Research On The Anti-apoptotic Pathway Of MiR-942 Target Gene IFI27 By Cryptosporidium Parvum Infection In HCT-8 Cells

Cryptosporidiosis caused by Cryptosporidium is one of the main causes of diarrhea in humans and livestocks.Cryptosporidium infection can cause up to three weeks of diarrhea in immune-normal individuals,while it can cause life-threatening weight loss and malnutrition in immunocompromised individuals.At present,little is known about the invasion mechanism of Cryptosporidium,the pathogenic mechanism and the immune regulation mechanism of the host against Cryptosporidium infection at domestic and foreign,which severely restricts the prevention and control of Cryptosporidium.miRNAs are a type of endogenous regulatory RNAs that regulate more than 60%of mammalian protein-encoded mRNAs after initiation of transcription,and are factors that regulate post-transcriptional gene expression.Studies have shown that miRNAs play an important role in the post-transcriptional regulation of host cells’ innate immune response against Cryptosporidium infection,including a series of biological processes such as regulation of cell signaling pathways,release of cytokines,and inflammatory responses.The previous experimental group studied the expression profile of related miRNAs after Cryptosporidium infection in HCT-8 cells and found that the genes that were significantly up-regulated and down-regulated at 4 hours after infection were 1 and 6,and the genes that were significantly up-regulated and down-regulated at 12 hours after infection were 3 and 10.Among them,Hsa-miR-942-5p was significantly up-regulated after Cryptosporidium parvum infection,and bioinformatics analysis speculated that it can involve in the apoptosis process of HCT-8 cells.In the early stage,our experimental group had carried out the mutual verification of miRNA-942 and the target gene interferon alpha inducible protein 27(IFI27).This project focuses on the study of the role and pathway of miRNA-942 target gene encoding protein IFI27 in regulating HCT-8 cell apoptosis,and provides a new strategy for the design and development of anti-cryptosporidiosis drug targets.By qPCR and Western Blotting methods,the effects of Cryptosporidium parvum on the expression of IFI27 after infection of HCT-8 cells were detected.The results showed that after Cryptosporidium infection HCT-8 cells,compared with blank cells,the expression of IFI27 did not change significantly at the mRNA level(P>0.05),and the protein level decreased in the early stage and increased in the later stage.Constructing IFI27 overexpression vector,synthesizing and screening siRNA that specifically interferes with IFI27.Using transfection reagent Lipofectamine3000 to transfect IFI27 overexpression vector,negative control empty vector,siRNA and negative control si-NC into HCT-8 cells,respectively.24 hours after transfection,the sporozoites of Cryptosporidium parvum were added,HCT-8 cells were stained with Annexin V and PI,and apoptosis was detected by flow cytometry;the worm count of each group of cells was detected by fluorescence quantitative PCR.The results showed that the percentage of apoptosis in the overexpression vector group was significantly higher than that in the empty vector group(P<0.01),while the percentage of apoptosis in the siRNA group was significantly lower than that in the NC group(P<0.01);there was no significant difference between the test group and the control group at 2 h after infection(P>0.05);compared with the empty vector group at 24 h after infection,the overexpression vector group had a lower cell load of insects(P<0.05),while the cell load of the siRNA group Higher than the NC group(P<0.05).In summary,the miR-942 target gene encoding protein IFI27 has no effect on the invasion of Cryptosporidium parvum into HCT-8 cells.After the invasion,IFI27 affects the development of Cryptosporidium parvum in HCT-8 cells by affecting cell apoptosis..The expression levels of Apaf-1,FasL,TRAIL,Caspase-3,Caspase-8 and Caspase-9 were detected by qPCR at different time points in the inactivated group and the infected group,and the expression of each molecule was differentially expressed(P<0.05),indicating that the microcryptography After spore worms infect HCT-8 cells,they can cause cell apoptosis through both endogenous and exogenous apoptotic pathways.Using transfection reagent Lipofectamine3000 to transfect IFI27 overexpression vector,negative control empty vector,siRNA and negative control si-NC into HCT-8 cells respectively,and detect the relative expression of TRAIL molecules of each infected cells at different time points.Compared with the control empty vector group,the expression of IFI27 overexpression vector increased significantly(P<0.05).On the contrary,the expression of the si-IFI27 group decreased compared with the control si-NC group(P<0.05).For further verification,the apoptotic proportions of the si-NC group,si-NC+rTRAIL group and si-IFI27+rTRAIL group were detected,and it was found that the apoptotic proportions of the si-NC group and si-IFI27+rTRAIL group were significantly lower than that of si-NC+rTRAIL group(P<0.001),and with the increase of rTRAIL concentration,the proportion of cell apoptosis also increased(P<0.05),further indicating that IFI27 affects cell apoptosis by regulating TRAIL.To sum up,after infection with Cryptosporidium parvum,hsa-miR-942-5p targeted to regulate the expression of its target gene encoding protein IFI27,and then affect cell apoptosis through the extrinsic TRAIL pathway.Hsa-miR-942-5p target gene encoding protein IFI27 plays a role in the endogenous development of Cryptosporidium parvum after invading host cells,that is,down-regulate the expression of IFI27 in the early stage of infection,inhibit cell apoptosis and promote the development of Cryptosporidium parvum;In the late stage of infection,up-regulate the expression of IFI27,promote cell apoptosis and inhibit the development of Cryptosporidium parvum.