Limin Liu1, Maoying Zhu2, Xiaoli Liu2, Lumin Fei2, Jianyun Shen2, Deyu Chen3. 1. Center for Reproductive Medicine, Huizhou Central People's Hospital, Huizhou, 516001, Guangdong, China. 2. College of Biological and Food Engineering, Fuyang Normal University, No.100 Qinghe Road, Fuyang, 236037, Anhui, China. 3. College of Biological and Food Engineering, Fuyang Normal University, No.100 Qinghe Road, Fuyang, 236037, Anhui, China. chendeyu7104@aliyun.com.
Abstract
BACKGROUND: Occludin protein is the primary assembling protein of TJs and the structural basis for tight junction formation between Sertoli cells in the spermatogenic epithelium. The expression of miR-122-5p and occludin are negatively correlated. In order to investigate the regulation mechanism of miR-122-5p on occludin and TJ, the present study isolated primary Sertoli cells from C57BL/6 mice, identified a transcription factor of miR-122-5p in testicle, studied the modulating loci of miR-122-5p on occludin using a dual-luciferase reporter assay, analyzed the regulate of miR-122-5p on the expression of occludin with real-time RT-PCR and Western blot, and studied the effect of miR-122-5p on the tight junction using a Millicell Electrical Resistance System. RESULTS: The relative luciferase activity in the pcDNA-Sp1 + pGL3-miR-122-5p promoter group was significantly higher than that in the pcDNA-Sp1 + pGL3-basic group, which suggests that transcript factor Sp1 promotes the transcription of miR-122-5p. The relative luciferase activity in the occludin 3'-UTR (wt) + miR-122-5p mimic group was significantly lower than that in the other groups (p < 0.01), which indicates that miR-122-5p modulates the expression of occludin via the ACACTCCA sequence of the occludin-3'UTR. The levels of occludin mRNA and protein in the miR-122-5p mimic group were significantly lower than that in the other groups (p < 0.05), which indicates that miR-122-5p reduces the expression of occludin. The trans-epithelial resistance of the miR-122-5p mimic group was significantly lower than that of the blank control group after day 4 (p < 0.05), which indicates that miR-122-5p inhibited the assembly of the inter-Sertoli TJ permeability barrier in vitro. CONCLUSION: These results displayed that miR-122-5p could regulate tight junctions via the Sp1-miR-122-5p-occludin-TJ axis.
BACKGROUND:Occludin protein is the primary assembling protein of TJs and the structural basis for tight junction formation between Sertoli cells in the spermatogenic epithelium. The expression of miR-122-5p and occludin are negatively correlated. In order to investigate the regulation mechanism of miR-122-5p on occludin and TJ, the present study isolated primary Sertoli cells from C57BL/6 mice, identified a transcription factor of miR-122-5p in testicle, studied the modulating loci of miR-122-5p on occludin using a dual-luciferase reporter assay, analyzed the regulate of miR-122-5p on the expression of occludin with real-time RT-PCR and Western blot, and studied the effect of miR-122-5p on the tight junction using a Millicell Electrical Resistance System. RESULTS: The relative luciferase activity in the pcDNA-Sp1 + pGL3-miR-122-5p promoter group was significantly higher than that in the pcDNA-Sp1 + pGL3-basic group, which suggests that transcript factor Sp1 promotes the transcription of miR-122-5p. The relative luciferase activity in the occludin 3'-UTR (wt) + miR-122-5p mimic group was significantly lower than that in the other groups (p < 0.01), which indicates that miR-122-5p modulates the expression of occludin via the ACACTCCA sequence of the occludin-3'UTR. The levels of occludin mRNA and protein in the miR-122-5p mimic group were significantly lower than that in the other groups (p < 0.05), which indicates that miR-122-5p reduces the expression of occludin. The trans-epithelial resistance of the miR-122-5p mimic group was significantly lower than that of the blank control group after day 4 (p < 0.05), which indicates that miR-122-5p inhibited the assembly of the inter-Sertoli TJ permeability barrier in vitro. CONCLUSION: These results displayed that miR-122-5p could regulate tight junctions via the Sp1-miR-122-5p-occludin-TJ axis.