MiR-625-3p promotes cell migration and invasion via inhibition of SCAI in colorectal carcinoma cells

Abstract

MicroRNAs (miRNAs) play a critical role in controlling tumor invasion and metastasis via regulating the expression of a variety of targets, which act as oncogenes or tumor suppressor genes. Abnormally expressed miR-625-3p has been observed in several types of human cancers. However, the molecular mechanisms of miR-625-3p-mediated tumorigenesis are largely elusive. Therefore, the aim of this study was to evaluate the biological function and molecular insight on miR-625-3p-induced oncogenesis in colorectal carcinoma (CRC). The effects of miR-625-3p in cell migration and invasion were analyzed by wound healing assay and transwell assay, respectively. In addition, the expression of miR-625-3p and its targets was detected in five human CRC cell lines. In the present study, we found that overexpression of miR-625-3p promoted migration and invasion in SW480 cells, whereas downregulation of miR-625-3p inhibited cell motility in SW620 cells. More importantly, we observed potential binding sites for miR-625-3p in the 3'-untranslated region of suppressor of cancer cell invasion (SCAI). Notably, we identified that overexpression of miR-625-3p inhibited the expression of SCAI, while depletion of miR-625-3p increased SCAI level, suggesting that SCAI could be a target of miR-625-3p. Additionally, we revealed that miR-625-3p exerts its oncogenic functions through regulation of SCAI/E-cadherin/MMP-9 pathways. Our findings indicate the pivotal role of miR-625-3p in invasion that warrants further exploration whether targeting miR-625-3p could be a promising approach for the treatment of CRC.

Keywords: SCAI; colorectal carcinoma; invasion; miR-625-3p; migration.

Figure 1. Over-expression of miR-625-3p promoted cell migration and invasion in SW480 cells

NC: negative control; miR-625-3p: miR-625-3p mimic. *P < 0.05; **P < 0.01 vs control. A. miR-625-3p level was measured by real-time RT-PCR in five CRC cell lines (left panel) and SW480 cells with miR-625-3p mimic treatment (right panel). B. Left panel, the cell motility was detected using wound healing assay in SW480 cells transfected with miR-625-3p. Right panel, Quantitative results are illustrated for left panels. C-D. Left panel, the cell migration and invasion were detected by uncoated (C) and coated (D) Transwell chambers assay. Right panel, Quantitative results are illustrated for left panel.

Figure 2. Down-regulation of miR-625-3p inhibited cell migration and invasion in SW620 cells

Anti-miR-625-3p: miR-625-3p inhibitor. *P < 0.05; **P < 0.01 vs control. Control: anti-miR-control. A. miR-625-3p level was determined by real-time RT-PCR in SW620 cells with miR-625-3p inhibitor treatment. B. Left panel, the cell motility was measured using wound healing assay in SW620 cells treated with miR-625-3p inhibitor. Right panel, Quantitative results are illustrated for left panels. C-D. Left panel, the cell migration and invasion were measured using uncoated (C) and coated (D) Transwell chambers assay. Right panel, Quantitative results are illustrated for left panel.

Figure 3. The expression of miR-625-3p, SCAI and E-cadherin was correlated in CRC cells

A. SCAI sequence analysis indicates that it harbors potential miR-625-3p target sites, which are nt 6845–6852 sequences of the SCAI 3′ UTR. B. SCAI mRNA level was detected by real-time RT-PCR in CRC cell lines. C. Western blotting analysis was performed to detect the SCAI protein level in CRC cell lines. D. Quantitative results are illustrated for panel C.

Figure 4. Over-expression of miR-625-3p inhibited SCAI expression in SW480

NC: negative control; miR-625-3p: miR-625-3p mimic. *P < 0.05; **P < 0.01 vs control. A. Real-time RT-PCR was conducted to detect the expression of SCAI, E-cadherin, and MMP-9 at the mRNA levels in SW480 cells transfected with miR-625-3p mimic. B. Western blotting analysis was performed to detect the expression of SCAI, E-cadherin, and MMP-9 in SW480 cells transfected with miR-625-3p mimic (left panel). Quantitative result of SCAI expression is presented (right panel). C. Quantitative results are illustrated for panel B.

Figure 5. Down-regulation of miR-625-3p increased SCAI expression

Anti-miR-625-3p: miR-625-3p inhibitor. *P < 0.05; **P < 0.01 vs control. A. Real-time RT-PCR was performed to detect the expression of SCAI, MMP-9, and E-cadherin at the mRNA levels in SW620 treated with miR-625-3p inhibitor. B. Western blotting analysis was used to measure the expression of SCAI, E-cadherin, and MMP-9 in SW620 cells with miR-625-3p inhibitor treatment (left panel). Quantitative result of SCAI expression is presented (right panel). C. Quantitative results are illustrated for panel B.

Figure 6. A schematic illustration of the signaling network showing how miR-625-3p promotes cell migration and invasion

MiR-625-3p inhibited SCAI expression and subsequently suppressed E-cadherin and upregulated MMP-9 expression, leading to enhanced cell migration, invasion and metastasis in CRC.