Abstract
BackgroundOccludin 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.ResultsThe 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.ConclusionThese results displayed that miR-122-5p could regulate tight junctions via the Sp1-miR-122-5p-occludin-TJ axis.
Highlights
MicroRNAs are a family of small noncoding ribonucleic acid (RNA) with a length of ~ 22 bp and carry a 5′-phosphate terminal and 3′-hydroxy terminal
MiR-122-5p is a transcript processed from the Hcr gene, and it plays an important role in modulation of the cell cycle, cell proliferation and apoptosis [9]. miR-122-5p is related to multiple diseases, e.g., it is a prognosis marker for acute heart stroke [10], affects the proliferation of keratinocytes [11], inhibits the migration of melanoma cells [12], suppresses cellular differentiation in nasopharyngeal carcinoma [13], regulates cell proliferation in gastric cancer [14]
Sertoli cells proliferation occurs between days 12–15 in rodents [30]
Summary
MicroRNAs (miRs) are a family of small noncoding ribonucleic acid (RNA) with a length of ~ 22 bp and carry a 5′-phosphate terminal and 3′-hydroxy terminal. The major functions of miRs include the modulation of transcription, RNA cutting and trimming, messenger ribonucleic acid (mRNA) stabilization and translation, protein stabilization and transportation, chromosome formation and structure stabilization, and cellular development [1]. Different miRs are involved in the proliferation [2,3,4] and differentiation of spermatogenic cells [5] and spermatogenesis [6]. 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
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