Aim: To determine the functional role of miR-490-5p in mast cell proliferation and apoptosis, and in the mast cell tryptase/PAR-2 signal pathway.
Methods: The 3rd generation of lentivirus vector systems containing enhanced green fluorescent protein (EGFP) (Ruisai Inc., Shanghai, China), which acts as a reporter gene was used to construct the mmu-miR-490-5p lentivirus expression vector pEGFP-antagomiR-490-5p, and the lentivirus vector pEGFP-negative was used as a negative control. The stably transfected mast cell line p815 was then constructed. GFP positive cells were successfully transfected cells. We determined the expression of miR-490-5p in p815 mast cells before and after transfection using quantitative real-time PCR (qRT-PCR). In addition, after transduction with the lentivirus vectors, the role of miR-490-5p in mast cell proliferation and apoptosis was investigated using the CCK-8 assay and flow cytometry, respectively. The mRNA levels of tryptase and PAR-2 were detected by qRT-PCR and the protein levels were detected by Western blot.
Results: The inhibition of miR-490-5p expression promoted apoptosis and inhibited proliferation of p815 mast cells. The mRNA levels of tryptase and PAR-2 were significantly increased after transfection compared with the control group, tryptase (P = 0.721, normal vs null; P = 0.001, siRNA vs normal; P = 0.002, siRNA vs null) and PAR-2 (P = 0.027, siRNA vs null; P = 0.353, normal vs null; P = 0.105, siRNA vs normal). The protein levels of tryptase and PAR2 were slightly higher in the siRNA group than those in the control group, but the difference was not statistically significant (P > 0.05).
Conclusion: miR-490-5p plays a vital role in the pathogenesis of irritable bowel syndrome by affecting mast cell proliferation and apoptosis; with down-regulation of miR-490-5p, the mRNA level of mast cell tryptase and PAR-2 increased, and the protein level increased, but the difference was not statistically significant.
Keywords: Irritable bowel syndrome; Mast cell tryptase; PAR-2; miR-490-5p.