MicroRNA-21 up-regulates expression of occludin by targeting ROCK1 in NCM460 cells

AIM: To investigate the role of microRNA-21 (miR-21) in regulation of tight junction (TJ)-associated protein occludin in human normal colon mucosal epithelial cell line NCM460, and to analyze related target genes. METHODS: Using miR-21 over-expression lentivirus, the NCM460 cells with miR-21 overexpression were established. The expression level of miR-21 was detected by qPCR. The expression of occludin was determined by Western blot. The target genes of miR-21 were predict by bioinformatic method. According to the scores and the appropriate literature search, a target gene was selected for further study. The miR-21 mimic and inhibitor were transfected into NCM460 cells, and the expression levels of miR-21 and ROCK1 were measured. Dual-luciferase reporter assay was also performed to verify ROCK1 as the target gene of miR-21. RESULTS: In miR-21-overexpressing NCM460 cells, the protein expression level of occludin was upregulated. Bioinformatics and literature analysis predicted that the target gene of miR-21 was ROCK1. The mRNA and protein levels of ROCK1 were down-regulated in the NCM460 cells transfected with miR-21 mimic. Accordingly, the mRNA and protein levels of ROCK1 were up-regulated in the NCM460 cells transfected with miR-21 inhibitor. Luciferase assay confirmed that miR-21-binding sequence was on the 3'UTR of ROCK1, indicating that ROCK1 is the target gene of miR-21. CONCLUSION: In NCM460 cells, miR-21 up-regulates the expression of occludin, which functions in the maintenance of intestinal epithelial mechanical barrier. ROCK1 is a target gene of miR-21, and is involved in the miR-21 regulation of occludin.