Prognostic roles of miR-124-3p and its target ANXA7 and their effects on cell migration and invasion in hepatocellular carcinoma

Abstract

Recent studies have indicated that ANXA7 promotes progression and metastasis of hepatocellular carcinoma (HCC). In this study we found a significant negative correlation between the levels of miR-124-3p and ANXA7 protein in HCC. Level of miR-124-3p in tumor tissues was negatively correlated, while ANXA7 protein was positively correlated, with TNM stage and tumor metastasis. Furthermore, we confirmed ANXA7 was a target gene of miR-124-3p by a dual luciferase reporter assay. In vitro, up-regulation of miR-124-3p promotes apoptosis and inhibits migration and invasion of Hca-F. Bcl-2 correlates X protein (Bax) protein level was up-regulated, while ANXA7, B-cell lymphoma-2 (Bcl-2), Matrix metalloproteinase (MMP-9) and C-X-C motif chemokine 12 (CXCL12) protein levels were suppressed relative to miR-124-3p over-expression. In vivo, up-regulation of miR-124-3p suppresses lymph node metastasis (LNM) and tumorigenicity of Hca-F cells. The expression of ANXA7, MMP-9, and CXCL12 protein in transplanted tumors was suppressed relative to miR-124-3p overexpression. In addition, we found the levels of Bcl-2, MMP-9, and CXCL12 in Hca-F cells decreased significantly after transfection of shRNA-Anxa7 in vitro. In conclusion, our study revealed miR-124-3p inhibits tumor growth, invasion, and lymphatic metastasis in HCC by down-regulation of ANXA7 gene, thereby reducing the expression of Bcl-2, MMP-9, and CXCL12.

Keywords: ANXA7; MiR-124-3p; hepatocellular carcinoma; invasion; lymphatic metastasis.

Figure 1

miR-124-3p was down-regulated while ANXA7 was up-regulated in Hca-F cells and HCC. A. Protein expression of ANXA7 in tumor tissues and related adjacent tissues from HCC patients by immunohistochemical stain revealed that miR-124-3p was down-regulated while ANXA7 protein was up-regulated in tumor tissue. B. miR-124-3p level in tumor tissues and related adjacent tissues from HCC patients by q-PCR. C. Protein expression of ANXA7 in tumor tissues and related adjacent tissues from HCC patients. D. Pearson analysis between level of ANXA7 protein and level of miR-124-3p. There was a significant negative correlation. E. miR-124-3p level in Hca-F cells, compared with a normal hepatic cell line NCTC1469 by q-PCR. F, G. Protein expression of ANXA7 in Hca-F cells, compared with a normal hepatic cell line NCTC1469 by western blot. The error bar represents the SD (n = 3). **P < 0.01.

Figure 2

ANXA7 is a target gene of miR-124-3p. A. Sequencing results of WT ANXA7 3’-UTR gene and MUT ANXA7 3’-UTR gene. The sequences were consistent with miRanda, Pubmed, and Targetscans databases and there were binding sites with miR-124-3p in the 155-162 bp sites, while the MUT ANXA7’-UTR 155-162 sites had no miR-124-3p binding sites. B. miR-124-3p binding site in WT ANXA7 3’-UTR and MUT ANXA7 3’-UTR mutation site. C. Dual-luciferase reporter gene assay. D. Comparison of ANXA7 protein levels in Hca-F cells after transfection of miR-124-3p mimics, and miR-124-3p inhibitor. The error bar represents the SD (n = 3). *P < 0.05, **P < 0.01.

Figure 3

Upregulation of miR-124-3p promotes Hca-F cell apoptosis. A. Annexin V-FITC/PI flow cytometry was used to detect the apoptosis of Hca-F cells, miR-124-3p mimic cells, and miR-124-3p inhibitor cells. B, C. The levels of ANAX7, Bax, and Bcl-2 proteins in Hca-F cells, miR-124-3p mimic cells, and miR-124-3p inhibitor cells were detected by western blot. The error bar represents the SD (n = 3). **P < 0.01.

Figure 4

Upregulation of miR-124-3p promotes Hca-F cell apoptosis. A. Transwell migration and invasion experiment in Hca-F cells, miR-124-3p mimic cells, and miR-124-3p inhibitor cells. B. The number of cells on the insert membrane after transwell migration and invasion experiments in Hca-F cells, miR-124-3p mimic cells, and miR-124-3p inhibitor cells. C, D. The levels of ANXA7, MMP-9, and CXCL12 protein in Hca-F cells, miR-124-3p mimic cells, and miR-124-3p inhibitor cells were detected by western blot. The error bar represents the SD (n = 3). *P < 0.05, **P < 0.01.

Figure 5

Upregulation of miR-124-3p suppresses LNM and tumorigenicity in 615 mice with Hca-F cells. A. The volume of transplanted tumors on the footpad of mice in each group at different time points. B. The transplanted tumors at the footpad of mice in each group at 28 days. C. The volume of normal lymph nodes smaller than the nodes infected with Hca-F cells. D. The average weight of transplanted tumors on the footpad of mice in each group at 28 days. E. The axillary and inguinal lymph node volume in each group on 28 days. F, G. The expression levels of ANXA7, MMP-9, and CXCL12 proteins in transplanted tumors in each group. The error bar represents the SD (n = 3). *P < 0.05, **P < 0.01.

Figure 6

The expression of ANXA7, MMP-9, and CXCL12 detected by immunohistochemical assay. (A) Immunostaining results of ANXA7, MMP-9, and CXCL12 in transplanted tumors of mice in each group, using a magnification of 40 × 10, (B) IOD values of positive results in each group. The error bar represents the SD (n = 3). **P < 0.01.

Figure 7

Silencing ANXA7 affects miR-124-3p related protein in Hca-F cells. A. The expression vector ANXA7 and its unrelated sequence were transfected into Hca-F cells by Lipofectamine 2000. The green fluorescent protein was observed under fluorescence microscope after incubation at 37°C for 24-48 hours. B. The levels of ANXA7 gene in Hca-F group, N-control group, and shRNA-Anxa7 group were detected by qPCR. C, D. Western blot was used to detect the levels of ANXA7 protein in Hca-F group, N-control group, and shRNA-Anxa7 group. E. q-PCR was used to detect the levels of Bax, Bcl-2, MMP-9, and CXCL12 gene in the Hca-F group, N-control group, and shRNA-Anxa7 group. F, G. Western blot was used to detect the levels of Bax, Bcl-2, MMP-9, and CXCL12 protein in the Hca-F group, N-control group, and shRNA-Anxa7 group. The error bar represents the SD (n = 3). *P < 0.05, **P < 0.01, ns P > 0.05.