Research On The Hsa-miR-181d Regulation Pathway And Target Gene Of HCT-8 Cells Infected With Cryptosporidium Parvum Ⅱd Subtype

Cryptosporidium is an important parasitic protozoan that can infect more than 260 animals such as humans,cattle,breeds,pigs,cats,dogs,etc.,and can cause self-limiting diarrhea and even death.There are currently no effective drugs and vaccines for Cryptosporidium control.Up to now,41 Cryptosporidium effective species and more than 70 genotypes have been identified.Among more than 20 species infected humans,Cryptosporidium has significant importance because of the wide range of parasitic hosts.In addition,it has obvious zoonotic significance.In China,the main subtype of Cryptosporidium is the Ⅱd subtype.Studies have reported that Cryptosporidium is the second largest diarrhea pathogen after rotavirus and poses a major threat to human public safety.After Cryptosporidium parvum infects the host epithelial cells,the interaction between the host epithelial cells and Cryptosporidium parvum can be accomplished by changing its miRNAs.Previous experiments have shown that:the expression levels of TLR2,TLR4,etc.are significantly increased after HCT-8 cells infected with Cryptosporidium parvum,and the expression profile of miRNAs is remarkably changed,such as the expression level of Hsa-miR-181d is extremely reduced.In order to understand the role of Hsa-miR-181d between Cryptosporidium parvum and host cells,this study investigated the relationship of Hsa-miR-181d and TLRs/NF-κB signal pathway of HCT-8 cells infected with Cryptosporidium parvum Ⅱd subtypes,and Bcl-2 can inhibit apoptosis were target gene of Hsa-miR-181d has been verified.It is hoped to obtain the key elements for the interaction between the parasite and the host,and provide reference for the development of effective drugs and vaccines.1.The mRNA and protein expression of key elements such as TLR2,TLR4,MyD88,and NF-κB in TLRs/NF-κB signal pathway of HCT-8 cells infected with Cryptosporidium parvum Ⅱd subtype were detected by qPCR and Western blotting.The expression of mRNA and protein of key signal elements of TLRs/NF-κB signal pathway in HCT-8 cells infected with Cryptosporidium parvum Ⅱd subtype were detected by qPCR and Western blotting.At 4 h,8 h,12 h,and 24 h,the mRNA and protein expression of TLR2,TLR4,MyD88,and NF-κB were significantly increased.The mRNA and protein expression of TLR2,TLR4,and NF-κB were significantly higher at 8 h.The mRNA and protein expression of MyD88 were significantly increased at 8 h and 12 h,the obove rusult indicating that Cryptosporidium parvumⅡd subtype infected HCT-8 cells by activating signal molecules such as TLR4,MyD88,NF-κB,etc.activates MyD88-dependent TLRs/NF-κB signal pathway participates in defense against Cryptosporidium parvum infection.2.Hsa-miR-181d was detected in HCT-8 cells infected with Cryptosporidium parvumⅡd subtype by qPCR,and apoptosis of HCT-8 cells infected with Cryptosporidium parvum Ⅱ d subtype was detected by flow cytometry after the inhibition of TLR2 and TLR4.The expression of Hsa-miR-181d were down-regulated at 4 h,8 h,and 12 h,and the down-regulation was extremely significant at 12 h,and significantly up-regulated at 48 h after infection of Cryptosporidium parvum Ⅱd subtype.Hsa-miR-181d expression were up-regulated at 4 h,8 h,12 h,and 24 h,and down-regulated at 48 h after the inhibition of TLR2 and TLR4.,which was the opposite results compared with before,this indicates that TLR2 and TLR4 are involved in the regulation of Hsa-miR-181d expression.In addition,the apoptosis reached a bottom peak at 12 h and reached its peak at 48 h after HCT-8 cells were infected with Cryptosporidium parvum Ⅱd subtype.The above results indicate that the TLRs/NF-κB signal pathway is involved in regulating the expression of Hsa-miR-181d after Cryptosporidium parvum Ⅱd subtype infection in HCT-8 cells,and there is a potential interaction effect between Hsa-miR-181d and apoptosis.3.The relationship between Hsa-miR-181d and its predicted target gene B-cell lymphoma-2(Bcl-2)were verificated through dual-luciferase reporter gene assay,then detected the gene and protein expression of Bcl-2 by qPCR and Western blotting when transfected the mimics and inhibitor to Hsa-miR-181d.Construct dual-luciferase reporter gene wild type vector(pmirGLO-Bcl-2-wild)and its mutant vector(pmirGLO-Bcl-2-mut)which containing the 3’UTR of Bcl-2 that is potential target gene of Hsa-miR-181d.Preliminary proof the potential targeting relationship between Hsa-miR-181d and Bcl-2 through dual-luciferase reporter gene assay.Then use the transfection reagent Lipofectamine(?)3000 to co-transform the mimics and negative control(NC)of Hsa-miR-181d and the inhibitor of Hsa-miR-181d and its corresponding negative control NC in HCT-8-stained cells,the changes of gene and protein expression of Bcl-2 were detected by qPCR and Western blotting.Compared with the mimics NC group,the mRNA and protein expression of Bcl-2 in the mimics group of miR-181d were significantly reduced.The inhibitor group was just the opposite,indicating that Bcl-2 is the target gene of Hsa-miR-181d.In summary,Cryptosporidium parvum Ⅱd subtype infected HCT-8 cells can activate MyD88-dependent TLRs/NF-κB signal pathway,and the level of Hsa-miR-181d showed opposite trends after the inhibition of TLR2 and TLR4,indicating that the TLRs/NF-κB signal pathway is involved in regulating the expression of Hsa-miR-181d.Apoptosis decreased significantly at 12 h and reached its peak at 48 h after Cryptosporidium parvum Ⅱd subtypes infected HCT-8 cells,and Bcl-2 is the target gene of Hsa-miR-181d.There is a negative targeting relationship between Bcl-2 and Hsa-miR-181d.Therefore,HCT-8 cells infect with Cryptosporidium parvum Ⅱd subtype activates the TLRs/MyD88/NF-κB signal pathway,which is involved in regulating the expression of Hsa-miR-181d and further negatively regulate the anti-apoptotic effect of Bcl-2,eventually causes the host cell to exhibit a two-way regulation of early anti-apoptosis and later pro-apoptosis in order to achieve protection against Cryptosporidium parvum infection.