miR-182-5p promotes hepatocellular carcinoma progression by repressing FOXO3a

Abstract

Background: High frequency of recurrence is the major cause of the poor outcomes for patients with hepatocellular carcinoma (HCC). microRNA (miR)-182-5p emerged as a high-priority miRNA in HCC and was found to be related to HCC metastasis. Whether the expression of miR-182-5p in tumor tissue correlated with early recurrence in HCC patients underwent curative surgery was unknown.

Methods: Real-time PCR (RT-PCR) and in situ hybridization (ISH) were conducted to assess the expression of miR-182-5p in HCC cells and tissues. Cell Counting Kit-8 (CCK-8), transwell assays were performed to detected cells proliferation and migration ability. Flow cytometry assays were used to detect cell apoptosis rate, and xenograft model was employed to study miR-182-5p in HCC growth and lung metastasis. The target of miR-182-5p was validated with a dual-luciferase reporter assay and western blotting. Immunohistochemistry, immumoblotting, and immunoprecipitation were performed to test relative protein expression.

Results: We showed that high expression of miR-182-5p in tumor tissues correlated with poor prognosis as well as early recurrence in HCC patients underwent curative surgery. miR-182-5p enhanced motility and invasive ability of HCC cells both in vitro and in vivo. miR-182-5p directly targets 3'-UTR of FOXO3a and repressed FOXO3a expression, activating AKT/FOXO3a pathway to promote HCC proliferation. Notably, miR-182-5p activated Wnt/β-catenin signaling by inhibiting the degradation of β-catenin and enhancing the interaction between β-catenin and TCF4 which was mediated by repressed FOXO3a.

Conclusions: Consistently, miR-182-5p can be a potential predictor of early recurrence for HCC patients underwent curative surgery, and FOXO3a plays a key mediator in miR-182-5p induced HCC progression.

Keywords: FOXO3a; HCC; Wnt signaling; miR-182-5p.

Fig. 1

miR182-5p can be regarded as predictor for early recurrence of HCC. a Relative expression of miR-182-5p in different HCC cell lines. b Relative expression of miR-182-5p in HCC and adjacent tissues of GEO database (GSE22058). c Relative miR-182-5p expression in paired HCC tissues and adjacent tissues. d The relationship between expression of miR-182-5p and overall survival of HCC patients in TCGA database. e ISH staining of U6 and miR-182-5p in HCC and adjacent tissues. f The relationship between expression of miR-182-5p and recurrence-free survival of HCC patients in Zhongshan hospital. (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 2

Effects of miR182-5p in HCC proliferation and invasion. a Relative miR-182-5p expression in HCC cells that overexpressed miR-182-5p in MHCC-97H cells and knockdown miR-182-5p in MHCC-97L cells. b CCK8 assays of miR-NC cells and miR-182-5p-overexpression cells of MHCC-97H, and CCK8 assays of Anti-NC cells and miR-182-5p-inhibition cells of MHCC-97L cells. c Transwell assays of miR-NC cells and miR-182-5p-overexpression cells of MHCC-97H, and transwell assays of anti-NC cells and miR-182-5p-inhibition cells of MHCC-97L cells. (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 3

The role of miR-182-5p in HCC growth and lung metastasis. a Orthotopic transplantation tumor formation with miR-NC and miR-182-5p overexpression in MHCC-97H cells, and tumor size analysis of the miR-NC and miR-182-5p overexpression groups. b Orthotopic transplantation tumor formation with anti-NC and miR-182-5p inhibition in MHCC-97L cells, and tumor size analysis of the anti-NC and miR-182-5p inhibition groups. c The images of lung metastasis of miR-NC and miR-182-5p-overexpression groups by stereo fluorescence microscope and lung metastasis analysis of miR-NC and miR-182-5p-overexpression groups. d The images of lung metastasis of anti-NC and miR-182-5p-inhibiton groups by stereo fluorescence microscope, and lung metastasis analysis of anti-NC and miR-182-5p-inhibition groups. (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 4

miR-182-5p targets FOXO3a to activate AKT pathway. a Predicted binding sites of 3′-UTR of FOXO3a to miR-182-5p, and the relative Rluc/Luc ratio of dual-luciferase reporter assay in different groups. b Western blot analysis of AKT/FOXO3a signaling-related proteins in control group, overexpression and knockdown miR-182-5p group of HCC cells. c Western blot analysis of p-AKT Ser473 expression after FOXO3a knockdown. d FASC analysis of cell apoptosis rate in miR-NC and miR-182-5p overexpression cells

Fig. 5

FOXO3a exerts an important mediator in miR-182-5p induced Wnt signaling activation. a Western blot analysis of Wnt signaling pathway related proteins following miR-182-5p overexpression and knockdown. b IHC staining of FOXO3a and β-catenin in orthotopic tumor tissues of negative control and overexpression of miR-182-5p. c Western blot analysis of c-Myc in miR-NC cells treated with different concentration of XAV939 (0, 1, 5, 10, 100, and 200 μM). d Transwell assay of HCC cells that transfected with miR-NC, overexpression of miR-182-5p and miR-182-5p overexpression cells that further treated with 10 μM XAV939. e Western blot analysis of Wnt signaling pathway-related proteins in miR-NC cells, overexpression of miR-182-5p cells and miR-182-5p overexpression cells further overexpress FOXO3a

Fig. 6

miR-182-5p inhibits β-catenin degradation and enhances the binding of β-catenin/TCF4. a Western blot analysis of β-catenin expression in miR-NC and miR-182-5p overexpression cells that treated with 100 μg/mL CHX at 0, 1.5, and 3 h. b Endogenous TCF4 was immunoprecipitated, and binding of β-catenin to TCF4 was analyzed by immunoblotting for β-catenin. c Endogenous FOXO3a was immunoprecipitated, and binding of β-catenin to FOXO3a was analyzed by immunoblotting for β-catenin