Long noncoding RNA MIR4435-2HG promotes hepatocellular carcinoma proliferation and metastasis through the miR-22-3p/YWHAZ axis

Abstract

Long noncoding RNAs (lncRNAs) play the critical biological role in many malignant tumours. MIR4435-2HG has been proven to be a novel oncogenic lncRNA. However, the exact role and mechanism of MIR4435-2HG in hepatocellular carcinoma (HCC) remain unclear. Here, we found that MIR4435-2HG is overexpressed in HCC tissue compared to normal controls and that high level of MIR4435-2HG indicates a poorer prognosis in HCC patients. MIR4435-2HG enhances the growth and metastasis ability of HCC cells. MIR4435-2HG promotes the expression of YWHAZ by sponging miR-22-3p to liberate YWHAZ mRNA transcripts. MIR4435-2HG facilitates the proliferation and metastasis of HCC by modulating the miR-22-3p/YWHAZ axis. These results demonstrated the role and mechanism of MIR4435-2HG in malignant progression of HCC. MIR4435-2HG may be used as the prognostic marker and treatment target for the patient with HCC.

Keywords: HCC; MIR4435-2HG; YWHAZ; miR-22-3p; proliferation and metastasis.

Figure 1

MIR4435-2HG is significantly upregulated in HCC. A. The level of MIR4435-2HG was analyzed in 22 HCC tissues and adjacent normal tissues. Statistical significance is evaluated by one-tailed t-test. B. StarBase (http://starbase.sysu.edu.cn/) was used to detect the expression of MIR4435-2HG in HCC and matched normal tissue of TCGA dataset. C. GEPIA (http://gepia.cancer-pku.cn/) was used to detect the expression of MIR4435-2HG in HCC and matched normal tissue of TCGA dataset. D. The expression profile of MIR4435-2HG in HCC cell lines (HepG2, Huh7, SMMC7721 and BEL-7402) and normal human hepatic epithelial cells (LO2). Statistical significance is evaluated by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

High MIR4435-2HG levels are a risk factor for the prognosis of HCC. A. The survival curves of 22 HCC patients with high and low MIR4435-2HG levels. High MIR4435-2HG expression group (n=11, MIR4435-2HG expression ratio ≥ median ratio), low MIR4435-2HG expression group (n=11, MIR4435-2HG expression ratio < median ratio). Kaplan Meier survival curve was used. B. The prognostic data of HCC patients with high and low MIR4435-2HG levels in TCGA were analyzed by using Starbase (http://http://www.sysu.edu.cn). Kaplan Meier survival curve was used. C. We analyzed the prognostic data of HCC patients in TCGA using GEPIA (http://gepia.cancer-pku.cn/). Kaplan Meier survival curve was used. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

ceRNA prediction of MIR4435-2HG. A. The 3’-UTR of YWHAZ has the same binding sites that MIR4435-2HG binds to miR-22-3p. B. miR-22-3p expression was detected in 22 HCC tissues and adjacent normal tissues. Statistical significance is evaluated by one-tailed t-test. C. The expression of YWHAZ was analyzed in 22 HCC tissues and adjacent normal tissues. Statistical significance is evaluated by one-tailed t-test. D. The expression of miR-22-3p was detected in HCC and matched normal tissue of TCGA dataset. E. The expression of YWHAZ was analyzed in HCC and matched normal tissue of TCGA dataset. F. The correlation of MIR4435-2HG and miR-22-3p in 22 HCC tissues was negative. Spearman correlation analysis was carried out. G. The correlation of MIR4435-2HG and YWHAZ in 22 HCC tissues was positive. Spearman correlation analysis was carried out. H. A negative correlation between MIR4435-2HG and miR-22-3p was found in the TCGA HCC dataset. Spearman correlation analysis was carried out. I. The positive correlation between MIR4435-2HG and YWHAZ mRNA levels in the TCGA HCC dataset. Spearman correlation analysis was carried out. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

MIR4435-2HG sponges miR-22-3p in HCC cells. A. qRT-PCR of nuclear and cytoplasmic RNA fractions were used to detect MIR4435-2HG expression in the cytoplasm and nucleus. B. The levels of miR-22-3p in HCC cells following transfection with MIR4435-2HG siRNA. Statistical significance is evaluated by two-tailed Student’s t-test. C. The binding sites of miR-22-3p on MIR4435-2HG and target sequences were mutated. D. Luciferase assay of HCC cells transfected with MIR4435-2HG-WT or MIR4435-2HG-MUT plasmid together with miR-22-3p mimic. Statistical significance is evaluated by two-tailed Student’s t-test. E. MS2-RIP was used to detect endogenous miR-22-3p associated with MS2-tagged MIR4435-2HG. Empty and mutated plasmids were used as the control. Statistical significance is evaluated by two-tailed Student’s t-test. F. HCC cells transfected with biotin-labeled miR-22-3p, mutated or NC, and subjected to an RNA pull-down assay. The expression of MIR4435-2HG was detected by qRT-PCR. Statistical significance is evaluated by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

MIR4435-2HG promotes YWHAZ expression by sponging miR-22-3p in HCC cells. A. The binding sites of miR-22-3p on the 3pg sites YWHAZ, and target sequences were mutated. B. Luciferase assay of HCC cells transfected with YWHAZ-WT or YWHAZ-MUT reporter together with miR-22-3p mimic. Statistical significance is evaluated by two-tailed Student’s t-test. C. Luciferase assay of HCC cells transfected with YWHAZ-WT or YWHAZ-MUT plasmid together with MIR4435-2HG siRNA or MIR4435-2HG siRNA plus miR-22-3p inhibitor. Statistical significance is evaluated by two-tailed Student’s t-test. D. The expression of YWHAZ mRNA in HCC cells transfected with miR-22-3p mimic, MIR4435-2HG siRNA or MIR4435-2HG siRNA plus miR-22-3p inhibitor. Statistical significance is evaluated by two-tailed Student’s t-test. E. Western blotting detected YWHAZ protein expression changes in NC, miR-22-3p mimic, si-MIR4435-2HG or si-MIR4435-2HG plus miR-22-3p inhibitor transfected HCC cells, GAPDH was used as a control. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

MIR4435-2HG promotes the proliferation and metastasis of HCC via the miR-22-3p/YWHAZ axis. A. Western blots identified YWHAZ protein expression changes in NC, miR-22-3p mimic, miR-22-3p mimic plus YWHAZ, si-MIR4435-2HG or si-MIR4435-2HG plus miR-22-3p inhibitor transfected HCC cells, GAPDH was used as a control. B. The effect of miR-22-3p mimic and si-MIR4435-2HG on the proliferative ability of HCC cells was determined by CCK8 assay. The results were further verified by cotransfection with miR-22-3p inhibitor and YWHAZ plasmid. Statistical significance is evaluated by two-tailed Student’s t-test. C. Effect of si-MIR4435-2HG and miR-22-3p mimic on the invasive capacity of HCC cells was assessed by transwell assay. The results were confirmed by the recovery experiment of cotransfection with miR-22-3p inhibitor and YWHAZ plasmid respectively. Statistical significance is evaluated by two-tailed Student’s t-test. Scale bar, 50 μm. D. The migration ability of HCC cells in different transfection groups was detected by scratch wound assay. YWHAZ plasmid and miR-22-3p inhibitor reversed the effect of miR-22-3p mimic and si-MIR4435-2HG on the migration capability of HCC cells. Statistical significance is evaluated by two-tailed Student’s t-test. Scale bar, 100 μm. E. The excision tumour of HepG2 xenografts in NC and si-MIR4435-2HG treated groups. F. The tumour volume of HepG2 xenografts in NC and si-MIR4435-2HG treated groups. Statistical significance is evaluated by two-tailed Student’s t-test. G. PCR identified miR-22-3p and MIR4435-2HG expression changes in NC and si-MIR4435-2HG treated groups. Statistical significance is evaluated by two-tailed Student’s t-test. H. The level of YWHAZ was detected by immunohistochemical staining of sections from the xenograft tumour tissues of NC and si-MIR4435-2HG treated groups. Scale bar, 25 μm. *P < 0.05, **P < 0.01, ***P < 0.001.