MicroRNA Expression Profile And TLRs Of HCT-8 Cells In The Early Phsae Of Cryptosporidium Parvum Subtype ?d Oocysts Infection

Cryptosporidium is one of the most important protozoan parasites infecting animals including humans and is the second microbe leading diarrhea in children.At present,there is no effective drugs and vaccines against cryptosporidiosis.The only FDA-approved nitazoxanide hsa a limited effect on cryptosporidiosis.miRNAs are a class of endogenous non-coding small RNA molecules that regulate the expression of genes,which in turn affect many biological processes,such as cell proliferation,differentiation,apoptosis,and organism development.Studies have shown that miRNAs can regulate more than 60% of mammalian protein encoding mRNAs and play an important role in the regulation of Cryptosporidium infection and host defense.TLRs?Toll-like receptor,TLRs?are a common pathogen recognition receptor that plays a very important role in pathogen invasion.Studies have shown that host cell TLRs play an important role in the identification of Cryptosporidium and the host's immune response to Cryptosporidium.The small intestine is the main parasitic site of Cryptosporidium parvum,but the effect of Cryptosporidium parvum subtype ?d on the miRNAs and TLRs of intestinal epithelial cells hsa not been reported.The aim of this study was to investigate micro RNA expression profile and TLRs of HCT-8 cells in the early phsae of Cryptosporidium parvum subtype ?d oocysts infection.Isolation and purification of Cryptosporidium parvum subtype ?d oocysts.In this study,oocysts of C.parvum??d subtype?were maintained in infected neonatal calves.We found the symptoms of diarrhea and Cryptosporidium parvum oocysts on the third day after infection.The results showed that the excrete peak of oocysts was the 5-9^th days post infection and the ovulation period was fifteen days.Cryptosporidium parvum subtype ?d oocysts were successfully isolated and purified by saturated saturated brine floatation method?saturated brine floatation method + cesium chloride density gradient centrifugation method?.We finally obtained a high purity of 3.5×10⁸ Cryptosporidium ?d subtype oocysts,76% of Cryptosporidium oocysts are active,can be used for the cell infection assays.Micro RNA expression profile of HCT-8 cells in the early phsae of Cryptosporidium parvum subtype ?d oocysts infection.In this study,miRNA microarray was used to analyze the effect of early stimulation?4 h and 12 h?of Cryptosporidium ?d subtype oocysts on HCT-8 cells.The results showed that there were 7 significant differences miRNAs at 4 h?1 up-regulation and 6 significant down-regulation?;13 differentially significant miRNAs at 12 h?3 up-regulated and 10 down-regulated?.hsa-miR-122-5p?4 h up-regulated?,hsa-miR-3591-3p?4 h down-regulated?,hsa-miR-454-5p?4 h down-regulated?,hsa-miR-181d-3p?12 h down-regulated?,hsa-let-7b-3p?12 h down-regulation?and hsa-miR-5580-3p?12 h up-regulation?.Six selected miRNAs and the housekeeping U6 mRNA were assayed with q PCR to confirm the expression profiles identified with the microarray analysis.The expression patterns determined with q PCR agreed well with those determined from the microarray data,which proves that the result of microarray analysis is reliable.Mi RNAs target gene prediction and GO classification and KEGG pathway analysis.The target genes of differentially expressed miRNAs were predicted by Targetscan human7.1 and miRDBR.Only 18 differentially expressed miRNAs could be predicted by Target Scan Human7.1 and miRDB.The results showed that the number of target genes ranged from 10 to 1034.The target gene was annotated by DAVID analysis method,the functional analysis of each miRNA target gene was analyzed by GO classification method.It was found that the miRNAs were involved in the biological process: the regulation of transcription process,cell adhesion,regulation of apoptosis,inflammation Response,natural immune response,adaptive immune response,calcium ion balance,potassium ion transport,regulatory protein phosphorylation / dephosphorylation,modulation of interferon synthesis and tyrosine kinase transmembrane signaling pathways,etc?P <0.05?;Cell components: Cell membrane,apical plasma membrane,mitochondrial membrane etc?P <0.05?;The molecular function were mainly composed of protein binding,ATP activation,RNA polymerase II transcription factor activation,tumor necrosis factor receptor activation,protein transport,Calcium ion channel activation,Serine kinase activation and Proteinase K to activation etc?P <0.05?.miR-34b-5p,miR-942-5p,miR-6721-5p,miR-3591-3p,miR-18b-3p,miR-18b-3p,miR-3976 and miR-3121-5p can regulate the process of apoptosis,let-7b-3p,miR-3591-3p,miR-34b-5p,miR-6721-5p and miR-3121-5p can regulate small intestinal epithelial cell immunity reaction.KEGG pathway analysis of miRNAs revealed significant differences miRNAs that modulate immune-related tumor necrosis factor signaling pathways,chemokine receptor interactions,regulatory signaling pathways and interferon signaling pathways;Apoptosis-related p53 signaling pathways,MAPK and Erb B signaling pathways;Cytoskeleton rearrangement of actin cytoskeleton and Wnt signaling pathways;cell adhesion molecules and calcium ion signaling pathways.Using the Cytoscape 3.4.0 to establish the miRNAs-target gene network,we found that hsa-miR-34b-5p and hsa-miR-3591-3p were the key miRNAs at 4 h,hsa-miR-942-5p,hsa-let-7b-3p and hsa-miR-6721-5p were the key miRNAs at 4 h.TLRs of HCT-8 cells in the early phsae of Cryptosporidium parvum subtype ?d oocysts infection and the relationship between miRNAs and TLRs by bioinformatics analysis.The expression of TLR1 TLR10 was analyzed by q PCR.The results showed that TLR1 TLR10 mRNA was expressed in HCT-8 cells,and TLR2 and TLR4 mRNA were significantly up-regulated at 4 h and 12 h after Cryptosporidium parvum infection.Down-regulated miR-34b-5p,miR-454-5p,miR-3591-3p,miR-4685-3p,miR-18b-3p,miR-3976,miR-1256,miR-1287-3p,miR-3121-5p,miR-6721-5p and let-7b-3p may increase the expression of IFN?,IFN?,IL-12,IL-1?,IL-6 and TNF? by up-regulating target genes in TLR signaling pathway.Up-regulated miR-942-5p may decrease the expression of IFN?,IFN?,IL-12,IL-1?,IL-6 and TNF? by down-regulating the expression of CHUK,MAPK10,PIK3 CD and TAB2.By analyzing the relationship between significant miRNA and TLR2 and TLR4 interacting proteins,it was found that Immune-related TRAF3 was the target gene of miR-6721-5p,and the DNA binding protein HMGB1 was the target gene of let-7b-3p.In summary,the expression of miRNAs in small intestinal epithelial cells were changed by Cryptosporidium parvum subtype ?d oocysts infection;these significantly altered miRNAs may play an important biological role in the early stages of Cryptosporidium parvum infection and affect the early propagation of Cryptosporidium parvum in the host cells;Up-regulated TLR2 and TLR4 may be related to the identification and immune response of the host cell to Cryptosporidium parvum;significant differences miRNAs may play an important role in immune regulation in TLR signaling pathway.