微小RNA-424-5p调控OTX1对结直肠癌细胞增殖、迁移和侵袭的影响

Abstract

Objective To investigate the correlation of the expression of microRNA-424-5p (miR-424-5p) and orthodenticle homeobox 1 (OTX1) gene in colorectal cancer (CRC) tissue, and the effects of miR-424-5p on the proliferation, migration and invasion of CRC cells HCT116. Methods A total of 60 patients with CRC were collected from June 2017 to June 2018 in Department of Colorectal Surgery of Third Affiliated Hospital of Kunming Medical University. Real time quantitative PCR (RT-qPCR) was used to detect the expression of miR-424-5p and OTX1 mRNA in 60 cases of CRC tissues, para-carcinoma tissues and CRC cell lines. The correlation between the expression of miR-424-5p and OTX1 gene was further analyzed. miR-NC (miR-NC group) and miR-424-5p-mimic (miR-424-5p-mimic group) were transfected into HCT116 cells. CCK-8 assay, scratch assay and Transwell assay were used to detect the effects of miR-424-5p on the proliferation, migration and invasion of HCT116 cells. The effect of miR-424-5p on the expression of OTX1 protein was detected by Western blotting. The luciferase report assay was used to detect the influence of miR-424-5p on the luciferase activity of OTX1-3′UTR vector. Results The relative expressions of OTX1 mRNA in CRC tissues and para-carcinoma tissues were 1.049±0.446 and 0.639±0.178 (t=-6.583, P<0.001); and the relative expression of miR-424-5p in CRC tissues and para-carcinoma tissues were 0.865±0.261 and 1.329±0.387 (t=7.705, P<0.001), with statistically significant differences. Negative correlation was found between the expression of miR-424-5p and OTX1 mRNA in CRC tissues (r=-0.439, P=0.015). The absorbance values of HCT116 cells transfected with miR-424-5p-mimic were 0.813±0.064, 0.960±0.098, 1.287±0.192 on 72, 96 and 120 hours respectively, and those of HCT116 cells transfected with miR-NC were 1.163±0.158, 1.645±0.117 and 2.043±0.236. The proliferation ability of miR-424-5p-mimic group was lower than that of miR-NC group and the differences between the two groups were statistically significant (t=3.538, P=0.024; t=7.778, P=0.001; t=4.257, P=0.013). The scratch assay showed that the migration of HCT116 cells in miR-424-5p-mimic group was inhibited as compared with miR-NC group. The numbers of cells permeating septum of miR-NC and miR-424-5p-mimic group were 177.104±17.834 and 35.667±13.634, and the difference was statistically significant (t=15.246, P<0.001). The relative expressions of OTX1 protein in miR-NC and miR-424-5p-mimic group were 0.862±0.121 and 0.342±0.103 respectively, and the difference was statistically significant (t=16.286, P<0.001). Luciferase report assay showed that the luciferase activities of wt-OTX1-3′UTR and mut-OTX1-3′UTR vector were 0.305±0.095 and 0.898±0.080 after over expression of miR-424-5p, and the difference was statistically significant (P<0.001). Conclusion The expressions of miR-424-5p and OTX1 mRNA are negatively correlated in CRC tissue. miR-424-5p can inhibit the proliferation, migration and invasion of CRC cells by down-regulating the expression of OTX1. Key words: Colorectal neoplasms; MicroRNAs; Cell proliferation; OTX1