Hepatitis B virus X protein (HBx) is recognized as an oncogene in hepatocellular carcinoma (HCC). HBx regulates microRNA expression, including down-regulating miR-338-3p in LO2 cells. Here, we investigated miR-338-3p function in HBx-mediated hepatocarcinogenesis. In 23 HBV-infected HCC clinical patient tumor and adjacent non-tumor control tissues, 17 and 19 tumors expressed HBx mRNA and protein, respectively. When considered as a group, HBV-infected HCC tumors had lower miR-338-3p expression than controls; however, miR-338-3p was only significantly down-regulated in HBx-positive tumors, indicating that HBx inversely correlated with miR-338-3p. Functional characterization of miR-338-3p indicated that miR-338-3p mimics inhibited cell proliferation by inducing cell cycle arrest at the G1/S phase as assessed by EdU and cell cycle assays in HBx-expressing LO2 cells. CyclinD1, containing two putative miR-338-3p targets, was confirmed as a direct target using 3′-UTR luciferase reporter assays from cells transfected with mutated binding sites. Mutating the 2397–2403 nt binding site conferred the greatest resistance to miR-338-3p suppression of CyclinD1, indicating that miR-338-3p suppresses CyclinD1 at this site. Overall, this study demonstrates that miR-338-3p inhibits proliferation by regulating CyclinD1, and HBx down-regulates miR-338-3p in HCC. This newly identified miR-338-3p/CyclinD1 interaction provides novel insights into HBx-mediated hepatocarcinogenesis and may facilitate therapeutic development against HCC.

Mesenchymal stem cells (MSCs) have potential ability to differentiate into osteocytes in response to in vitro specific induction. However, the molecular basis underlying this biological process remains largely unclear. In this study, we identify lncRNA HOTAIRM1 as a critical regulator to promote osteogenesis of MSCs. Loss of HOTAIRM1 significantly inhibits the calcium deposition and alkaline phosphatase activity of MSCs. Mechanistically, we find that HOTAIRM1 positively modulates the activity of JNK and c‐Jun, both of which are widely accepted as crucial regulators of osteogenic differentiation. More importantly, c‐Jun is found to be functionally involved in the regulation of RUNX2 expression, a master transcription factor of osteogenesis. In detail, c‐Jun can help recruit the acetyltransferase p300 to RUNX2 promoter, facilitating acetylation of histone 3 at K27 site, therefore epigenetically activating RUNX2 gene transcription. In summary, this study highlights the functional importance of HOTAIRM1 in regulation of osteogenesis, and we characterize HOTAIRM1 as a promising molecular target for bone tissue repair and regeneration.

Hepatitis B Virus (HBV) DNA integration and HBV X (HBx) deletion mutation occurs in HBV-positive liver cancer patients, and C-terminal deletion in HBx gene mutants are highly associated with hepatocarcinogenesis. Our previous study found that the HBx-d382 deletion mutant (deleted at nt 382–400) can down-regulate miR-338-3p expression in HBx-expressing cells. The aim of the present study is to examine the role of miR-338-3p in the HBx-d382-mediated liver-cell proliferation. We established HBx-expressing LO2 cells by Lipofectamine 2000 transfection. A miR-338-3p mimics or inhibitor was transfected into LO2/HBx-d382 and LO2/HBx cells using miR-NC as a control miRNA. In silico analysis of potential miR-338-3p targets revealed that miR-338-3p could target the cell cycle regulatory protein CyclinD1. To confirm that CyclinD1 is negatively regulated by miR-338-3p, we constructed luciferase reporters with wild-type and mutated CyclinD1-3′UTR target sites for miR-338-3p binding. We examined the CyclinD1 expression by real-time PCR and western blot, and proliferation activity by flow cytometric cell cycle analysis, Edu incorporation, and soft agar colony. HBx-d382 exhibited enhanced proliferation and CyclinD1 expression in LO2 cells. miR-338-3p expression inhibited cell proliferation in LO2/HBx-d382 cells (and LO2/HBx cells), and also negatively regulated CyclinD1 protein expression. Of the two putative miR-338-3p binding sites in the CyclinD1-3′UTR region, the effect of miR-338-3p on the second binding site (nt 2397–2403) was required for the inhibition. miR-338-3p can directly regulate CyclinD1 expression through binding to the CyclinD1-3′UTR region, mainly at nt 2397–2403. Down-regulation of miR-338-3p expression is required for liver cell proliferation in both LO2/HBx and LO2/HBx-d382 mutant cells, although the effect is more pronounced in LO2/HBx-d382 cells. Our study elucidated a novel mechanism, from a new miRNA-regulation perspective, underlying the propensity of HBx deletion mutants to induce hepatocarcinogenesis at a faster rate than HBx.

Dengue virus (DENV) is an arthropod-borne viral pathogen and a global health burden. Knowledge of the DENV-host interactions that mediate virus pathogenicity remains limited. Host lipid metabolism is hijacked by DENV for virus replication in which lipid droplets (LDs) play a key role during the virus lifecycle. In this study, we reveal a novel role for phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in LDs-mediated DENV infection. We demonstrate that PTEN expression is downregulated upon DENV infection through post-transcriptional regulation and, in turn, PTEN overexpression enhances DENV replication. PTEN lipid phosphatase activity was found to decrease cellular LDs area and number through Akt/FoxO1/Maf1 signaling, which, together with autophagy, enhanced DENV replication and virus production. We therefore provide mechanistic insight into the interaction between lipid metabolism and the DENV replication cycle.

The aim of the present study was to investigate the effect of hepatitis B virus X protein (HBx) on the expression of DNA methyltransferase (DNMT)3A/3B and suppressors of cytokine signaling‑1 (SOCS‑1), as well as promoter CpG island methylation of the SOCS‑1 gene. Stable hepatocyte cell lines expressing the HBx gene (pcDNA‑X/QSG7701) or an empty gene (pcDNA3.0/QSG7701) were established. Reverse transcription quantitative polymerase chain reaction (PCR) was used to detect the mRNA expression levels of DNMT3A/3B and SOCS‑1. Immunohistochemistry was used to detect the protein expression of DNMT3A/3B. Methylation‑specific PCR (MSP) was used to detect the methylation status of the SOCS‑1 gene promoter. The mRNA and protein expression levels of DNMT3A/3B were significantly higher in the pcDNA‑X/QSG7701‑transfected cells, compared with those in the pcDNA3.0/QSG7701 or non‑transfected QSG7701 cells (P<0.05), whereas the relative mRNA expression of SOCS‑1 was significantly lower in the pcDNA‑X/QSG7701 cells compared with the pcDNA3.0/QSG7701 and non‑transfected QSG7701 cells (F=19.6; P<0.05). Western blot analysis showed that the protein expression of SOCS‑1 was significantly lower in the pcDNA‑X/QSG7701 cells, compared with the pcDNA3.0/QSG7701 or non‑transfected QSG7701 cells (F=19.4; P<0.05). The results of the MSP analysis showed that SOCS‑1 promoter region methylation was present only in the pcDNA‑X/QSG7701 cells. The HBV‑X gene upregulated the mRNA and protein expression levels of DNMT3A/3B, downregulated the expression of SOCS‑1 and increased SOCS‑1 gene promoter CpG island methylation. This may provide a potential explanation of the mechanism underlying HBx-associated hepatocellular carcinoma.