Identification and functional characterization of exosomal microRNAs associated with HBV reactivation after liver transplantation

Abstract

Hepatitis B virus (HBV) reactivation after liver transplantation (LT) is a risk factor of graft failure and poor prognosis. Although administrations of nucleos(t)ides analogs (NAs) and hepatitis B immunoglobin (HBIG) have achieved effective potent in suppressing HBV replication, a proportion of patients still suffer from post-LT HBV reactivation because this prophylaxis strategy cannot completely remove HBV. Therefore, it is a pressing need to develop novel predictive and preventive strategies against post-LT HBV reactivation. MicroRNAs(miRNAs), as exosome-mediated secretory molecules, play important regulatory roles in HBV expression, replication, and HBV-host interaction. We hypothesized that the initiation and progression of post-LT HBV reactivation should alter and secret specific regulatory miRNAs. In this study, we identified and characterized novel exosomal miRNAs associated with post-LT HBV reactivation, aiming to develop novel miRNA-based predictive and preventive strategies against post-LT HBV reactivation.

Serum samples from the first timepoint of HBV reactivation (HBV-reactive) and the last timepoint before HBV reactivation (HBV-inactive) in HBV-reactivated LT patients were recruited. Differentially expressed miRNAs were identified using the low-density array (LDA). Differential exosomal miRNAs were identified and validated in serum exosomes. The correlations were analyzed between exosomal miRNAs and HBV-related clinical parameters. An in vitro HBV infection model was established by generating an NTCP-overexpressing HepG2 cell line (HepG2-NTCP) as the HBV-recipient and adopting the HepG2.2.15 cell line as the HBV-producing donor. The efficiency of HBV infection was evaluated by examining the HBV DNA and HBsAg. The functions of miR-766-3p in HBV infection and replication were characterized through overexpression and suppression approaches. Downstream targets of miR-766-3p were identified by computational and experimental methods. Molecular experiments were performed to reveal the underlying mechanisms of miR-766-3p in regulating HBV infection.

From the LDA analysis, twenty-six circulating miRNAs were differentially upregulated at HBV-reactive status compared to HBV-inactive status. Three novel exosomal miRNAs, including miR-151-3p, miR-625-3p, and miR-766-3p, were significantly elevated at HBV-reactive status. The deregulations of exosomal miR-151-3p and miR-766-3p were significantly and positively associated with HBV DNA copies at the HBV-reactive status. In addition, the expression of exosomal miR-766-3p in HBV-positive patients and cells was significantly higher than in negative ones. An in vitro HBV infection model was established after several steps of optimization. Exosomal miR-766-3p was significantly upregulated in the HepG2-NTCP cells after HBV infection, indicating that miR-766-3p was an HBV-infection-responding target. Targeted inhibition of miR-766-3p in HepG2-NTCP cells significantly reduced its intracellular and extracellular levels of HBV DNA and HBsAg after HBV infection, suggesting that miR-766-3p might be a potential therapeutic target in preventing HBV infection. Inhibition of miR-766-3p could restore the effect of HBV-induced inactivation of MAPK signaling pathways. We identified that mitogen-activated protein kinase 1(MAPK1/ERK2) was the downstream target of miR-766-3p during HBV infection. Importantly, inhibition of miR-766-3p could suppress an ERK2-downstream transcriptional factor hepatocyte nuclear factor homeobox 1A (HNF1α), leading to the repression of HBV infection, indicating a novel mechanism of the miR-766-3p-MAPK1- HNF1α axis in regulating HBV infection.

In conclusion, exosomal miR-766-3p is not only a circulating biomarker but also a therapeutic target for HBV reactivation after liver transplantation.