Overexpression of microRNA-495 improves the intestinal mucosal barrier function by targeting STAT3 via inhibition of the JAK/STAT3 signaling pathway in a mouse model of ulcerative colitis

Abstract

We aim to investigate the role of microRNA-495 (miR-495) in the intestinal mucosal barrier by indirectly targeting signal transducer and activator of transcription 3 (STAT3) through the Janus kinase-signal transducer and activator of transcription (JAK)/STAT3 signaling pathway in a mouse model of ulcerative colitis (UC). BALB/c mice were selected for establishing mice model of UC, and intestinal tissues of normal and UC mice were collected. ELISA was conducted for detecting levels of TNF-α, IL-6, IFN-γ and IL-10. The levels of SOD, MPO, MDA and NO were tested in the intestinal tissues. Dual luciferase reporter gene assay was applied to determine whether miR-495 directly targets STAT3. Cells were cultured, transfected and assigned into: normal group, blank group, NC group, miR-495 mimic group, miR-495 inhibitor group, siRNA-STAT3 group and miR-495 inhibitor+siRNA-STAT3 group. MTT was used for testing cell proliferation, flow cytometry for cell cycle and apoptosis. Northern blotting and Western blotting were performed to detect miR-495 expression and expressions of STAT3, JAK and Claudin-1. Results show that the UC group had higher expression levels of TNF-α, IL-6, IFN-γ, MPO, MDA, NO, STAT3 and JAK and lower expression levels of IL-10, SOD, miR-495 and Claudin-1, compared to the normal group. Dual luciferase reporter gene assay confirmed that STAT3 was the target gene of miR-495. The miR-495 mimic and siRNA-STAT3 groups had higher expressions of Claudin-1, higher cell proliferation and increased amount of cells in S phase, but lower expressions of STAT3 and JAK, decreased amount of cells in G0/G1 phase and cell apoptotic rate compared with the blank, NC groups. We also found that the miR-495 inhibitor+siRNA-STAT3 group had reduced miR-495 expression. No significant differences were found in mRNA and protein expressions of STAT3, JAK and Claudin-1, cell proliferation, apoptosis and cycle amongst the miR-495 inhibitor+siRNA-STAT3 groups. Our study provides evidence that miR-495 improves the intestinal mucosal barrier function by targeting STAT3 through inhibiting the JAK/STAT3 signaling pathway in UC mice.