Mechanism Of MicroRNA-3976 Regulating The HCT-8 Cell Apoptosis Induced By Cryptosporidium Parvum Infection

Cryptosporidium is an emerging zoonotic parasitic protozoan that is second only to rotavirus as a cause diarrhea and death in children younger.Cryptosporidium infection can lead to self-limited diarrhea in immune-normal individuals,while it can cause persistent diarrhea and death in immunocompromised individuals,especially AIDS patients.Currently,there are no fully efficacious treatment drugs or vaccines for cryptosporidiosis.The pathogenesis of Cryptosporidium and the regulatory mechanism of host defense against Cryptosporidium are not clear,which is the key bottlenecks restricting the prevention and control of Cryptosporidium.Studies have shown that miRNAs play an important role in host innate immunity against Cryptosporidium infection.The research group analyzed the expression profile of miRNAs in HCT-8 cells at the early stage of Cryptosporidium parvum infection and obtained some significantly up-regulated or down-regulated miRNAs,among which miR-3976 was significantly down-regulated at 12 h after Cryptosporidium parvum infection.Biological function analysis suggested that miR-3976 was involved in the apoptosis/anti-apoptosis process of host cell defense against Cryptosporidium parvum infection.The purpose of this study was to investigate the mechanism of miR-3976 regulating the HCT-8 cell apoptosis induced by Cryptosporidium parvum infection,in order to provide a new strategy for the design and development of anti-cryptosporidiosis drug targets.The expression levels of miR-3976 at different time points after Cryptosporidium parvum infection of HCT-8 cells were detected by q PCR,and the results showed that miR-3976 was significantly decreased at 8 h and 12 h(P8 hpi<0.05,P12 hpi<0.01),and significantly increased at 24 h and 48 h(P24 hpi-48 hpi<0.01)after Cryptosporidium parvum infection.The expression of miR-3976 was changed by transfection of miR-3976 mimics and miR-3976 inhibitor,and samples were collected for apoptosis detection at 24 h after infection with Cryptosporidium parvum.Flow cytometry results showed that miR-3976 mimics transfected group significantly increased apoptosis rate than miR-3976 mimics NC group(P<0.01),while the apoptosis rate of miR-3976 inhibitor group was significantly lower than that of miR-3976 inhibitor NC group(P<0.05);Under the condition of ensuring the transfection efficiency of miR-3976,q PCR was used to detect the burden of parasite infection in different groups.The results showed that there was no difference in the burden of parasite infection among groups at 2 h after Cryptosporidium parvum infection(P>0.05),48 h after infection,the burden of parasite infection in miR-3976 mimics group was significantly lower than miR-3976 mimics NC group(P<0.05),whereas the miR-3976 inhibitor group had the opposite effect(P<0.01);These results suggest that miR-3976 is involved in regulating the apoptosis of HCT-8 cells after Cryptosporidium parvum infection and the burden of parasite infection.In order to verify the potential mechanism of miR-3976 regulating the apoptosis of HCT-8 cells after Cryptosporidium parvum infection,the target genes of miR-3976 were predicted by bioinformatics software,and the potential binding site of 3’UTR of B-cell lymphoma 2-associated protein A1(BCL2A1)related to apoptosis function of miR-3976 was found.Design and synthesis of BCL2A1 dual-luciferase reporter gene vectors(pmi GLO-BCL2A1-wild and pmi GLO-BCl2A1-mut)containing miR-3976 binding site and mutation site.After co-transfection of dual-luciferase reporter gene vector with miR-3976 mimics and miR-3976 mimics NC,dual-luciferase activity of different groups was detected.The results showed that the relative lucifase activity of the pmi GLO-BCL2A1-wild and miR-3976 mimics group was significantly lower than that of the pmi GLO-BCl2A1-wild and miR-3976 mimics NC group(P<0.05),there was no change in other groups.q PCR was used to detect the expression of BCL2A1 in HCT-8 cells after Cryptosporidium parvum infection.The results showed that BCL2A1 was significantly upregulated at different time points of Cryptosporidium parvum infection(P4 hpi-8 hpi<0.05,P12 hpi-48 hpi<0.01);After overexpression or inhibition of miR-3976,the expression level of BCL2A1 was detected by q PCR and Western Blotting,and the results showed that the overexpression of miR-3976 significantly inhibited the m RNA and protein expression of BCL2A1(P<0.001),decreased expression of miR-3976 significantly increased the expression of BCL2A1(P<0.05).These results suggest that BCL2A1 is the target gene of miR-3976,and miR-3976 binds to the 3’UTR of BCL2A1 and negatively regulating the expression of BCL2A1 at the post-transcriptional level.In order to accurately verify miR-3976 targeting BCL2A1 to regulate HCT-8 cell apoptosis,this study constructed BCL2A1 overexpression vector(pc DNA3.1-BCl2A1)and synthesized and screened specific si RNA(si-BCl2A1),verified its overexpression and interference effect.After transfection of HCT-8 cells with BCL2A1 overexpression vector and empty vector,si-BCl2A1 and si-NC,the apoptosis rate of BCL2A1 cells in different groups was detected by flow cytometry 24 h after Cryptosporidium parvum infection.The results showed that the apoptosis rate of BCL2A1 overexpression group was significantly lower than that of the empty vector group(P<0.01),the apoptosis rate of si-BCl2A1 group was significantly higher than si-NC group(P<0.01);The results of the parasite burdens showed that there was no difference among different groups at 2 h after infection(P>0.05),at 48 h after infection,the parasite burdens of BCL2A1 overexpression group was significantly higher than that of empty vector group(P<0.01),the results were opposite in si-BCl2A1 group;In order to further verify the mechanism of miR-3976 regulating the apoptosis of HCT-8 cells after Cryptosporidium parvum infection,HCT-8 cells were co-transfected with BCL2A1 overexpression vector and empty vector,miR-3976 mimics and miR-3976 NC.The apoptosis and parasite burdens were measured after infection.The results showed that the apoptosis of miR-3976 mimics and empty vector group was significantly higher than that of miR-3976 NC and empty vector group after 24 h of infection(P<0.01),co-transfection of miR-3976 mimics and BCL2A1 overexpressed vector group significantly down-regulated apoptosis rate of miR-3976 mimics and empty vector group(P<0.01);At 2 h after infection,there was no difference in the parasite burdens in each group(P>0.05),at 48 h after infection,the parasite burdens in the co-transfected miR-3976 mimics and empty vector group was significantly lower than that in the co-transfected miR-3976 NC and empty vector group(P<0.05),and the co-transfection of miR-3976 mimics and overexpression vector group significantly increased the parasite burdens of miR-3976 mimics and empty vector group(P<0.05).In conclusion,BCL2A1 is involved in regulating the apoptosis of HCT-8 cells after Cryptosporidium parvum infection and the burden of parasite infection.miR-3976 regulates the HCT-8 cell apoptosis after Cryptosporidium parvum infection and the burden of parasite infection by targeting BCL2A1.In conclusion,the infection of HCT-8 cells by Cryptosporidium parvum induces the expression of miR-3976,and miR-3976 regulating the HCT-8 cells apoptosis and the parasites burdens after Cryptosporidium parvum infection by targeting BCL2A1.