AIM To determine whether cell division cycle(Cdc)42 is regulated by microRNA(miR)-15 a in the development of pediatric inflammatory bowel disease(IBD).METHODS We cultured 293 T cells, used plasmids and performed dual-...AIM To determine whether cell division cycle(Cdc)42 is regulated by microRNA(miR)-15 a in the development of pediatric inflammatory bowel disease(IBD).METHODS We cultured 293 T cells, used plasmids and performed dual-luciferase assay to determine whether Cdc42 is a miR-15 a target gene. We cultured Caco-2 cells, and stimulated them with tumor necrosis factor(TNF)-α. We then employed lentiviruses to alter the expression of miR-15 a and Cdc42. We performed quantitative reverse transcription polymerase chain reaction(qRTPCR) and immunofluorescence to determine whether Cdc42 is regulated by miR-15 a in Caco-2 cells. Finally, we collected ileocecal tissue by endoscopy from patients and performed qRT-PCR to examine the expression of miR-15 a and Cdc42 in pediatric IBD patients.RESULTS Target Scan and dual-luciferase assay revealed thatCdc42 was a miR-15 a target gene. MiR-15 a expression increased(P = 0.0038) and Cdc42 expression decreased(P = 0.0013) in cells stimulated with TNF-α, and the expression of the epithelial junction proteins zona occludens(ZO)-1(P < 0.05) and E-cadherin(P < 0.001) decreased. Cdc42 levels decreased in miR-15 a-mimic cells(P < 0.001) and increased in miR-15 a inhibitor cells(P < 0.05). ZO-1 and E-cadherin decreased in mi R-15 a-mimic cells(P < 0.001) but not in the miR-15 a inhibitor + TNF-α cells. In Lv-Cdc42 + TNF-α cells, ZO-1 and E-cadherin expression increased compared to the Lv-Cdc42-NC + TNF-α(P < 0.05) or miR-15 a-mimic cells(P < 0.05). Fifty-four pediatric IBD patients were included in this study, 21 in the control group, 19 in the Crohn's disease(CD) active(AC) group, seven in the CD remission(RE) group, and seven in the ulcerative colitis(UC) group. MiR-15 a increased and Cdc42 decreased in the CD AC group compared to the control group(P < 0.05). miR-15 a decreased and Cdc42 increased in the CD RE group compared to the CD AC group(P < 0.05). miR-15 a was positively correlated with the Pediatric Crohn's disease Activity Index(PCDAI)(P = 0.006), while Cdc42 was negatively correlated with PCDAI(P = 0.0008). Finally, miR-15 a expression negatively correlated with Cdc42 in pediatric IBD patients(P = 0.0045).CONCLUSION Mi R-15 a negatively regulates epithelial junctions through Cdc42 in Caco-2 cells and pediatric IBD patients.展开更多
基金Supported by the National Natural Science Foundation of Shanghai,No.201540068
文摘AIM To determine whether cell division cycle(Cdc)42 is regulated by microRNA(miR)-15 a in the development of pediatric inflammatory bowel disease(IBD).METHODS We cultured 293 T cells, used plasmids and performed dual-luciferase assay to determine whether Cdc42 is a miR-15 a target gene. We cultured Caco-2 cells, and stimulated them with tumor necrosis factor(TNF)-α. We then employed lentiviruses to alter the expression of miR-15 a and Cdc42. We performed quantitative reverse transcription polymerase chain reaction(qRTPCR) and immunofluorescence to determine whether Cdc42 is regulated by miR-15 a in Caco-2 cells. Finally, we collected ileocecal tissue by endoscopy from patients and performed qRT-PCR to examine the expression of miR-15 a and Cdc42 in pediatric IBD patients.RESULTS Target Scan and dual-luciferase assay revealed thatCdc42 was a miR-15 a target gene. MiR-15 a expression increased(P = 0.0038) and Cdc42 expression decreased(P = 0.0013) in cells stimulated with TNF-α, and the expression of the epithelial junction proteins zona occludens(ZO)-1(P < 0.05) and E-cadherin(P < 0.001) decreased. Cdc42 levels decreased in miR-15 a-mimic cells(P < 0.001) and increased in miR-15 a inhibitor cells(P < 0.05). ZO-1 and E-cadherin decreased in mi R-15 a-mimic cells(P < 0.001) but not in the miR-15 a inhibitor + TNF-α cells. In Lv-Cdc42 + TNF-α cells, ZO-1 and E-cadherin expression increased compared to the Lv-Cdc42-NC + TNF-α(P < 0.05) or miR-15 a-mimic cells(P < 0.05). Fifty-four pediatric IBD patients were included in this study, 21 in the control group, 19 in the Crohn's disease(CD) active(AC) group, seven in the CD remission(RE) group, and seven in the ulcerative colitis(UC) group. MiR-15 a increased and Cdc42 decreased in the CD AC group compared to the control group(P < 0.05). miR-15 a decreased and Cdc42 increased in the CD RE group compared to the CD AC group(P < 0.05). miR-15 a was positively correlated with the Pediatric Crohn's disease Activity Index(PCDAI)(P = 0.006), while Cdc42 was negatively correlated with PCDAI(P = 0.0008). Finally, miR-15 a expression negatively correlated with Cdc42 in pediatric IBD patients(P = 0.0045).CONCLUSION Mi R-15 a negatively regulates epithelial junctions through Cdc42 in Caco-2 cells and pediatric IBD patients.