| Cryptosporidium parvum is a zoonotic intestinal parasite that can cause severe diarrhea and even death in infants,neonatal animals and immunocompromised individuals.It is the secondary leading cause of diarrhea-associated mortalities in children younger than 2 years old globally,and the main cause of diarrhea in calves of less than 1 month old.So far,only nitazoxanide has been approved for the treatment of cryptosporidiosis.However,this drug is 56%-96% efficiency in immunocompetent hosts,and has no efficiency in immunocompromised individuals and infants.There have not been vaccines available to prevent the infection of Cryptosporidium.Circular RNA(circRNA)has been reported to be involved in many kinds of physiological and pathological processes by regulating gene expression at different levels.Previous studies showed that infections of several parasites,including Cryptosporidium,could induce significantly differential expression of host circRNAs,but the functions of these differentially expressed circRNAs(DE circRNAs)are still unknown.The present study performed microarray analysis to detect the expression profiles of circRNAs in HCT-8cells infected with C.parvum for 24 h,then screened the DE circRNAs,and investigated the potential regulatory role and mechanism of DE circRNAs in C.parvum-infecting HCT-8 cells by using quantitative real-time polymerase chain reaction,overexpression and interference assays,western blot and dual luciferase reporter assay.The following results were obtained.1.The expression profiles of circRNAs in HCT-8 cells during C.parvum infection were obtained.A total of 178 DE circRNAs were detected in HCT-8 cells infected with C.parvum for 24 h by using microarray analysis,including 128 up-and 50 downregulated circRNAs,with fold change(FC)> 2 and P < 0.05.GO enrichment analysis indicated that genes producing DE circRNAs were enriched in important biological processes and molecular functions.KEGG pathway analysis showed that genes producing DE circRNAs were involved in vital signal pathways,especially metabolic pathways.2.The up-regulated circRNA ciRS-7 significantly promoted the proliferation of C.parvum in HCT-8 cells.circRNA ciRS-7(hsa＿circ＿0001946＿CBC1,also known as CDR1as)was remarkably upregulated(FC = 7.96530932,P < 0.05)in microarray results.q RT-PCR analysis showed that the expression levels of ciRS-7 were significantly increased in HCT-8 cells from 12 h post-infection(pi)to 48 h pi.Overexpression of ciRS-7 significantly increased the mRNA level of C.parvum hsp70 gene in infected cells,while interfering the expression of ciRS-7 had the opposite effect.3.The potential target miRNAs of circRNA ciRS-7 were obtained.A total of 22 potential miRNA targets of ciRS-7 were predicted by using starbase v3.0.q RT-PCR analysis found that the expression levels were significantly decreased for miR-1270,miR-219a-5p,miR-135b-5p,miR-135a-5p,miR-139-3p,miR-601 and miR-7 in HCT-8 cells at 24 h pi.Overexpression of ciRS-7 markedly decreased the expression levels of miR-1270,miR-219a-5p,miR-135b-5p,miR-135a-5p and miR-139-3p in infected cells,while interfering the expression of ciRS-7 markedly increased the expression levels of miR-1270,miR-219a-5p,miR-135b-5p,miR-135a-5p and miR-7 in infected cells.Dual luciferase reporter assays showed that ciRS-7 had the binding capacity to miR-1270,miR-219a-5p and miR-135a-5p in HCT-8 cells.Thus,ciRS-7 could target miR-1270,miR-219a-5p and miR-135a-5p in HCT-8 cells.4.circRNA ciRS-7 affected the propagation of C.parvum in HCT-8 cells by sponging miR-1270 to regulate NF-κB signaling pathway.It was predicted and confirmed that miR-1270 had the binding capacity to the 3’-untranslated region(3’-UTR)of rel A(p65)gene,a subunit of the nuclear transcription factor-κB(NF-κB)signal pathway.The mRNA levels of the rel A gene were significantly upregulated in HCT-8 cells from 12 h pi to 48 h pi and the protein level of RELA was remarkably increased at 24 h pi.Additionally,the mRNA levels of nos2 and cxlc2 genes,two downstream molecules of the NF-κB signal pathway,were also significantly increased in cells at 24 h pi.Transfection of miR-1270 mimics markedly inhibited the mRNA and protein levels of the rel A gene and the mRNA levels of nos2 and cxlc2 genes in infected cells,while miR-1270 inhibitor had the reverse effects.Overexpression of ciRS-7markedly promoted the mRNA and protein levels of the rel A gene and the mRNA levels of nos2 and cxlc2 genes in infected cells,while interfering the expression of ciRS-7obtained the reverse effects.The upregulated effects of overexpression of ciRS-7 on the mRNA levels of rel A,nos2 and cxlc2 genes in infected cells were reversed by miR-1270 mimics.Futhermore,miR-1270 mimics significantly inhibited the mRNA level of C.parvum hsp70 gene in infected cells,whereas miR-1270 inhibitor had the reverse effect.The upregulated effect of overexpression of ciRS-7 on the mRNA level of C.parvum hsp70 gene in infected cells was attenuated by miR-1270 mimics.5.circRNA ciRS-7 affected the propagation of C.parvum in HCT-8 cells by sponging miR-135a-5p to regulate the expression of STAT1 and p-STAT1.It was predicted and confirmed that miR-135a-5p had the binding capacity to the 3’-UTR region of signal transducer and activator of transcription1(stat1)gene in HCT-8 cells.The mRNA levels of the stat1 gene were significantly upregulated in HCT-8 cells from12 h pi to 72 h pi,and the protein level of STAT1 and phosphorylated STAT1(p-STAT1)were significantly upregulated at 24 h pi.Transfection of miR-135a-5p mimics significantly inhibited the protein levels of STAT1 and p-STAT1 in infected cells,while miR-135a-5p inhibitor obtained the opposite effects.Overexpression of ciRS-7markedly promoted the protein levels of STAT1 and p-STAT1 in infected cells,while interfering the expression of ciRS-7 obtained the opposite effects.The upregulated effects of overexpression of ciRS-7 on the protein levels of STAT1 and p-STAT1 in infected cells were reversed by miR-135a-5p mimics.Futher studies found that although miR-135a-5p mimics had no effect on the mRNA level of C.parvum hsp70 gene in infected cells,but miR-135a-5p inhibitor notably upregulated the mRNA level of C.parvum hsp70 gene in infected cells.The downregulated effect of interfering the expression of ciRS-7 on the mRNA level of C.parvum hsp70 gene in infected cells was reversed by miR-135a-5p inhibitor.6.circRNA ciRS-7 affected the propagation of C.parvum in HCT-8 cells by sponging miR-219a-5p to regulate cell autophagy.The protein levels of the key autophagy associated-proteins of LC3B-II and p62 were remarkably increased in HCT-8 cells from 8 h pi to 48 h pi,indicating that C.parvum infection induced incomplete autophagy in HCT-8 cells.Transfection of miR-219a-5p mimics significantly increased the protein level of LC3B-II but decreased the protein level of p62 in infected cells,while miR-219a-5p inhibitor had the opposite effects.Overexpression of ciRS-7remarkably inhibited the protein level of LC3B-II but promoted the protein level of p62 in infected cells,whereas interfering the expression of ciRS-7 obtained the opposite effects.The inhibition effect on the protein level of LC3B-II and the promoted effect on the protein level of p62 by overexpression of ciRS-7 in infected cells were reversed by miR-219a-5p mimics.16 potential target genes of miR-219a-5p were predicted to be related to autophagy pathways,and the mRNA levels of akt1,pik3 ca and p62 genes were significantly upregulated in infected cells.However,there were no binding capacities between miR-219a-5p and these three genes in HCT-8 cells,suggesting that ciRS-7/miR-219a-5p axis might regulate cell autophagy in C.parvum-infecting HCT-8 cells via other mechanisms.Furthermore,miR-219a-5p mimics significantly inhibited the mRNA level of C.parvum hsp70 in infected cells,while miR-219a-5p inhibitor had the opposite effect.The upregulated effect of overexpression of ciRS-7 on the mRNA level of C.parvum hsp70 gene in infected cells was attenuated by miR-219a-5p mimics.In conclusion,C.parvum infection markedly altered the circRNAs expression profiles of HCT-8 cells,and the significantly up-regulated circRNA ciRS-7 promoted the propagation of C.parvum in HCT-8 cells by sponging miR-1270,miR-135a-5p and miR-219a-5p to regulate the NF-κB signaling pathway,the expression of STAT1 and p-STAT1,and cell autophagy,respectively.The present study preliminarily investigated the role of host circRNA ciRS-7 during Cryptosporidium infection,and provided a novel insight for further clarifying the infection and pathogenesis of Cryptosporidium. |
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