Mechanisms Of The Host Cell Mir-4521 Targeting Foxm1 To Affect The Propagation Of Cryptosporidium Parvum In HCT-8 Cells

Cryptosporidium parvum,a common zoonotic parasite,causes cryptosporidiosis in humans and a variety of animals with diarrhea as the main symptom,and lethal diarrhea can occur in immunocompromised hosts such as young animals and HIV/AIDS patients.However,up to now,there are still no effective drugs and vaccines for the prevention and control of C.parvum infection.The host intestinal epithelial cells are the main parasitic sites of C.parvum,therefore,analyzing the interactions between C.parvum and host epithelial cells is crucial for the development of drugs and vaccines to prevent and control cryptosporidiosis.Studies have found that C.parvum could regulate the expression of microRNAs(miRNAs)to affect various biological processes(e.g.metabolism,proliferation,apoptosis,and autophagy)of epithelial cells to facilitate intracellular proliferation of the parasite.miR-4521 has been found to play important implications in tumors(including cancers)and infectious diseases(e.g.Schistosoma infection)through regulating its targets to affect cell proliferation,apoptosis,and autophagy.In order to clarify the role of miR-4521 during Cryptosporidium infection,using C.parvum infecting human ileocecal adenocarcinoma cells(HCT-8)as the model,the present study explored the effect and the mechanisms of miR-4521 on the propagation of C.parvum in HCT-8 cells.The following results were obtained.1.C.parvum infection induced upregulation of host miR-4521 to facilitate intracellular propagation of parasites in vitro.Quantitative real-time polymerase chain reaction(qRT-PCR)found that the expression of miR-4521 was up-regulated in HCT-8 cells from 8 h post infection(pi)to 48 hpi of C.parvum,and was also significantly up-regulated stimulated by lipopolysaccharide(LPS)at 24 hpi.PDTC,an inhibitor of the NF-κB signaling pathway,reversed the effect of up-regulation of miR-4521 expression caused by C.parvum infection,suggesting that C.parvum infection would upregulate the expression of miR-4521 in HCT-8 cells through NF-κB signaling pathway.Further study using the immunofluorescence assay showed that transfection of miR-4521 mimics significantly promoted the propagation of C.parvum in HCT-8 cells,while transfection of miR-4521 inhibitor significantly inhibited the propagation of C.parvum in HCT-8 cells.The same results were obtained for the detection of C.parvum hsp70 in HCT-8 cells by using qRT-PCR.2.miR-4521 regulated the expression of its target foxml in HCT-8 cells during C.parvum infection.A total of 3010 potential targets of miR-4521 were predicted by TargetScan online software.qRT-PCR analysis showed that the expression of the target genes foxml,igf2,and klf6 were down-regulated during C.parvum infection,but the transfection of miR-4521 mimics only significantly reduced the expression of foxm1 in HCT-8 cells infected with C.parvum.Furthermore,transfection of miR-4521 inhibitor significantly increased the protein level of FOXM1.The dual luciferase reporter assays verified the target interaction between miR-4521 and foxm1.3.foxml inhibited the propagation of C.parvum in HCT-8 cells.qRT-PCR analysis found that the mRNA level of foxml gene was significantly down-regulated in HCT-8 cells infected with C.parvum at 24 hpi to 48 hpi,and the protein level of FOXM1 was significantly down-regulated at 24 hpi.Immunofluorescence assay showed that overexpression of foxm1 significantly inhibited the propagation of C.parvum in HCT-8 cells.The same results were obtained for the detection of C.parvum hsp70 in HCT-8 cells by using qRT-PCR.4.miR-4521 regulated the apoptosis of HCT-8 cells through targeting foxm1 during C.parvum infection.CCK-8 kit detection found that transfection of miR-4521 mimics and miR-4521 inhibitor,and overexpression of foxml didn’t affect the proliferation of HCT-8 cells infected with C.parvum at 24 hpi.Western Blot analysis showed that transfection of miR-4521 mimics significantly decreased the expression of LC3B protein but had no effect on the expression of P62 protein in HCT-8 cells infected with C.parvum.miR-4521 inhibitor significantly increased the expression of LC3B protein and suppressed the expression of P62 protein in HCT-8 cells infected with C.parvum,and overexpression of foxml significantly increased the expression of LC3B but had no effect on the expression of P62 protein in HCT-8 cells infected with C.parvum,suggesting that miR-4521/foxm1 axis would regulate autophagy of HCT-8 cells during C parvum infection.However,further studies should be conducted in the future.Flow cytometry analysis showed that the infection of C.parvum significantly inhibited the apoptosis of HCT-8 cells,and transfection of miR-4521 mimics further inhibited the apoptosis induced by C.parvum infection.In the other hand,transfection of miR-4521 inhibitor significantly reversed the apoptosis induced by C.parvum infection,and the effect of overexpression of foxm1 on the apoptosis of infected cells was consistent with the results of transfection of miR-4521 inhibitor.These results suggested miR-4521 targeting foxm1 to regulate the apoptosis of HCT-8 cells during C.parvum infection.5.The miR-4521/foxm1 axis affects the apoptosis of HCT-8 cells during C.parvum infection by regulating the expression of BCL-2 protein.Western Blot analysis found that infection of C.parvum could significantly promote the expression of BCL-2 at 24 hpi,and transfection of miR-4521 mimics further promoted the up-regulation of BCL-2 protein caused by C.parvum infection.However,transfection with miR-4521 inhibitor significantly reversed the up-regulation of BCL-2 protein caused by C.parvum infection,and the effect of overexpression of foxml on BCL-2 protein expression was consistent with the results of transfection with miR-4521 inhibitor,indicating that the miR-4521/foxm1 axis affected the apoptosis of HCT-8 cells through regulating the BCL-2 protein during C.parvum infection.In summary,C.parvum hijacked the miR-4521/foxm1 axis to regulate cell apoptosis and promote the propagation of the parasite in HCT-8.The results in the present study provide a novel target for developing new strategies to prevent and control C.parvum infection.