miR-557 inhibits hepatocellular carcinoma progression through Wnt/β-catenin signaling pathway by targeting RAB10

Abstract

Accumulating evidence suggests that aberrant miRNAs participate in carcinogenesis and progression of hepatocellular carcinoma (HCC). Abnormal miR-557 expression is reported to interfere with the progression of several human cancers. However, the potential roles of miR-557 in HCC remain largely unknown. In the current study, we found that miR-557 was down-regulated in HCC tissues and cell lines, and was closely related to recurrence and metastasis of HCC. Notably, overexpression of miR-557 inhibited proliferation, migration, invasion, epithelial-to-mesenchymal transition (EMT) progression, blocked cells in G0/G1 phase of MHCC-97H cells in vitro, and suppressed tumor growth in vivo. However, loss of miR-557 facilitated these parameters in Huh7 cells both in vitro and in vivo. Moreover, RAB10 was identified as a direct downstream target of miR-557 through its 3'-UTR. Furthermore, RAB10 re-expression or knockdown partially abolished the effects of miR-557 on proliferation, migration, invasion, and EMT progression of HCC cells. Mechanistically, overexpression of miR-557 suppressed Wnt/β-catenin signaling by inhibiting GSK-3β phosphorylation, increasing β-catenin phosphorylation, and decreasing β-catenin transport to the nucleus, while knockdown of miR-557 activated Wnt/β-catenin signaling. Moreover, the TOP/FOP-Flash reporter assays showed that miR-557 overexpression or knockdown significantly suppressed or activated Wnt signaling activity, respectively. Additionally, low expression of miR-557 and high expression of RAB10 in HCC tissues was closely associated with tumor size, degree of differentiation, TNM stage and poor prognosis in HCC patients. Taken together, these results demonstrate that miR-557 blocks the progression of HCC via the Wnt/β-catenin pathway by targeting RAB10.

Keywords: RAB10; Wnt/β-catenin pathway; epithelial-to-mesenchymal transition; hepatocellular carcinoma; miR-557.

Figure 1

MiR-557 down-regulation in HCC predicts poor prognosis and metastasis of HCC. (A) Relative expression of miR-557 in HCC tissues and adjacent non-tumor tissues of GEO database (GSE108724). (B) Relative miR-557 expression in HCC tissues and adjacent non-tumor tissues. (C) Relative expression of miR-557 in primary HCC tissues and pulmonary metastasis of HCC tissues of GEO database (GSE26323). (D) Relative miR-557 expression in primary HCC tissues and paired pulmonary metastasis of HCC tissues. (E) The relationship between the expression level of miR-557 and overall survival of HCC patients. (F) The relationship between the expression level of miR-557 and disease-free survival of HCC patients. (^*P<0.05, ^**P<0.01).

Figure 2

Effect of miR-557 on HCC cell proliferation, migration, invasion, cell cycle, and EMT. (A) Relative expression of miR-557 in different HCC cells lines. (B–E) Relative miR-557 expression in HCC cells that overexpressed miR-557, in MHCC-97H cells, and knockdown miR-557 in Huh7 cells. (F, G) CCK8 assays of mimic-NC cells and miR-557-overexpression cells of MHCC-97H, and CCK8 assays of inhibitor-NC cells and miR-557-inhibition cells of Huh7 cells. (H, I) Transwell assays of mimic-NC cells and miR-557-overexpression cells of MHCC-97H, and transwell assays of inhibitor-NC cells and miR-557-inhibition cells of Huh7 cells, Scale bar: 100 μm. (J–M) The protein and mRNA of EMT process of mimic-NC cells and miR-557-overexpression cells of MHCC-97H, and of inhibitor-NC cells and miR-557-inhibition cells of Huh7 cells. (N, O) Clone formation assay of Lv-miR-NC cells and Lv-miR-557-overexpression cells of MHCC-97H, and Clone formation assay of Lv-sponge-NC cells and Lv-sponge-miR-557-inhibition cells of Huh7 cells. (P) Orthotopic transplantation tumor formation with Lv-miR-NC and Lv-miR-557 overexpression in MHCC-97H cells or Lv-sponge-NC and Lv-sponge-miR-557-inhibition cells of Huh7 cells. (Q, R) The distribution phases of cell cycle of mimic-NC cells and miR-557-overexpression cells of MHCC-97H, and of inhibitor-NC cells and miR-557-inhibition cells of Huh7 cells. All blots were cut prior to hybridisation with antibodies during western blotting. (S, T) The tumor weight analysis of mimic-NC cells and miR-557-overexpression cells of MHCC-97H, and of inhibitor-NC cells and miR-557-inhibition cells of Huh7 cells. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 3

RAB10 is a downstream target gene of miR-557, is overexpressed in HCC, and is related to poor prognosis. (A) Venn diagram of TargetScan, miWalk, miRDB, and miRTarBase. (B) Meta analysis of RAB10 expression from Oncomine database. (C) Predicted binding sites of 3′-UTR of RAB10 to miR-557. (D, E) The immunohistochemical images of relative RAB10 expression in HCC tissues and paired non-tumor tissues stem from the Human Protein Atlas database (Version 23.0, https://www.proteinatlas.org/ENSG00000084733-RAB10/pathology/liver+cancer#img). (F) The relationship between RAB10 expression and overall survival from the Human Protein Atlas database. (G, H) The relationship between the expression level of RAB10 and overall survival or disease-free survival from the Kaplan–Meier plotter online database. (I–L) The transfection efficiency of pmirGLO-NC, pmirGLO-RAB10 of MHCC-97H cells, and of siNC and siRAB10 of Huh7 cells. All blots were cut prior to hybridisation with antibodies during western blotting. (^***P<0.001, ^****P<0.0001).

Figure 4

MiR-557 targets RAB10 to affect the biological behaviour of cells. (A–D) Relative expression of RAB10 in mimic-NC cells and miR-557-overexpression cells of MHCC-97H, and in inhibitor-NC cells and miR-557-inhibition cells of Huh7 cells. (E) The relationship between the expression level of miR-557 and the expression level of RAB10 in patients. (F) The relative dual-luciferase reporter assays in four different groups. (G) CCK-8 assays of NC cells, miR-557-overexpression cells, RAB10-overexpression cells, co-transfected miR-557 mimics and pmirGLO-RAB10 cells of MHCC-97H. (H) CCK-8 assays of NC cells, miR-557-down-expression cells, RAB10-down-expression cells, co-transfected miR-557 inhibitors and siRAB10 cells of Huh7. (I, J) Transwell assays of NC cells, miR-557-overexpression cells, RAB10-overexpression cells, co-transfected miR-557 mimics and pmirGLO-RAB10 cells of MHCC-97H, Scale bar: 100 μm. (K, L) Transwell assays of NC cells, miR-557-down-expression cells, RAB10-down-expression cells, co-transfected miR-557 inhibitors and siRAB10 cells of Huh7, Scale bar: 100 μm. (M, N) The distribution phases of cell cycle of NC cells, miR-557-overexpression cells, RAB10-overexpression cells, co-transfected miR-557 mimics and pmirGLO-RAB10 cells of MHCC-97H. (O, P) The distribution phases of cell cycle of NC cells, miR-557-down-expression cells, RAB10-down-expression cells, co-transfected miR-557 inhibitors and siRAB10 cells of Huh7. All blots were cut prior to hybridisation with antibodies during western blotting. (^*P<0.05, ^**P<0.01, ^***P<0.001, ^****P<0.0001).

Figure 5

MiR-557 targets RAB10 to affect the process of EMT and inhibit Wnt/β-catenin signaling. (A) The protein of EMT process of NC cells, miR-557-overexpression cells, RAB10-overexpression cells, co-transfected miR-557 mimics and pmirGLO-RAB10 cells of MHCC-97H. (B) The protein of EMT process of NC cells, miR-557-down-expression cells, RAB10-down-expression cells, co-transfected miR-557 inhibitors and siRAB10 cells of Huh7. (C) GSEA of RAB10 expression and Wnt/β-catenin signaling. (D, E) TOP-flash/FOP-flash assays of mimic-NC cells and miR-557-overexpression cells of MHCC-97H, and TOP-flash/FOP-flash assays of inhibitor-NC cells and miR-557-inhibition cells of Huh7 cells. (F) Western blot analysis of AKT/FOXO3a signaling-related proteins in NC cells, miR-557-overexpression cells, RAB10-overexpression cells, co-transfected miR-557 mimics and pmirGLO-RAB10 cells of MHCC-97H. (G) Western blot analysis of AKT/FOXO3a signaling-related proteins in NC cells, miR-557-down-expression cells, RAB10-down-expression cells, co-transfected miR-557 inhibitors and siRAB10 cells of Huh7. All blots were cut prior to hybridisation with antibodies during western blotting. (^****P<0.0001).