MiR-425-5p promotes invasion and metastasis of hepatocellular carcinoma cells through SCAI-mediated dysregulation of multiple signaling pathways

Abstract

MicroRNAs (miRNAs) play critical roles in hepatocellular carcinoma (HCC) progression and are key determinants of prognosis. In this study, we found that miR-425-5p was elevated in HCC and correlated with poor prognostic clinicopathological features and low post-operative long-term survival. Multivariate survival analysis indicated that miR-425-5p expression was an independent risk factor for overall and disease-free survival. Interestingly, miR-425-5p promoted invasion and metastasis by HCC cells, but not HCC cell proliferation or apoptosis in vitro. SCAI and PTEN were determined to be downstream targets of miR-425-5p. miR-425-5p-mediated effects were inhibited by ectopic expression of SCAI, and PTEN exhibited a smaller inhibitory effect. SCAI also suppressed PTEN expression. In addition, miR-425-5p promoted epithelial-to-mesenchymal transition (EMT), which was antagonized by SCAI. miR-425-5p also promoted HCC cell invasion and metastasis via SCAI-mediated dysregulation of integrin β1-Fak/Src-RhoA/CDC42, PTEN-AKT, and TIMP2-MMP2/MMP9 signaling. Finally, miR-425-5p promoted metastasis in a xenograft mouse model of HCC. These results indicate that miR-425-5p facilitates EMT and extracellular matrix degradation and promotes HCC metastasis through SCAI-mediated dysregulation of multiple signaling pathways. MiR-425-5p is therefore a potential prognostic biomarker and novel therapeutic target in HCC.

Keywords: PTEN; SCAI; hepatocellular carcinoma; integrin β1; miR-425-5p.

Figure 1. MiR-425-5p is up-regulated in HCC and is correlated with OS and DFS

(A) Quantification of mature miR-425-5p expression in 110 paired HCC and ANLT samples using qRT-PCR. (B) MiR-425 expression in HCC tissue with (n = 52) or without (n = 58) microvascular invasion. Expression was compared using ANOVA. (C and D) The OS and DFS of patients with high or low miR-425-5p expression.

Figure 2. OS and DFS of HCC patients

Patients were stratified into different subgroups according to pathological characteristics of HCC, including TNM stage (A, B), BCLC stage (C, D), tumor number (E, F), and microvascular invasion (G, H).

Figure 3. MiR-425-5p promotes HCC cell migration and invasion in vitro

Wound healing (A) and transwell (B) assays were performed to analyze the effects of miR-425-5p on HCC cell migration and invasion. The percentage of wound closure and percentage of cells that migrated through the transwell membranes are shown. *, P < 0.05. (C) Representative immunofluorescence images showing miR-425-5p-induced changes in HCC cellular morphology. Cell nuclei were stained with DAPI (blue), and the cytoskeleton was stained with actin-tracker FITC (red). Original magnification ×400.

Figure 4. MiR-425-5p suppresses SCAI expression

(A) Analysis of candidate target gene expression by western blotting. (B) Reporter plasmids with wild-type or mutant 3′ UTR sequences of PTEN, SCAI, and TIMP2 were transfected into HCCLM3 and SMMC-7721 cells infected with anti-miR-425-5p or miR-425-5p lentiviruses and relative luciferase activity analyzed. To investigate whether SCAI expression either interfered with or mimicked the function of miR-425-5p, HCC cells were infected with lentiviral vectors expressing SCAI siRNA or SCAI to inhibit or restore SCAI expression, respectively. Wound healing (C) and invasion (D) assays were performed using the above cells. * P < 0.01.

Figure 5. Representative immunofluorescence images of the cytoskeleton showing the cellular morphology in each group

Cell nuclei were stained with DAPI (blue), while the cytoskeleton was stained with actin-tracker FITC (red). Original magnification ×400.

Figure 6. Western blot analysis of protein expression in HCC cells following ectopic expression or silencing of miR-425-5p, as well as ectopic expression or silencing of SCAI

Figure 7. PTEN partially blocks the effects of miR-425-5p on the migration and invasion of HCC cells

Wound healing (A) and transwell (B) assays were performed to analyze the effects of PTEN on miR-425-5p-induced HCC cell motility and invasion. The percentage of wound closure and percentage of cells that migrated through the transwell membranes are shown. * P < 0.05.

Figure 8

(A) SCAI stimulates PTEN expression Western blot analysis of PTEN expression in response to ectopic expression or silencing of SCAI. The relative expression of PTEN in these cells is. (B) MiR-425-5p promotes EMT and ECM degradation in HCC through suppression SCAI-mediated deregulation of the ITGB1-Fak/Src-RhoA/CDC42, PTEN-AKT, and TIMP2-MMP2/MMP9 signaling pathways.

Figure 9. MiR-425-5p promotes metastasis in vivo

(A) Representative image of the tumor nodules observed at the primary (spleen) and metastatic (liver) sites. The black arrows indicate the locations of the primary tumor and metastatic nodules. (B, D) The number of mice with intrahepatic metastatic nodules. (C, E) The total number of intrahepatic metastatic nodules in each group.