miR-296 inhibits the metastasis and epithelial-mesenchymal transition of colorectal cancer by targeting S100A4

Abstract

Background: Dysregulation of microRNAs (miRNAs) is actively involved in the pathogenesis and tumorigenicity of colorectal cancer (CRC). miR-296 was found to play either oncogenic or tumor suppressive role in human cancers. However, the status of miR-296 and its function in CRC remain unknown.

Methods: The expression of miR-296 was confirmed by qRT-PCR in CRC tissues and cells, and its level was altered by corresponding miRNA vectors. Wound healing and Transwall assays were performed to detect the migration and invasion of CRC cells. The levels of proteins were measured using immunoblotting, immunohistochemistry and immunofluorescence.

Results: Underexpression of miR-296 was disclosed in CRC tissues and cells. Its decreased level was evidently correlated with adverse clinical parameters and poor prognosis of CRC patients. In vitro experiments indicated that miR-296 inhibited CRC cell migration and invasion. Mechanically, miR-296 inhibited the epithelial-mesenchymal transition (EMT) of CRC cells. A negative correlation between miR-296 and S100A4 expression was observed in CRC tissues. Luciferase reporter assays indicated that miR-296 inversely regulated the luciferase activity of 3'-UTR of S100A4. Herein, S100A4 was found to be a downstream molecule of miR-296 in CRC. Furthermore, S100A4 mediated the anti-metastatic effects of miR-296 on EMT, migration and invasion of CRC cells.

Conclusions: miR-296 functions as an anti-metastatic factor mainly by suppressing S100A4 in CRC. It potentially acts as a prognostic predictor and a drug-target for CRC patients.

Keywords: Colorectal cancer; Epithelial-mesenchymal transition; Metastasis; S100A4; miR-296.

Fig. 1

The status and prognostic value of miR-296 expression in CRC. a The expression differences of miR-296 between CRC tissues and normal tumor-adjacent tissues. n = 90. b The expression differences of miR-296 between 5 different CRC cells lines (HCT116, Caco2, HT29, SW620, SW480) and HIEC cells. *P < 0.05 versus HIEC cells. c and d Compared with those of high miR-296 level (n = 45), miR-296 low-expressing patients (n = 45) had significantly reduced overall survival and recurrence-free survival

Fig. 2

miR-296 overexpression inhibits the mobility of HT29 cells. a HT29 cells that were transduced with negative control mimics (miR-control) or miR-296 mimics were confirmed by qRT-PCR. n = 3, *P < 0.05. b Wound healing assays indicated that miR-296 overexpression reverses the migration of HT29 cells. c Transwell assays confirmed that miR-296 overexpression inhibited HT29 cell migration and invasion. n = 3, *P < 0.05

Fig. 3

miR-296 knockdown facilitates the metastasis of SW480 cells. a SW480 cells that were transduced with negative control inhibitors (NC) or miR-296 inhibitors (anti-miR-296) were confirmed by qRT-PCR. n = 3, *P < 0.05. b miR-296 knockdown notably facilitated the migration of SW480 cells. c miR-296 knockdown prominently promoted SW480 cell migration and invasion. n = 3, *P < 0.05

Fig. 4

miR-296 inhibits the epithelial-mesenchymal transition of CRC cells. a Western blot showed overexpression of miR-296 decreased S100A4 and Vimentin expression, and increased E-cadherin expression in HT29 cells. b Immunofluorescence showed miR-296 overexpression increased E-cadherin expression and decreased Vimentin expression in HT29 cells. c Western blot showed knockdown of miR-296 increased S100A4 and Vimentin expression, and decreased E-cadherin expression in SW480 cells. d Immunofluorescence showed knockdown of miR-296 decreased E-cadherin expression and increased Vimentin expression in SW480 cells

Fig. 5

The expression of S100A4, E-cadherin, and Vimentin in CRC tissues. In representative immunohistochemical staining, miR-296 low expressing tumors showed strong staining of (a) S100A4 and (e) Vimentin, and weak staining of (c) E-cadherin. However, miR-296 high expressing tumors showed weak staining of (b) S100A4 and (f) Vimentin, and strong staining of (d) E-cadherin. Scale bar: 50 μm

Fig. 6

miR-296 binds to the complementary sequence in S100A4 3’-UTR. a The binding sites for miR-296 in wild type (wt) and mutant (mt) 3’-UTR of S100A4. b Overexpression of miR-296 decreased while inhibition of miR-296 increased the luciferase activity of wt 3’-UTR of S100A4. Alteration of miR-296 had no effect on the luciferase activity of mt 3’-UTR of S100A4. n = 3, *P < 0.05

Fig. 7

S100A4 restoration reverses the effects of miR-296. a miR-296 overexpressing HT29 cells that were infected with empty vector (EV) or S100A4 retroviruses were confirmed by western blotting for S100A4, E-cadherin and Vimentin. b S100A4 restoration significantly promoted the migration of miR-296 overexpressing HT29 cells. c S100A4 restoration evidently facilitated cell migration and invasion in miR-296 overexpressing HT29 cells. n = 3, *P < 0.05

Fig. 8

S100A4 knockdown abrogates the effects of miR-296 inhibition on EMT, migration and invasion of CRC cells. a miR-296 silenced SW480 cells that were transfected with S100A4 siRNA and scrambled siRNA (scr siRNA) were subjected to western blot. S100A4 siRNA significantly decreased S100A4 in miR-296 silenced SW480 cells, and led to increased E-cadherin expression and decreased Vimentin expression. b S100A4 knockdown significantly inhibited the migration of miR-296 silenced SW480 cells. c S100A4 knockdown significantly inhibited the migration and invasion of miR-296 silenced SW480 cells. n = 3, *P < 0.05