microRNAs (miRNAs) can play a role in a variety of physiological and pathological regulation, and its role is achieved by regulating the expression of target genes. Our previous high-throughput sequencing found that ssc-miR-185 plays an important regulatory role in piglet diarrhea, but its specific target genes and functions in intestinal porcine epithelial cell (IPEC-J2) are still unclear. We intended to verify the target relationship between porcine miR-185 and cell division cycle 42 (CDC42) gene in IPEC-J2, and explored the effect of miR-185 on the proliferation of IPEC-J2 cells.
The TargetScan, miRDB and miRanda softwares were used to predict the target genes of porcine miR-185, and CDC42 was selected as a candidate target gene. The CDC42-3’UTR-wild type (WT) and CDC42-3’UTR-mutant type (MUT) segments were successfully cloned into pmirGLO luciferase vector, and the luciferase activity was detected after co-transfection with miR-185 mimics and CDC42-3’UTR. The expression level of CDC42 was analyzed using qPCR and Western blot. The proliferation of IPEC-J2 was detected using CCK-8, MTT and EdU assays.
Double enzyme digestion and sequencing confirmed that CDC42-3’UTR-WT and CDC42-3’UTR-MUT were successfully cloned into pmirGLO luciferase reporter vector, and the luciferase activity was significantly reduced after co-transfection with miR-185 mimics and CDC42-3’UTR (WT). Further we found that the mRNA and protein expression level of CDC42 were down-regulated after transfection with miR-185 mimics, while the opposite trend was observed after transfection with miR-185 inhibitor (p < 0.01). In addition, the CCK-8, MTT and EdU results demonstrated that miR-185 promotes IPEC-J2 cells proliferation by targeting CDC42.
These findings indicate that porcine miR-185 can directly target CDC42 and promote the proliferation of IPEC-J2 cells. However, the detailed regulatory mechanism of miR-185/CDC42 axis in piglets resistance to diarrhea is yet to be further investigation.