HBx-related long non-coding RNA DBH-AS1 promotes cell proliferation and survival by activating MAPK signaling in hepatocellular carcinoma

Abstract

Accumulating evidence supports an important role for the hepatitis B virus x protein (HBx) in the pathogenesis of hepatitis B virus (HBV)-induced hepatocellular carcinoma (HCC), but the underlying mechanisms are not entirely clear. Here, we identified a novel long noncoding RNA (lncRNA) DBH-AS1 involved in the HBx-mediated hepatocarcinogenesis. The levels of DBH-AS1 were positively correlated with hepatitis B surface antigen (HBsAg) and tumor size in HCC tissues. Functionally, transgenic expression of DBH-AS1 significantly enhanced cell proliferation and tumorigenesis, whereas short hairpin RNA knockdown of DBH-AS1 caused an inhibition of cell proliferation. Mechanistically, overexpression of DBH-AS1 induced cell cycle progression by accelerating G1/S and G2/M transition concomitantly with upregulation of CDK6, CCND1, CCNE1 and downregulation of p16, p21 and p27. We also found that enhanced DBH-AS1 expression inhibited serum starvation-induced apoptosis of HCC cells. In contrast, suppressed DBH-AS1 expression had opposite effects. Furthermore, DBH-AS1 was shown to activate MAPK pathway. We also provide evidence that DBH-AS1 could be significantly induced by HBx protein and markedly down-regulated by p53. Thus, we concluded that DBH-AS1 can be induced by HBx and inactivated by p53, and consequently promote cell proliferation and cell survival through activation of MAPK signaling in HCC. Our study suggests that DBH-AS1 acts as an oncogene for HCC.

Keywords: DBH-AS1; HBx; HCC; lncRNA; proliferation.

Figure 1. LncRNA DBH-AS1 promotes HCC cell proliferation in vitro

A. HepG2 and SMMC-7721 cells were infected with lentivirus carrying the DBH-AS1 gene, and HepG2 and SMMC-7721 cells stably overexpressing DBH-AS1 were screened by qRT-PCR. B. Short hairpin RNA against DBH-AS1 stably decreased the expression of DBH-AS1 in sh-DBH-AS1 Hep3B and SK-Hep1 cells compared with sh-control cells by qRT-PCR. C. After overexpression of DBH-AS1 in HepG2 and SMMC-7721 cells, the cell viability was assessed by CCK-8 assays daily for 3 days. D. Cell viability was assessed by CCK-8 assays daily for 3 days in Hep3B and SK-Hep1 cells with silenced DBH-AS1 expression. E. Colony formation assays were performed on HepG2 and SMMC-7721 cells stably overexpressing DBH-AS1 for 2 weeks. F. In vitro proliferative ability of Hep3B and SK-Hep1 cells was significantly decreased in DBH-AS1-suppressed cells compared to sh-control cells by colony formation assays. Data are presented as mean ± SD for at least three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2. LncRNA DBH-AS1 accelerates tumor growth in vivo

A. Photographs of tumors that developed in xenograft-transplanted nude mouse tumor models 5 weeks after injection of DBH-AS1-overexpressing or control SMMC-7721 cells. B. In vivo subcutaneous tumor growth curves were shown for SMMC-7721 cells of Lv-DBH-AS1 and Lv-control vectors. Images C. and weights D. of xenografts established by subcutaneous transplantation with Lv-DBH-AS1-overexpressing and Lv-control SMMC-7721 cells 5 weeks after cell injection. E. H&E-stained paraffin-embedded sections obtained from xenografts. IHC staining shows that the expression of Ki67 was enhanced in the Lv-DBH-AS1 group compared to the Lv-control group. The higher magnification for the selected region in each part was shown in the right of each part. Original magnification 400×.

Figure 3. LncRNA DBH-AS1 induces cell-cycle progression in HCC cells

A. HepG2 and SMMC-7721 cells with elevated DBH-AS1 expression were seeded on 96-well plates, and cell proliferation was examined by EdU immunofluorescence staining. Effect of DBH-AS1 knockdown on Hep3B and SK-Hep1 cell proliferation was also measured by EdU immunofluorescence staining. The graph on the right shows the percentage of EdU-positive nuclei. B. Cell-cycle analysis of HepG2 and SMMC-7721 cells overexpressing DBH-AS1 and Hep3B and SK-Hep1 cells with stably silenced DBH-AS1 expression. C. Proportion of cells in various phases of the cell cycle. D.-E. The relative expression levels of cell cycle associated genes, including CDK6, CCND1, CCNE1, P16, P21 and P27, were detected in HepG2 cells overexpressing DBH-AS1 and Hep3B cells with stably down-regulated DBH-AS1 expression by qRT-PCR D. and western blot with quantitative analysis E.. The results show the means ± SD from at least 3 separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4. LncRNA DBH-AS1 inhibits serum starvation-induced apoptosis of HCC cells

A. Cells were cultured in serum-free medium for 48h. The apoptosis rate was measured by FACS-based Annexin-V/7AAD double staining. Cells positive for annexin V staining were counted as apoptotic cells. B. The bar graph shows the percentage of apoptotic cells. C. The levels of Caspase3 and cleaved Caspase3 were detected by western blot with quantitative analysis in HepG2 cells with elevated DBH-AS1 expression and Hep3B cells with silenced DBH-AS1 expression. The experiments were performed in triplicate; the data are expressed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5. LncRNA DBH-AS1 activates MAPK signaling pathways

The levels of ERK, p38, JNK, p-ERK, p-p38 and p-JNK were examined by western blot analysis in HepG2 cells overexpressing DBH-AS1 A. and Hep3B cells with silenced DBH-AS1 expression B.. The experiments were performed in triplicate; the data are expressed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6. HBx induces the expression of lncRNA DBH-AS1

A. Ectopic re-expression of HBx was detected in Lv-HBx-transfected HepG2 and LO2 cells by qRT-PCR and western blot. β-actin was used as a loading control. B. The relative expression of lncRNA DBH-AS1 in HepG2 and LO2 cells re-expressing HBx compared with controls by qRT-PCR. Data are shown as the mean±SD based on at least three independent experiments. C. Comparison of levels of DBH-AS1 in HCC patients with and without HBV infection (independent t test). D. The correlation between DBH-AS1 transcript level and HBx mRNA level in 31 HCC tissues. The ΔCt values were subjected to Pearson correlation analysis. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7. LncRNA DBH-AS1 is inactivated by p53

A. The potential p53-binding site upstream of DBH-AS1 predicted by JASPAR database. B. Western blot analysis showed the reduced levels of p53 protein in HepG2 cells and LO2 cells transfected with siRNAs. C. Reduced p53 mRNA expression by siRNAs in HepG2 cells and LO2 cells was shown by qRT-PCR. D. Expression of DBH-AS1 transcripts was quantified by qRT-PCR. Data shown are the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.