Endogenous antiviral microRNAs determine permissiveness for hepatitis B virus replication in cultured human fetal and adult hepatocytes

Abstract

Superior cell culture models for hepatitis B virus (HBV) will help advance insights into host-virus interactions. To identify mechanisms regulating HBV replication, this study used cultured human HepG2 cells and adult or fetal hepatocytes transduced with adenoviral vector to express HBV upstream of green fluorescent protein. The vector efficiently transduced all cell types. In HepG2 cells, replicative viral intermediates, nucleocapsid-associated HBcAg, and HBsAg were expressed. However, in fetal or adult hepatocytes, pregenomic HBV RNA and viral RNAs were expressed, but nucleocapsid-associated HBcAg in cells or HBsAg in culture medium were absent, indicating interruptions in viral replication due to possible microRNA-related interference. MicroRNA profiling demonstrated that a large number of microRNAs with antiviral potential were differentially expressed in hepatocytes after culture. In transfection assays using HepG2 cells, candidate antiviral microRNAs, e.g., hsa-miR-24 or hsa-miR-638 decreased the levels of HBV transcripts or HBV gene products. Since candidate microRNAs could have targeted interferon response genes as an alternative explanation interferon signaling was examined. However, HBV replication in cultured hepatocytes was not restored despite successful inhibition of JAK1/2-STAT signaling by the inhibitor, ruxolitinib. Therefore, HBV was unable to complete replication in cultured hepatocytes due to expression of multiple antiviral microRNAs. This mechanism should help understand restrictions in HBV replication for developing HBV models in cultured cells while providing frameworks for pathophysiological studies of HBV replication in subsets of hepatocytes or stem/progenitor cells during hepatitis.

Differentiation in stem/progenitor cells along fetal or adult hepatic stages requires transcriptional regulators independently of oscillations in microRNA expression

“…3). In AH-PP after culture, which alters hepatic gene expression [16,17], miRNA profile resembled that in FH-P3 (Table 3), including abundant hsa-miR-638, −26a and −15b. Since these miRNA were also present in hESC, this rapid return of their expression in mature hepatocytes seemed remarkable.…”

“…From translational perspectives, inducing or maintaining hepatic differentiation in stem cell-derived hepatocytes or natural hepatocytes is critical for drug development or toxicology tools, disease models, stem cell biology, cell/gene therapy, etc. Previously, cell culture altered an array of miRNA networks in human hepatocytes, which could not be overcome for HBV model development [16,17]. In hESC-MEC, greater expression of stemness-associated miRNA altered cellular and molecular processes, TR networks actually restricted differentiation.…”

“…Remarkably, constitutive expression of miRNA similarly regulated genomes in mammalian cells, e.g., in case of hepatitis B virus (HBV), with dominant-negative factors guiding viral replication in permissive/nonpermissive cell fusions [15]. Later, these were found to concern miRNA networks [16,17].…”

Dicer suppresses the malignant phenotype in VHL-deficient clear cell renal cell carcinoma by inhibiting HIF-2α

“…High-quality total RNAs extracted from eight ccRCC VHL-deficient tissue samples and eight ccRCC tissue samples with wild-type VHL were analyzed for expression of 2,019 miRNAs (Sanger miRBase 20.0) at LC Sciences (Houston, TX) using their in-house developed μParaflo ™ technology platform. Methods and data analysis were performed as previously described [ 46 ].…”

Sensitive to the effects of environmental factors miR-638 and common diseases

“…Однако в исследовании X. Liu et al [14] показано, что вирус гепатита С ингибировал экспрессию данной микроРНК в клетках гепатомы человека. С другой стороны, miR-638 может влиять на уровень инфицирования, в частности приводить к снижению уровня транскрипции генов вируса гепатита В [19,20]. miR-638 также чувствительна к окислительному стрессу [21] и температурным условиям (при высоких температурах уровень снижается) [22].…”