Research On The Apoptotic Regulated By MiR-181d Mediating Bcl2 In HCT-8 Cells Infected With Cryptosporidium Parvum

Cryptosporidium is an important zoonotic pathogen in the world,which leads to severe diarrhea in human and animals and affects human health and the healthy development of animal husbandry.Increasing evidence supports that miRNA may regulate many response stages after Cryptosporidium infection,such as host cell signal pathway,production of antibacterial molecules,expression of cytokines/chemokines,and feedback regulation of immune homeostasis.The research group showed that the expression of miR-181d in HCT-8 cells was significantly down regulated in the early stage of C.parvum infection,and speculated that it may participate in the regulation of apoptosis and affect the parasite burden in HCT-8 cell.The research group has previously verified the interaction between miR-181d and Bcl2 gene,confirming that Bcl2 molecule is the target gene of miR-181d in the mitochondrial apoptosis pathway.Therefore,this study will rearch on the apoptosis pathway of HCT-8 cells infected with C.parvum regulated by miR-181d and its target gene Bcl2,so as to provide a basis for the design and development of anti-cryptosporidiosis drug targets.1.Study on the function of miR-181d and its target gene Bcl2 against C.parvum infectionIn order to study the relationship between miR-181d,Bcl2 and Cryptosporidium infection,their effects on intracellular proliferation and apoptosis of Cryptosporidium were detected.Firsty the pcDNA3.1-Bcl2 overexpression vector was constructed,specific siRNA interfered with Bcl2 and miR-181d specific mimic and inhibitor were designed and synthesized,then Bcl2 overexpression vector,siBcl2,miR-181d mimic,inhibitor and their control reagents were transfected into HCT-8 cells using Lipofectamine 3000 transfection reagent respectively,and followed by HCT-8 cells infected with C.parvum sporozoites after 24 h transfection respectively.The intracellular proliferation of C.parvum in HCT-8 cells was detected at 2 h and 24 h post infection.qPCR analysis showed that there was no significant change in the number of C.parvum in each group at 2 h post infection.However,there were differences in each group infected with C.parvum at 24 h.The parasite burden in HCT-8 cell were significantly increased in the miR181d inhibitor group and Bcl2 overexpression group,while the siBcl2 and miR-181d mimic group significantly were decreased.Apoptosis of HCT-8 cells infected with C.parvum were detected by flow cytometry at 24 h.The results showed that the apoptosis rate were decreased significantly in miR-181d inhibitor group and Bcl2 overexpression group,and the apoptosis rate of siRNA and mimic group increased significantly.2.Study on the apoptosis pathway of HCT-8 cells infected with C.parvum regulated by miR181d and its target gene Bcl2To study whether C.parvum regulates apoptosis through mitochondrial apoptosis pathway after infecting host cells,the expressions of miR-181d,Bcl2 and mitochondria apoptosis pathway-related apopotic molecules such as Bax,puma,caspase 9,and caspase 3 were detected by qPCR at different hours(0、4、8、12、24、36、48 h),it was found that in the early stage of infection(0-24 h),the expression of miR-181d decreased and the expression of Bcl2 increased significantly,while in the late stage of infection(36-48 h),the expression of miR-181d increased and the expression of Bcl2 decreased.The expressions trend of pro-apoptotic molecules Bax,Puma,Caspase 9,and Caspase 3 were completely opposite to Bcl2.The relative expression levels of Bcl2,Bax,Puma,Caspase 9 and Caspase 3 were detected in each group above after Bcl2 overexpression vector,siBcl2,miR-181d mimic,inhibitor and control reagents were transfected into HCT-8 cells using Lipofectamine 3000 transfection reagent respectively,and followed by HCT-8 cells infected with C.parvum sporozoites after 24 h transfection respectively.qPCR and WB analysis showed that the expression of Bcl2 was increased significantly in the transfected Bcl2 overexpression group and the miR-181d inhibitor group both at the early stage of infection(12 h)and at the late stage of infection(48 h),while the Bax,Puma,Caspase 9 and Caspase 3 expression were decreased.The expression of molecules in transfected Bcl2 overexpression group and the miR-181d inhibitor group were opposite to those of the siBcl2 group and the miR181d mimic group.The results indicated that miR-181d and targets Bcl2 expression can affect the expression of mitochondrial apoptosis pathway molecules lead to cell apoptosis.In conclusion,miR-181d plays an important role in the endogenous development of C.parvum invading host cells.miR-181d targets Bcl2 can regulate cell apoptosis through mitochondrial apoptosis pathway,and then affects the development of C.parvum in infected HCT-8 cells.