Effect And Mechanisms Of Plant-derived MiRNAs(MIR156a-5p And MIR167e-5p) On Intestinal Development

As an essential organ for digestion and absorption,the intestine is the largest immune organ and playing a key role in maintaining animal health and growth.Mi RNA is an important negative regulatory factor in genes regulation during the processes of physiological and pathological both in plants and animals.Recent studies indicate that mi RNAs can regulate gene expression among different species.If plant mi RNA regulate intestinal development directely remains unclear.The present study aims to explore how plant mi RNA regulate the intestinal proliferation and its molecular mechanisms.In the first trail,maize in mice diet were replaced with maize starch,and seven maize highly expressed mi RNAs were detected by real-time PCR.The results showed that detected mi RNAs were significantly reducted in starch diet.21-day-old weaned mice were fed with starch diet and maize diet for 30 days respectively.The results showed that MIR156a-5p was detected in the intestine of mice by western blot,PCR,real-time PCR and in situ hybridization.At the same time,body weight,small intestinal weight,villus length and crypt depth ratio were recorded and the intestinal epithelial specific gene Cdx2 m RNA level were detected,which indicated the maize mi RNA cloud be relate to regulate intestinal proliferation.Bioinformatics analyses showed that the target genes of MIR156a-5p,MIR166a-3p MIR167e-5p and MIR159a-5p were enriched into the Wnt/?-catenin signaling pathway.Wnt/?-catenin pathway related genes further analysis showed the m RNAs and proteins expression of the upstream gene Wnt10 b,the key gene ?-catenin,the Wnt/?-catenin pathway target genes c-Myc and cyclin D1 were significantly down-regulated.All those results indicated that plant mi RNAs could inhibit the mice's intestinal proliferation by inhibit Wnt/?-catenin pathway's activity.In the second trial,to determine the proliferation function of plant mi RNAs on the intestine,different MIR156a-5p and MIR167e-5p mimics concentrations were transfected to IPEC-J2 cells.The cell counting showed that the 10 pmol significantly inhibit the cell numbers' increased,and the inhibitory effect was displayed the dose-dependent.Moreover,the inhibitory effect was most robust at 24 h.Ed U incorporation showed that the DNA replication ability was significantly inhibited,and the mimics treated groups weresignificantly down-regulate the PCNA m RNA's expression.These results indicate that MIR156a-5p and MIR167e-5p are capable to inhibit the proliferation of intestinal epithelial cells.In the third trail,we firstly constructed a dual luciferase reporter system to identify the MIR156a-5p and MIR167e-5p can target the 3'UTR of Wnt10 b and ?-catenin,respectively.Secondly,we design the in vitro experiment on IPEC-J2 cells to explore those two mi RNAs' regulated mechanism on intestinal epithelial cell proliferation.The in vitro results showed that the MIR156a-5p can significantly down-regulate the Wnt10 b,c-Myc and cyclin D1 expressions both in m RNA and protein levels,while the ?-catenin m RNA level and its phosphorylation at serine33 protein and the total protein levels were down-regulated.The c-Myc and cyclin D1 expressions can rescue by the MIR156a-5p inhibitor.For MIR167e-5p result showed that it not only inhibit the ?-catenin m RNA and protein'expression but also inhibit the downstream c-Myc and cyclin D1 genes expressions.And the inhibitor could also rescue the levels.These results indicate that MIR156a-5p and MIR167e-5p can inhibited the activity of the Wnt/?-catenin classical signaling pathway.In the fourth trail,to further determine the regulation of Wnt/?-catenin signaling pathway by plant-deried mi RNA.The IPEC-J2 cells were co-treated by MIR156a-5p and Li Cl activators of Wnt/?-catenin signaling pathway.The results showed that MIR156a-5p significantly inhibited the number of cells and the expression of PCNA m RNA,and the effect can deteriorate by Li Cl activators.Meanwhile,the Li Cl activators cloud also restore MIR156a-5p inhibition of ?-catenin protein and up-regulate the m RNA expression of cyclin D1.Those results were further confirmed the inhibition function of MIR156a-5p on Wnt/?-catenin signaling pathway.In the fifth trail,to determine the maize MIR156a-5p can be intestine absorbed by and play a regulatory role in vivo,mature single-stranded MIR156a-5p(300 pmol/d)was administrated to 21-day-old mice for 7 days.Northern blot and q PCR results showed the MIR156a-5p has a high expression in mice intestinal tract.Double-label fluorescence in situ hybridization assay showed that MIR156a-5p was enriched in the intestinal crypt and fluorescence intensity of the coincident position Wnt10 b was significantly decreased,indicating that MIR156a-5p can bind Wnt10 b m RNA to reduce its expression.The decreased Cdx2 expression level suggesting that MIR156a-5p can inhibit the intestinal epithelial cells proliferation.And the down-regulated Wnt10 b,?-catenin and cyclin D1 were indicated that the Wnt/?-catenin pathway activity was inhibited.Results above confirmed that MIR156a-5p could inhibit the activity of the Wnt/?-catenin pathway and theproliferation of intestinal epithelial cells in vivo.In summary,we confirmed that plant mi RNAs can be absorbed by the intestine and regulate intestinal development.It is also confirmed MIR156a-5p and MIR167e-5p could inhibit intestinal proliferation via Wnt/?-catenin pathway in vitro.Our results suggested dietary plant mi RNAs may serve as a new functional component of feed.