eight miRNAs were validated by qRT-PCR, and the expression levels of six miRNAs (hsa-miR-21-5p, hsa-miR-34c-5p, hsa-miR-143-3p, hsa-miR-143-5p, hsa-miR-374a-3p and hsa-miR-542-3p) were significantly increased in patients with HBV-ACLF, which was consistent with the next-generation sequencing (NGS) results, while the other two miRNAs didn’t have significantly differential expression in HBV-ACLF patients. The Gene Ontology (GO) enrichment analysis showed that the target genes of aberrant miRNAs in HBV-ACLF mainly participated in system development, regulation of signaling, regulation of localization, cellular metabolic process, RNA metabolic process and gene expression, etc. In addition, five novel miRNAs were validated by qRT-PCR and two of them were significantly differentially expressed in HBV-ACLF patients.In the second section, the initial screen of an antibody array showed that 15 cytokines were significantly differentially expressed in patients with HBV-ACLF and chronic hepatitis B (CHB). Six of these cytokines, including hepatocyte growth factor (HGF), macrophage inflammatory protein 3a (MIP-3a), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), growth differentiation factor 15 (GDF15), E-selectin and osteopontin, were significantly increased in HBV-ACLF subjects compared to CHB subjects. These results were confirmed by ELISA in 279 HBV-ACLF patients,40 CHB patients and 20 normal adults. Moreover, high HGF and GDF15 expression levels could distinguish the HBV-ACLF and CHB patients, and 116 CHB patients could be distinguished from 279 HBV-ACLF patients accurately. The MIP-3a level was closely related to the mortality of HBV-ACLF patients, as confirmed using the external validation group. Immunohistochemistry showed that HGF, GDF15 and MIP-3a were positive in HBV-ACLF-derived liver tissues and negative in CHB and Healthy-derived liver tissues.Conclusion:The miRNA expression profile of PBMC alters in patients with HBV-ACLF and a six-miRNA signature may serve as promising biomarkers for representing progression of HBV-ACLF. HGF and GDF15 represent potential novel biomarkers for the early diagnosis of HBV-ACLF, and MIP-3a might be useful to predict the mortality of HBV-ACLF.