Hepatitis B virus (HBV) enters the host and survives by using several mechanisms. One of the ways that HBV survives and replicates in the host cells is by inducing autophagy. Previous reports have shown that microRNA (miRNA)-30a inhibits autophagosome formation in cancer cells. Hence, we hypothesized that overexpression of miRNA-30a could inhibit HBVinduced autophagosome formation in hepatic cells. To study this, both HepG2 cells and HepG2.2.1.5 cells (HBV-expressing stable cell line) were transfected with miRNA-30a, and the cells were collected either for RNA isolation or protein isolation after 72 h of transfection. Beclin-1 expression was significantly higher in untransfected HepG2.2.1.5 cells than in HepG2 cells. Western blots showed that miRNA-30a overexpression resulted in a significant decrease in beclin-1 expression (eight-fold and four-fold in HepG2 and HepG2.2.1.5 cells, respectively) and c-myc expression, whereas the numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells were increased. In contrast, overexpression of HBV X protein (HBx) in HepG2 cells resulted in the enhancement of beclin-1 (six-fold increase as compared with the empty vector-transfected cells) and c-myc expression, whereas the numbers of TUNEL-positive cells were reduced. To confirm these findings, HBx and miRNA-30a were coexpressed in HepG2 cells, and the results showed significant inhibition of autophagosome formation and beclin-1 and c-myc expression, whereas apoptosis increased. These data demonstrate that HBx induces autophagosome formation via beclin-1 expression, whereas miRNA-30a overexpression could successfully inhibit the beclin-1 expression induced by HBx, thereby modulating autophagosome formation in hepatic cells.
JAK2 is cytokine-activated non-receptor tyrosine kinase. Although JAK2 is mainly localized at the plasma membrane, it is also present on the centrosome. In this study, we demonstrated that JAK2 localization to the centrosome depends on the SH2 domain and intact kinase activity. We created JAK2 mutants deficient in centrosomal localization ΔSH2, K882E and (ΔSH2, K882E). We showed that JAK2 WT clone strongly enhances cell proliferation as compared to control cells while JAK2 clones ΔSH2, K882E and (ΔSH2, K882E) proliferate slower than JAK2 WT cells. These mutant clones also progress much slower through the cell cycle as compared to JAK2 WT clone and the enhanced proliferation of JAK2 WT cells is accompanied by increased S −> G2 progression. Both the SH2 domain and the kinase activity of JAK2 play a role in prolactin-dependent activation of JAK2 substrate STAT5. We showed that JAK2 is an important regulator of centrosome function as the SH2 domain of JAK2 regulates centrosome amplification. The cells overexpressing ΔSH2 and (ΔSH2, K-E) JAK2 have almost three-fold the amplified centrosomes of WT cells. In contrast, the kinase activity of JAK2 is dispensable for centrosome amplification. Our observations provide novel insight into the role of SH2 domain and kinase activity of JAK2 in centrosome localization of JAK2 and in the regulation of cell growth and centrosome biogenesis.
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