The Signaling Pathway Of MiR-942 Up-regulation Induced By Cryptosporidium Parvum In HCT-8 Cells And Identification Of MiR-942 Target Genes

Cryptosporidium is one of the main causes of diarrhea in humans and livestock.Cryptosporidium infections in normal immune individuals can cause diarrhea for up to three weeks,but can cause life-threatening wasting and malnutrition in immunocompromised individuals;Cryptosporidium infections can lead to decreased animal performance and even death.Studies have shown that miRNAs play an important role in post-transcriptional regulation of innate immune responses against Cryptosporidium infection.The previous research of the research group showed that the expression of miR-942 significantly increased at 12 h after Cryptosporidium parvum infection in HCT-8 cells.It is speculated that miR-942 is involved in the early antiapoptotic process of cells.Previously,the research team used q PCR,Western blotting and other technical methods to verify the upregulation of miR-942 expression induced by Cryptosporidium parvum through TLR2/TLR4-NF-?B signaling pathway.In this study,siRNA technology was used to further verify that Cryptosporidium parvum induced miR-942 expression through this signaling pathway.At the same time,the target gene of miR-942 in HCT-8 cells was identified.In this study,three TLR2 and TLR4 siRNA were designed,and the siRNA with the strongest inhibitory effect was selected through cell transfection.The sequences used are TLR2: GGA AGA UAA UGA ACA CCA ATT(sense)and UUG GUG UUC AUU AUC UUC CTT(antisense);TLR4: CCA GGU GCA UUU AAA GAA ATT(sense)and UUG GUG UUC AUU AUC UUC CTT(antisense).Transfect the selected siRNA to specifically inhibit the expression of TLR2 and TLR4 in HCT-8 cells.After infection with Cryptosporidium parvum at 0h,4h,8h,12 h,24h and 48 h,extract the total RNA and then use q PCR and other methods.To study the effect of TLRs on miR-942 expression.The results showed that miR-942 expression was significantly upregulated at 4 and 8 h after Cryptosporidium infection(P<0.001).After using TLR2 siRNA and TLR4 siRNA to silence the expression of TLR2 and TLR4,the upregulation of miR-942 expression was significantly suppressed at the corresponding time compared with the control group(P<0.001).These results indicate that HCT-8 cells initiate the up-regulation of miR-942 gene induced by Cryptosporidium parvum through TLR2 and TLR4.By using bioinformatics software to predict the potential target genes of miR-942,we combined whether the target protein is expressed in mesenchymal cells and the possible biological functions of the target protein.We found that interferon alphainducible protein 27 in the target protein of miR-942 is related to apoptosis.This study designed and synthesized a sequence containing IFI27-3'UTR that predicted a complementary binding site to miR-942 and a mutation at the binding site.We cloned two synthetic genes into a dual luciferase reporter vector.The transfection reagent was used to co-transfect the miR-942 mimics and the constructed dual luciferase reporter vector into HCT-8 cells,and the dual luciferase detection system was used to detect the fluorescent activity.The results showed that the size and orientation of the inserted sequences of the constructed wild-type and mutant double-luciferase reporter vectors were correctly identified.In the experimental group transfected with an empty double luciferase reporter vector and a reporter site containing a mutation site,the fluorescent activity test results showed that there was no significant change in luciferase activity in the miR-942 group and the negative control group.By over-expression and decreasing the expression of miR-942,it was found that over-expression of miR-942 can reduce the protein level of interferon alpha-induced protein 27.Decreasing the expression level of miR-942,the protein level of interferon alpha-inducing protein 27 will increase,but neither overexpression nor decreasing the expression level of miR-942 will affect the interferon alpha-inducing protein 27 m RNA.In summary,after Cryptosporidium parvum infection of cells,cells can up-regulate the expression of miR-942 through TLR2 and TLR4.Interferon alpha-inducing protein 27 is a target gene of miR-942 and miR-942 binds to the 3'-UTR region of interferon alpha-inducible protein 27 m RNA.miR-942 inhibits the expression of IFI27 protein by non-degrading m RNA at the post-transcriptional level.