MicroRNA-15a - cell division cycle 42 signaling pathway in pathogenesis of pediatric inflammatory bowel disease

Figure 1 Cell division cycle 42 is a microRNA-15a target gene in 293T cells. A: Bioinformatics analysis using Target Scan (http://www.targetscan.org/) revealed the putative seed region of miR-15a in the wild-type (WT) 3'-UTR of Cdc42. The putative binding sites of miR-15a on WT Cdc42 3’-UTR is highlighted. A MUT Cdc42 3’-UTR sequence was generated for luciferase assays; B: In a dual-luciferase reporter assay, 293T cells were transfected with firefly luciferase reporter inserted with WT Cdc42 3’-UTR plasmid (Cdc42-WT) or MUT Cdc42 3’-UTR Plasmid (Cdc42-MUT). Cells were co-transfected with either miR-15a mimics Plasmid (miR-15a) or its negative control plasmid (miR-NC). At 48 h after transfection, relative luciferase activities were evaluated and normalized to the values in cells transfected with Cdc42-MUT (^bP < 0.01 vs Cdc42-MUT). Cdc42: Cell division cycle 42; MUT: Mutation; WT: Wildtype; miR: MicroRNA; NC: Negative control.

Figure 2 Tumor necrosis factor-α induced microRNA-15a upregulation and cell division cycle 42 downregulation in Caco-2 cells. Caco-2 cells were stimulated with tumor necrosis factor-α (TNF-α) (35 ng/mL) (TNF-α group) or medium with no TNF-α (control) for 60 h. Quantitative reverse transcription polymerase chain reaction (commonly known as qRT-PCR) was used to evaluate the relative mRNA expression of miR-15a in A: the control and TNF-α groups; B: qRT-PCR was used to evaluate the relative mRNA expression Cdc42 in the control and TNF-α groups (^aP < 0.05 vs control). Cdc42: Cell division cycle 42; miR: MicroRNA; TNF-α: Tumor necrosis factor-α.

Figure 3 MicroRNA-15a negatively regulates cell division cycle 42 expression in Caco-2 cells. Caco-2 cells were transfected with microRNA-15a (miR-15a) mimic lentivirus and its negative controls or miR-15a inhibitor lentivirus and its negative controls. A: Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to evaluate the relative mRNA expression of miR-15a in different groups; B: qRT-PCR was used to evaluate the relative mRNA expression of cell division cycle 42 in different groups (^aP < 0.05, ^bP < 0.01 vs negative control cells). Cdc42: Cell division cycle 42; miR: MicroRNA; LV: Lentivirus; NC: Negative control.

Figure 4 MicroRNA-15a negatively regulates epithelial junctions through cell division cycle 42 in Caco-2 cells. Caco-2 cells were stimulated by Tumor necrosis factor-α (TNF-α) (35 ng/mL) [wild type (WT) + TNF-α group] or medium with no TNF-α (WT). Caco-2 cells were transfected with miR-15a mimic lentivirus or its negative controls (miR-NC group). Caco-2 cells were transfected with microRNA-15a (miR-15a) inhibitor lentivirus, and after passage, were stimulated by TNF-α (35 ng/mL) (miR-15a inhibitor + TNF-α). Caco-2 cells were transfected with Cdc42 lentivirus (Lv-Cdc42) or its negative control (Lv-Cdc42-NC), and after passage, stimulated by TNF-α (35 ng/mL) (Lv-Cdc42 +TNF-α or Lv-Cdc42nc + TNF-α group). A: Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to evaluate the relative mRNA expression of E-cadherin (E-Cad) in every group; B: Expression of epithelial marker E-cad was examined by immunofluorescence in every group. Cells were stained with E-cad antibodies and counter stained with DAPI; C: qRT-PCR was used to evaluate the relative mRNA expression of ZO-1 in every group; D: Expression of epithelial marker ZO-1 was examined by immunofluorescence in every group. Cells were stained with E-cad antibodies and counterstained with DAPI (^aP < 0.05, ^bP < 0.01 vs WT, ^cP < 0.05, ^dP < 0.01 vs miR-15a mimic, ^eP < 0.05 vs Lv-Cdc42-NC +TNF-α) (magnification 40 ×, bar = 25 μm). Cdc42: Cell division cycle 42; WT: Wildtype; miR: MicroRNA; NC: Negative control; TNF-α: Tumor necrosis factor-α; LV: Lentiviral; NC: Negative control; qRT-PCR: Quantitative reverse transcription polymerase chain reaction; ZO-1: Zona occludens-1.

Figure 5 MicroRNA-15a negatively regulates cell division cycle 42 in pediatric inflammatory bowel disease patients. A: Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to evaluate the relative mRNA expression of MicroRNA-15a (miR-15a) in every group; (B) qRT-PCR was used to evaluate the relative mRNA expression of cell division cycle 42 (Cdc42) in every group. C: Pearson r test was used to test the correlation of miR-15a and Pediatric Crohn’s disease Activity Index (PCDAI); D: Pearson r test was used to test the correlation between Cdc42 and PCDAI; E: Pearson r test was used to test the correlation between miR-15a and Cdc42 [^aP < 0.05, ^bP < 0.01 vs control, ^cP < 0.05 vs Crohn’s disease active group]. miR: MicroRNA; Cdc42: Cell division cycle 42; qRT-PCR: Quantitative reverse transcription polymerase chain reaction; CD AC: Crohn’s disease active; CD RE: Crohn’s disease remission; UC: Ulcerative colitis; PCDAI: Pediatric Crohn’s Disease Activity Index.