miR-3928v is induced by HBx via NF-κB/EGR1 and contributes to hepatocellular carcinoma malignancy by down-regulating VDAC3

Abstract

Background: Hepatitis B virus (HBV) plays a critical role in the tumorigenic behavior of human hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) have been reported to participate in HCC development via the regulation of their target genes. However, HBV-modulated miRNAs involved in tumorigenesis remain to be identified. Here, we found that a novel highly expressed miRNA, TLRC-m0008_3p (miR-3928v), may be an important factor that promotes the malignancy of HBV-related HCC.

Methods: Solexa sequencing was applied to profile miRNAs, and RT-qPCR was used to identify and quantitate miRNAs. We studied miR-3928v function in HCC cell lines by MTT, colony formation, migration/invasion, and vascular mimicry (VM) assays in vitro and by a xenograft tumor model in vivo. Finally, we predicted and verified the target gene of miR-3928v by a reporter assay, studied the function of this target gene, and cloned the promoter of miR-3928v and the transcription factor for use in dual-luciferase reporter assays and EMSAs.

Results: A variant of miR-3928 (miR-3928v) was identified and found to be highly expressed in HBV (+) HCC tissues. Voltage-dependent anion channel 3 (VDAC3) was validated as a target of miR-3928v and found to mediate the effects of miR-3928v in promoting HCC growth and migration/invasion. Furthermore, HBx protein increased early growth response 1 (EGR1) expression and facilitated its translocation into the nucleus to enhance miR-3928v promoter activity in an NF-κB signaling-dependent manner.

Conclusions: miR-3928v is induced by HBx through the NF-κB/EGR1 signaling pathway and down-regulates the tumor suppressor gene VDAC3 to accelerate the progression of HCC.

Keywords: EGR1; HBx; Hepatocellular carcinoma; VDAC3; miR-3928v; miRNA.

Fig. 1

HBV induces miR-3928v expression in HCC cells and tissues. a The secondary structure of the miR-3928v precursor and its sequence. b The relative miR-3928v mRNA level in HBV-related HCC cell lines. c The relative miR-3928v mRNA level in HBV (−) cell lines with pHBV1.3copy overexpression. d The effect of the HBx expression plasmid (pHBx) on miR-3928v expression. e The miR-3928v expression level was determined in 20 pairs of tissues from patients with liver cancer. f The miR-3928v mRNA level was determined by RT-qPCR in 60 serum samples from patients with HCC and 30 serum samples from healthy people. (**: P < 0.01)

Fig. 2

miR-3928v acts as an oncogene. Huh7 and HepG2 cells were transfected with pri-miR-3928v or controls. HepG2.2.15 cells were transfected with ASO-miR-3928v or controls. Twenty-four hours after transfection, cells were seeded into cell culture inserts. a MTT assays were performed to examine cell viability at 48 h or 72 h after transfection. b FACS was used to assess cell apoptosis. c Colony formation assays were used to evaluate cell growth. HepG2 and HepG2.2.15 cells were grown for 14 days, and the colony formation rate was calculated. d FACS was used to analyze changes in cell cycle distribution. e Twenty-four hours after transfection, cells were seeded into cell culture inserts to analyze migration/invasion. The cells were counted in five random fields. Then, the expression levels of EMT-associated markers were examined by Western blot. f The effect of miR-3928v on VM. VM-associated markers were also examined. g A nude mouse tumor xenograft model was utilized to study the effect of miR-3928v on tumor growth in vivo. The miR-3928v level in tumors (left) and the tumor weight (right) are shown underneath the picture. (*: P < 0.05, **: P < 0.01)

Fig. 3

miR-3928v directly targets the 3′UTR of VDAC3 mRNA. a The predicted miR-3928v binding site in the VDAC3 3′UTR and the introduced mutations in the binding site (VDAC3 mut) (left). Dual-luciferase reporter assays were performed to analyze the effect of miR-3928v on the VDAC3 3′UTR (right). # indicates no statistically significant difference. b RT-qPCR (left) and Western blot (right) were performed to examine VDAC3 mRNA and protein expression levels after miR-3928v overexpression or inhibition in HCC cells. c-d The relative VDAC3 mRNA level was determined in HCC cells (c) and tissues (d). e Representative IHC images showing the magnitude of VDAC3 expression in tissues isolated from liver cancer patients (left) and nude mice (right). f VDAC3 mRNA level in tissues isolated from nude mice. (*: P < 0.05, **: P < 0.01)

Fig. 4

VDAC3 acts as a tumor suppressor. Huh7 and HepG2 cells were transfected with pshR-VDAC3 or controls. HepG2.2.15 cells were transfected with pVDAC3 or controls. Twenty-four hours after transfection, cells were seeded into cell culture inserts. a MTT assays were performed to evaluate cell viability. b Colony formation assays were used to estimate cell proliferation. c Migration/invasion assays and f Western blot analyses were used to evaluate EMT. d VM and g molecular markers of VM were examined to assess tumor angiogenesis. e FACS assays were used to examine changes in cell cycle distribution. (*: P < 0.05)

Fig. 5

VDAC3 is a functional target of miR-3928v. HepG2 cells were co-transfected with pCD3/Flag or pVDAC3 and pri-miR-3928v. RT-qPCR and Western blot analyses (a) and MTT (b), colony formation (c), migration (d), invasion (e) and tube formation assays (f) were performed to analyze whether VDAC3 could functionally rescue the miR-3928v-induced phenotype. (*: P < 0.05, **: P < 0.01, ***: P < 0.001)

Fig. 6

HBx up-regulates miR-3928v expression via EGR1. a Schematic diagram of the location of miR-3928v in the human genome; the relative positions of the miR-3928v promoter constructs are shown (left). Luciferase reporter assays were used to examine miR-3928v promoter activity in Huh7 cells (right). b Promoter activity in HBV (+) and HBV (−) cells (left). Then, the promoter activity in Huh7 cells was examined after HBV or HBx overexpression (right). c The effect of EGR1 on promoter activity (left). The EGR1 binding site in the miR-3928v promoter was mutated, and the luciferase activity induced by EGR1 was examined (right). d ChIP analysis (left) and EMSAs (right) revealed that EGR1 directly binds to the miR-3928v promoter. e The miR-3928v expression level was measured by RT-qPCR after EGR1 overexpression or knockdown. f Blocking EGR1 inhibited HBx-induced miR-3928v promoter activity. g Representative IHC images showing the magnitude of EGR1 expression in tissues isolated from liver cancer patients. h Total and nuclear expression of EGR1 and P65 in Huh7 cells after transfection with HBx was analyzed by Western blot. i An immunofluorescence assay was used to detect the induction of EGR1 expression in the nucleus by HBx in Huh7 cells. j Mechanism by which HBx-induced miR-3928v expression contributes to HCC malignancy. (*: P < 0.05, **: P < 0.01)