AIM To determine the functional role of mi R-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 enhanc...AIM To determine the functional role of mi R-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-mi R-490-5p lentivirus expression vector p EGFP-antagomi R-490-5p,and the lentivirus vector p EGFP-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 mi R-490-5p in p815 mast cells before and after transfection using quantitative real-time PCR(q RT-PCR).In addition,after transduction with the lentivirus vectors,the role of mi R-490-5p in mast cell proliferation and apoptosis was investigated using the CCK-8 assay and flow cytometry,respectively.The m RNA levels of tryptase and PAR-2 were detected by q RT-PCR and the protein levels were detected by Western blot.RESULTS The inhibition of mi R-490-5p expression promoted apoptosis and inhibited proliferation of p815 mast cells.The m RNA levels of tryptase and PAR-2 were significantly increased after transfection comparedwith the control group,tryptase(P=0.721,normal vs null;P=0.001,si RNA vs normal;P=0.002,si RNA vs null)and PAR-2(P=0.027,si RNA vs null;P=0.353,normal vs null;P=0.105,si RNA vs normal).The protein levels of tryptase and PAR2 were slightly higher in the si RNA group than those in the control group,but the difference was not statistically significant(P>0.05).CONCLUSION mi R-490-5p plays a vital role in the pathogenesis of irritable bowel syndrome by affecting mast cell proliferation and apoptosis;with down-regulation of mi R-490-5p,the m RNA level of mast cell tryptase and PAR-2 increased,and the protein level increased,but the difference was not statistically significant.展开更多
基金the National Natural Science Foundation of China,No.81160053(to Zhang FC)
文摘AIM To determine the functional role of mi R-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-mi R-490-5p lentivirus expression vector p EGFP-antagomi R-490-5p,and the lentivirus vector p EGFP-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 mi R-490-5p in p815 mast cells before and after transfection using quantitative real-time PCR(q RT-PCR).In addition,after transduction with the lentivirus vectors,the role of mi R-490-5p in mast cell proliferation and apoptosis was investigated using the CCK-8 assay and flow cytometry,respectively.The m RNA levels of tryptase and PAR-2 were detected by q RT-PCR and the protein levels were detected by Western blot.RESULTS The inhibition of mi R-490-5p expression promoted apoptosis and inhibited proliferation of p815 mast cells.The m RNA levels of tryptase and PAR-2 were significantly increased after transfection comparedwith the control group,tryptase(P=0.721,normal vs null;P=0.001,si RNA vs normal;P=0.002,si RNA vs null)and PAR-2(P=0.027,si RNA vs null;P=0.353,normal vs null;P=0.105,si RNA vs normal).The protein levels of tryptase and PAR2 were slightly higher in the si RNA group than those in the control group,but the difference was not statistically significant(P>0.05).CONCLUSION mi R-490-5p plays a vital role in the pathogenesis of irritable bowel syndrome by affecting mast cell proliferation and apoptosis;with down-regulation of mi R-490-5p,the m RNA level of mast cell tryptase and PAR-2 increased,and the protein level increased,but the difference was not statistically significant.
