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MicroRNA-15a - cell division cycle 42 signaling pathway in pathogenesis of pediatric inflammatory bowel disease
Wen-Juan Tang1, Kai-Yue Peng1, Zi-Fei Tang1
1Department of Gastroenterology, Children's Hospital of Fudan University, Shanghai 201102, China.
Aim:
To determine whether cell division cycle (Cdc)42 is regulated by microRNA (miR)-15a in the development of pediatric inflammatory bowel disease (IBD).
Methods:
We cultured 293T cells, used plasmids and performed dual-luciferase assay to determine whether Cdc42 is a miR-15a 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-15a and Cdc42. We performed quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunofluorescence to determine whether Cdc42 is regulated by miR-15a in Caco-2 cells. Finally, we collected ileocecal tissue by endoscopy from patients and performed qRT-PCR to examine the expression of miR-15a and Cdc42 in pediatric IBD patients.
Results:
Target Scan and dual-luciferase assay revealed that Cdc42 was a miR-15a target gene. MiR-15a 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-15a-mimic cells (P < 0.001) and increased in miR-15a inhibitor cells (P < 0.05). ZO-1 and E-cadherin decreased in miR-15a-mimic cells (P < 0.001) but not in the miR-15a 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-15a-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-15a increased and Cdc42 decreased in the CD AC group compared to the control group (P < 0.05). miR-15a decreased and Cdc42 increased in the CD RE group compared to the CD AC group (P < 0.05). miR-15a 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-15a expression negatively correlated with Cdc42 in pediatric IBD patients (P = 0.0045).
Conclusion:
MiR-15a negatively regulates epithelial junctions through Cdc42 in Caco-2 cells and pediatric IBD patients.
Insights
MicroRNA (miR)-15a regulates cell division cycle (Cdc)42, impacting epithelial junctions in pediatric inflammatory bowel disease (IBD). This study reveals miR-15a
Area of Science:
- Gastroenterology
- Molecular Biology
- Pediatric Inflammatory Bowel Disease (IBD) Research
Background:
- Pediatric inflammatory bowel disease (IBD) involves complex molecular dysregulation.
- MicroRNAs (miRNAs) play critical roles in cellular processes and disease pathogenesis.
- The specific role of miR-15a in IBD development requires further elucidation.
Purpose of the Study:
- To investigate the regulatory relationship between miR-15a and cell division cycle (Cdc)42.
- To determine the impact of miR-15a and Cdc42 on epithelial junction integrity in IBD.
- To examine the expression patterns of miR-15a and Cdc42 in pediatric IBD patients.
Main Methods:
- Dual-luciferase assays and TargetScan to identify miR-15a targets.
- In vitro studies using Caco-2 cells with manipulated miR-15a and Cdc42 expression.
- Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunofluorescence for gene and protein expression analysis.
- Endoscopic tissue collection from pediatric IBD patients for expression profiling.
Main Results:
- Cdc42 was confirmed as a direct target of miR-15a.
- Tumor necrosis factor-α (TNF-α) stimulation increased miR-15a and decreased Cdc42, ZO-1, and E-cadherin expression.
- miR-15a mimicry reduced Cdc42, ZO-1, and E-cadherin, while miR-15a inhibition partially restored them.
- In pediatric IBD patients, miR-15a was upregulated and Cdc42 downregulated, correlating with disease activity.
Conclusions:
- miR-15a negatively regulates epithelial junction proteins, including ZO-1 and E-cadherin, via Cdc42.
- The miR-15a/Cdc42 axis is implicated in the pathogenesis of pediatric IBD.
- Targeting the miR-15a/Cdc42 pathway may offer therapeutic potential for pediatric IBD.
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