MicroRNA-301b-3p contributes to tumour growth of human hepatocellular carcinoma by repressing vestigial like family member 4

Abstract

MicroRNAs (miRNAs) are key regulators in the tumour growth and metastasis of human hepatocellular carcinoma (HCC). Increasing evidence suggests that miR-301b-3p functions as a driver in various types of human cancer. However, the expression pattern of miR-301b-3p and its functional role as well as underlying molecular mechanism in HCC remain poorly known. Our study found that miR-301b-3p expression was significantly up-regulated in HCC tissues compared to adjacent non-tumour tissues. Clinical association analysis revealed that the high level of miR-301b-3p closely correlated with large tumour size and advanced tumour-node-metastasis stages. Importantly, the high miR-301b-3p level predicted a prominent poorer overall survival of HCC patients. Knockdown of miR-301b-3p suppressed cell proliferation, led to cell cycle arrest at G2/M phase and induced apoptosis of Huh7 and Hep3B cells. Furthermore, miR-301b-3p knockdown suppressed tumour growth of HCC in mice. Mechanistically, miR-301b-3p directly bond to 3'UTR of vestigial like family member 4 (VGLL4) and negatively regulated its expression. The expression of VGLL4 mRNA was down-regulated and inversely correlated with miR-301b-3p level in HCC tissues. Notably, VGLL4 knockdown markedly repressed cell proliferation, resulted in G2/M phase arrest and promoted apoptosis of HCC cells. Accordingly, VGLL4 silencing rescued miR-301b-3p knockdown attenuated HCC cell proliferation, cell cycle progression and apoptosis resistance. Collectively, our results suggest that miR-301b-3p is highly expressed in HCC. miR-301b-3p facilitates cell proliferation, promotes cell cycle progression and inhibits apoptosis of HCC cells by repressing VGLL4.

Keywords: G2/M phase arrest; VGLL4; apoptosis; cell proliferation; hepatocellular carcinoma; miR-301b-3p.

Figure 1

The expression and prognostic value of miR‐301b‐3p in hepatocellular carcinoma (HCC). A, The expression of miR‐301b‐3p was detected by qRT‐PCR between HCC tissues (n = 80) and adjacent non‐tumour tissues (n = 80). P = 0.0038 by t test. B, Analysis of HCC (n = 370) and normal liver samples (n = 50) in TCGA dataset from starBase V3.0 platform showed that miR‐301b‐3p was over‐expressed in HCC tissues. P < 0.0001 by t test. C, HCC patients were divided into two subgroups (low or high miR‐301b‐3p level) using the median level of miR‐301b‐3p as a cut‐off value. HCC patients with high miR‐301b‐3p level (n = 40) showed a significant poorer overall survival compared to low miR‐301b‐3p group (n = 40). P = 0.0039 by log‐rank test. D, TCGA data from starBase V3.0 platform indicated that low miR‐301b‐3p level predicted poor prognosis of HCC patients. Low miR‐301b‐3p level (n = 181), high miR‐301b‐3p level (n = 181), P = 0.0460 by log‐rank test

Figure 2

miR‐301b‐3p knockdown suppresses hepatocellular carcinoma (HCC) cell proliferation, results in G2/M phase arrest and induces apoptosis. A, miR‐301b‐3p inhibitors or negative control (NC) were transduced into Huh7 and Hep3B cells, and qRT‐PCR was performed to detect miR‐301b‐3p expression. n=three independent repeats, *P < 0.05 by t test. B and C, CCK‐8 and colony formation assays revealed that miR‐301b‐3p knockdown suppressed the proliferation of HCC cells. n = three independent repeats, *P < 0.05 by ANOVA or t test. D, The percentage of apoptotic HCC cells was significant increased after miR‐301b‐3p knockdown. n = three independent repeats, *P < 0.05 by t test. E, miR‐301b‐3p knockdown led to G2/M phase arrest in both Huh7 and Hep3B cells. n = three independent repeats, *P < 0.05 by t test

Figure 3

Knockdown of miR‐301b‐3p represses tumour growth of hepatocellular carcinoma (HCC) in vivo. A, Huh7 and Hep3B cells that were transfected with lentivector‐mediated miR‐301b‐3p inhibitors or negative control (NC) were implanted into nude mice. The tumour volume in miR‐301b‐3p knockdown group (n = 5) was markedly less compared to control group (n = 5). *P < 0.05 by ANOVA. B, The expression of miR‐301b‐3p in xenograft tumour tissues collected from miR‐301b‐3p knockdown group (n = 5) was prominently lower compared to control group (n = 5). *P < 0.05 by t test. C, Immunostaining of Ki‐67 indicated that the percentage of Ki‐67 positive tumour cells in xenograft tumour tissues collected from miR‐301b‐3p knockdown group (n = 5) was significantly lower compared to control group (n = 5). *P < 0.05 by t test. Scale bar: 50 μm

Figure 4

Vestigial like family member 4 (VGLL4) is identified as a novel target of miR‐301b‐3p. A, Bioinformatics analysis based on starBase V3.0 platform predicted VGLL4 as a candidate target of miR‐301‐3p. B, miR‐301b‐3p inhibitors or negative control (NC) were transduced into Huh7 and Hep3B cells, and qRT‐PCR and immunoblotting were performed to detect VGLL4 expression. n=three independent repeats, *P < 0.05 by t test. C, Wild type (wt) or mutated (mt) 3′UTR of VGLL4 and miR‐301b‐3p mimics or inhibitors were co‐transfected into Huh7 cells and the relative fluorescence intensity was detected. n=three independent repeats, *P < 0.05 by t test. D, The expression of VGLL4 mRNA was detected by qRT‐PCR between HCC tissues (n = 80) and adjacent non‐tumour tissues (n = 80). P < 0.0001 by t test. E, An inverse correlation between miR‐301b‐3p level and the expression of VGLL4 mRNA was found in HCC tissues. n = 80, P = 0.0041 by Pearson correlation test

Figure 5

Vestigial like family member 4 (VGLL4) overexpression represses hepatocellular carcinoma (HCC) cell growth. A, pcDNA3.1‐VGLL4 or empty vector (EV) was transfected into Huh7 and Hep3B cells and immunoblotting was performed to detect the expression of VGLL4 protein. B, CCK‐8, (C) colony formation, (D) flow cytometry‐based apoptosis detection and (E) cell cycle distribution analysis was performed to determine the proliferation, apoptosis and cell cycle progression of HCC cells. n = three independent repeats, *P < 0.05 by t test or ANOVA

Figure 6

Vestigial like family member 4 (VGLL4) silencing rescues miR‐301b‐3p knockdown‐induced inhibition of proliferation, G2/M phase arrest and apoptosis of Huh7 cells. A, VGLL4 was knocked down by a specific siRNA in Huh7 cells with miR‐301b‐3p knockdown and immunoblotting was performed to detect VGLL4 protein. B, CCK‐8, (C) colony formation, (D) flow cytometry‐based apoptosis detection and (E) cell cycle distribution analysis was performed to determine the proliferation, apoptosis and cell cycle progression of Huh7 cells transfected with corresponding vectors. n = three independent repeats, *P < 0.05 by ANOVA