miR-129-5p could directly regulate the expression of VCP.

<p>A: miR-129-5p could significantly suppress the luciferase activities of pGL3-VCP-3′UTR in HepG2 cells. Five predicted miRNAs (miR-103, miR-107, miR-129-5p, miR-136, miR-339-5p) were transfected in HepG2 cells, respectively, with VCP 3′UTR report. 72 h after the transfection, the cells were harvested for luciferase activity. Shown data are representative from three independent experiments. **:P<0.01. B: Bioinformatic analysis of miR-129-5p predicted binding sites in the VCP 3′UTR. There were two putative miR-129-5p target sites located in the VCP 3′UTR (162–168 and 505–511). Three mutant reporter vectors were constructed with the deletion of two target sites individually (pGL3-VCP-3′UTRm1, pGL3-VCP-3′UTRm2) or both (pGL3-VCP-3′UTRm3). C: The luciferase activity of the mutant VCP 3′UTR report genes were not regulated by miR-129-5p in HepG2 cells. Shown data are representative from three independent experiments.**:P<0.01. D: miR-129-5p could significantly suppress the luciferase activities of pGL3-VCP-3′UTR in MHCC-LM3 cells, while has no effect on three mutant reporter vectors. Shown data are representative from three independent experiments.**:P<0.01. E: The inhibiter of miR-129-5p could increase the luciferase activities of pGL3-VCP-3′UTR in SK-HEP1 cells, while has no effect on three mutant reporter vectors. Shown data are representative from three independent experiments. **:P<0.01. F: Analysis of the level of miR-129-5p in HCC tissues by qRT-PCR. The result showed that the level of miR-129-5p is frequently reduced in human HCC tissues.*:P<0.05.**:P<0.01. G: The level of miR-129-5p was negatively correlated with the expression of VCP in HCC. The 39 tissue samples of HCC were divided into two groups according to the level of VCP. The level of miR-129-5p in each sample from the two groups (level 1,n = 17;level 2,n = 22) was measured by qRT-PCR and compared. U6 snRNA was used as internal control gene. Dot plots showed an inverse relationship between U6 snRNA and miR-129-5p expression in HCC. Significant differences were determined using Student's t tests. **: P<0.01; Bars: mean±SD.</p

VCP positively regulated the progress of HCC.

<p>A: Analysis of the level of VCP in HCC tissues by qRT-PCR. The result showed that the level of VCP is frequently increased in human HCC tissues. *:P<0.05.**:P<0.01. B,C: Effect of si-VCP and miR-129-5p on tumor growth in nude mice model. si-VCP, miR-129-5p and NC-transfected HepG2 cells were suspended and then injected subcutaneously into either side of the posterior flank of the male BALB/c athymic nude mouse. Tumor growth was examined every four days. Tumor volume (V) was monitored by measuring the length (L) and width (W) of the tumor with calipers and was calculated with the formula V = (L×W²)×0.5. Photographs of dissected tumors from nude mice (B) and the curve of tumor growth (C) are shown. *:P<0.05. Results are representative of three animals in every time point per group. D:The level of VCP and Bcl-2 in the transplanted tumors was detected by IHC.</p

miR-129-5p could regulate the cell growth, apopotosis and migration of HCC cells dependent on the regulation of VCP expression.

<p>A: HepG2 or SK-HEP1 cells were transfected and in the indicated time periods posttransfection, cell growth rate was evaluated using CCK8 assay. The result showed miR-129-5p and si-VCP could repress cell growth. The cell growth rate was restored after VCP expression vector were transfected into HepG2 or SK-HEP1 cells. Shown data are representative from three independent experiments. **:P<0.01. B: HepG2 cells were transfected and the apoptosis of cell was detected with FACS. Left panel showed miR-129-5p and si-VCP could increase the apoptosis of HepG2 cells at 48 h and 72 h after the transfection. Right panel showed the apoptosis of cell after miR-129-5p with/without VCP expression vector were transfected into HepG2 cells for 72 h. The experiment was repeated three times independently. NC: negative control RNA duplex; miR-129-5p: miR-129-5p mimic; miR-129-5p/pcDNA3.1: cells were co-transfected with miR-129-5p mimic and blank pcDNA3.1; miR-129-5p/VCP: cells were co-transfected with miR-129-5p mimic and VCP expression vector. The experiments were repeated three times independently.*:P<0.05. **:P<0.01. C: SK-HEP1 cells were transfected and the apoptosis of cells was detected with FACS. miR-129-5p and si-VCP could increase the apoptosis of SK-HEP1 cells at 72 h after the transfection. After the level of VCP was enhanced by transfecting VCP expression vector, the apoptosis rate of cells were returned. D:Detection the level of Bcl-2 and XIAP by RT-PCR and western blot. GAPDH served as the internal control. E:Transwell assay was performed to assess cell migration. Upper panel represented the photographs of treated and untreated cells at 24 h (×40 magnification). Lower panel showed the number of cells invaded at 24 h. Shown data are representative from four replicates per group. *P<0.05.</p