| The intestinal mucosal epithelial barrier is an important protective barrier of the body,and its integrity can effectively prevent the invasion of harmful substances and antigens in the intestinal lumen.Related research shows that intestinal mucosal damage weakens self-repairing ability under stress conditions such as severe infection,trauma,shock and major surgery,result in destruction of intestinal mucosal epithelial integrity.Recent researches have emphasized the critical role of molecular signaling in mucosal repair and barrier function reconstruction after intestinal mucosal epithelium injury.The intestinal mucosa has developed a self-repairing mechanism against the damage,including rapid repair mechanism and slow repair mechanism.After injury,the intestinal mucosal repair mechanisms are rapidly activated to avoid the invasion of bacteria and endotoxin.Compared with cell proliferation,cell migration is relatively fast.First,epithelial cells surrounding the wound lose their columnar polarity,and rapidly migrate into the lesion area to restore barrier integrity within minutes to hours.Subsequently,the intestinal epithelium initiates slow repair mechanism.Intestinal stem cells proliferate and differentiate,and migrate upward through migration to make up for the reduced number of cells.Cell migration involves the regulation of a series of transcriptional and post-transcriptional gene modifications,in which miRNA plays a crucial role.However,the role and mechanism of miRNAs involved in intestinal epithelial cell migration is not completely clear.Studies have shown that licorice,a traditional Chinese herb for strengthening spleen and qi,can accelerate the repair of mucosal damage by promoting the proliferation,differentiation and migration of intestinal epithelial cells.Studies have shown that glycyrrhizic acid,the active ingredient in licorice,can reduce intestinal mucosal permeability,reduce inflammation and promote intestinal mucosal barrier damage repair.The purpose of this study was to observe the effect and mechanism of miR-30c-2-3p on the intestinal epithelial migration,meanwhile,the effects of glycyrrhizic acid on intestinal epithelial cell migration and related signaling pathways were studied.ObjectiveTo observe the effect of miR-30c-2-3p on IEC-6 cells proliferation and migration.Confirming the targeting between miR-30c-2-3p and STIM2.To investigate the effect and mechanism of miR-30c-2-3p on intestinal epithelial cell migration and related signaling pathways,and the role of glycyrrhizic acid on PYK2/MYLK/MLC2 signaling pathway is observed,so as to illuminate the mechanism of miR-30c-2-3p and Glycyrrhizic acid on repairing intestinal mucosal injury,and to provide experimental basis for clinical applications.Methods1.After transfecting miR-30c-2-3p mimic and inhibitor in IEC-6 cells for 48h,cell proliferation was measured by MTT and EdU method,and the cell migration ability was measured by wound healing assay.2.The target genes of miR-30c-2-3p were predicted and analyzed by bioinformatics prediction software,and to detect the targeting of miR-30c-2-3p and STIM2 using Biotinylated-miRNA pull down method and luciferase reporter assay.The expression of STIM2 protein and mRNA were detected by RT-PCR and western blot after overexpression or knockdown of miR-30c-2-3p in IEC-6 cells,so as to verify that miR-30c-2-3p can target STIM2.3.After transfecting miR-30c-2-3p mimic and inhibitor in IEC-6 cells for 48h,the intracellular calcium concentration was detected by calcium fluorescence probe Fura-2 AM,the protein expression of MYLK?P-MLC2 and MLC2 was measured by western blot.4.Effect of miR-30c-2-3p on FITC-labeled phalloidin labeled F-actin by cellular immunofluorescence.5.To verify that miR-30c-2-3p promotes the migration of intestinal epithelial cells by targeting STIM2 by silencing STIM2 with siRNA and transfecting with overexpression plasmid STIM2.6.To measure the effect of glycyrrhizic acid on migration of intestinal epithelial cells by wound healing assay.After treating intestinal epithelial cells with glycyrrhizic acid at 50 ?g/mL for 48h,the intracellular calcium concentration was detected by calcium fluorescence probe Fura-2 AM,the protein expression of PYK2?MYLK?P-MLC2 and MLC2 was detected by western blot.Results1.miR-30c-2-3p mimic and inhibitor were transfected into IEC-6 cells,there was no obvious impact of miR-30c-2-3p on cell proliferation but it could significantly promote cell migration,transfection with miR-30c-2-3p mimic for 48h resulted in promoting the migration of TEC-6 cells when compared with miR-30c-2-3p mimic NC(P<0.05),conversely,transfection with the inhibitor showed opposite results.The differences were statistically significant(P<0.05).2.Bioinformatics analysis suggested STIM2 was a target of miR-30c-2-3p.Pull down assay showed that the mRNA binding rate of miR-30c-2-3p mimic group with STIM2 was significantly higher than the control group(P<0.05).Compared with the group of miR-30c-2-3p mimic NC co-transfected with pEZ-STIM2-W,the relative luciferase activity in group of miR-30c-2-3p mimic co-transfected with pEZ-STIM2-W showed a significant decrease(P<0.05),no difference was found in group of miR-30c-2-3p mimic co-transfected with pEZ-STIM2-M.The luciferase reporter assay confirmed that miR-30c-2-3p directly combined with the 3' UTR of STIM2 mRNA in IEC-6 cells.Real-Time PCR and Western blot analysis showed that STIM2 was decreased in cells with overexpressed miR-30c-2-3p.On the contrary,down regulation of miR-30c-2-3p showed opposite results.It was confirmed that miR-30c-2-3p could target STIM2.3.miR-30c-2-3p increased the intracellular calcium concentration,and up-regulated the protein expression of MYLK?p-MLC2 and MLC2,making the cells stretch due to changes in the cytoskeleton.4.SiRNA silencing of STIM2 expression in IEC-6 increased the intracellular calcium concentration,and up-regulated the protein expression of MYLK?p-MLC2 and MLC2,reduing intestinal epithelial cell migration.Transfecting with overexpression plasmid STIM2 decreased the intracellular calcium concentration,and down-regulated the protein expression of MYLK?p-MLC2 and MLC2,to reverse the effect of miR-30c-2-3p on intestinal epithelial cell migration.5.After treating intestinal epithelial cells with glycyrrhizic acid at 50 ?g/mL for 48h,Glycyrrhizic acid can promote the migration of IEC-6 cells,activate PYK2,promote Ca2+ influx,and increase the protein expression of MYLK?p-MLC2 and MLC2.ConclusionIn this study,miR-30c-2-3p can target the expression of STIM2 in intestinal epithelial cells,increase intracellular calcium concentration and the protein expression of MYLK?p-MLC2 and MLC2,and increase the migration of IEC-6 cells.Glycyrrhizic acid can activate PYK2/MYLK/MLC2 signaling pathway to promote the migration of IEC-6 cells. |
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