microRNA-20a enhances the epithelial-to-mesenchymal transition of colorectal cancer cells by modulating matrix metalloproteinases

Abstract

The mortality rates associated with colorectal cancer (CRC) are high due to metastasis. Epithelial-to-mesenchymal transition (EMT) is a key step in tumor metastasis. The aim of the present study was to investigate the function of microRNA-20a (miR-20a) in EMT. The expression of miR-20a was analyzed in CRC tissues and cell lines using the reverse transcription-quantitative polymerase chain reaction. Plasmids containing miR-20a short hairpin RNA and miR-20a mimics were transfected into SW620 and LS174T cell lines, respectively. Cell counting kit-8, Transwell® and wound healing assays were performed to assess the effects of miR-20a on cell proliferation, invasion and migration. EMT markers and matrix metalloproteinases (MMPs) were identified using western blotting. The results showed that increased expression of miR-20a in CRC tissues was associated with tumor invasion and lymph node metastasis (P<0.05). Further experiments indicated that miR-20a-knockdown inhibited the proliferation, invasion and migration of CRC cells, upregulated the expression of vimentin and tissue inhibitor of metalloproteinases-2 (TIMP-2) and downregulated the expression of E-cadherin, MMP-2 and MMP-9. The opposite effects were observed in CRC cell lines overexpressing miR-20a. In conclusion, these results have shown that the upregulation of miR-20a suppresses TIMP-2 expression, which subsequently increases the expression of MMP-2 and MMP-9, thereby promoting the EMT of CRC cells. These findings suggest that miR-20a represents a potential therapeutic target for patients with CRC.

Keywords: colorectal cancer; epithelial-to-mesenchymal transition; microRNA-20a.

Figure 1.

Relative expression level of miR-20a in CRC tissues and cell lines. The reverse transcription-quantitative polymerase chain reaction was used to detect the relative expression of miR-20a in CRC (A) tissues and (B) cell lines. U6 was used as an internal control. Error bars represent the mean ± standard deviation from three independent experiments. *P<0.05, **P<0.01. miR-20a, microRNA-20a; CRC, colorectal cancer; T, tumor tissues; N, adjacent non-tumor tissues.

Figure 2.

Expression level of miR-20a in knocked down and overexpressed colorectal cancer cell lines. The reverse transcription-quantitative polymerase chain reaction was used to detect the expression of miR-20a in (A) SW620-NC and SW620-Si20a and (B) LS174T-NC and LS174T-OV20a cell lines. U6 was used as an internal control. Error bars represent the mean ± standard deviation from three independent experiments. *P<0.05. miR-20a, microRNA-20a; SW620-NC, SW620 cells transfected with miR-20a inhibitor negative control plasmid; SW620-Si20a, SW620 cells transfected with miR-20a inhibitor plasmid; LS174T-NC, LS174T cells transfected with miR-20a mimic negative control plasmid; LS174T-OV20a, LS174T cells transfected with miR-20a mimic plasmid.

Figure 3.

Effect of miR-20a on cell proliferation. The proliferative ability was detected by performing the cell counting kit-8 assay using colorectal cancer cell lines with (A) knocked down and (B) overexpressed miR-20a. *P<0.05. miR-20a, microRNA-20a; OD, optical density; SW620-NC, SW620 cells transfected with miR-20a inhibitor negative control plasmid; SW620-Si20a, SW620 cells transfected with miR-20a inhibitor plasmid; LS174T-NC, LS174T cells transfected with miR-20a mimic negative control plasmid; LS174T-OV20a, LS174T cells transfected with miR-20a mimic plasmid.

Figure 4.

Effect of miR-20a on cell migration. (A) The Transwell® assay was used to assess the invasive ability of the SW620-NC, SW620-Si20a, LS174T-NC and LS174T-OV20a cell lines (x400). (B) A wound healing assay was used to assess the mobility of the SW620-NC, SW620-Si20a, LS174T-NC and LS174T-OV20a cell lines (x400). (C and D) The expression of the epithelial-to-mesenchymal transition markers E-cadherin and vimentin in the (C) SW620-NC and SW620-Si20a and (D) LS174T-NC and LS174T-OV20a cell lines was examined by western blot analysis. miR-20a, microRNA-20a; SW620-NC, SW620 cells transfected with miR-20a inhibitor negative control plasmid; SW620-Si20a, SW620 cells transfected with miR-20a inhibitor plasmid; LS174T-NC, LS174T cells transfected with miR-20a mimic negative control plasmid; LS174T-OV20a, LS174T cells transfected with miR-20a mimic plasmid.

Figure 5.

Expression of the epithelial-to-mesenchymal transition-related proteins. The effect of miR-20a on the protein expression of TIMP-2, MMP-2 and MMP-9 in colorectal cancer cell lines with (A) knocked down and (B) overexpressed miR-20a was analyzed using western blotting. miR-20a, microRNA-20a; TIMP2, tissue inhibitor of metalloproteinases-2; MMP, matrix metalloproteinase; SW620-NC, SW620 cells transfected with miR-20a inhibitor negative control plasmid; SW620-Si20a, SW620 cells transfected with miR-20a inhibitor plasmid; LS174T-NC, LS174T cells transfected with miR-20a mimic negative control plasmid; LS174T-OV20a, LS174T cells transfected with miR-20a mimic plasmid.