| Cryptosporidium parvum(C.parvum)is an important zoonotic protozoan,and mainly causes human or animal diarrhea.It is harmful to young animals and patients with immune deficiency,and leads to growth disorders,malnutrition and other symptoms,even fatal diarrhea for infants.At present,the main drug used to treat cryptosporidiosis is Nitazoxanide,but the its efficiency is limited,Cryptosporidium vaccine is not available currently.Therefore,it is necessary to promote the research of Cryptosporidium vaccines and drugs.Exploring host immune response during C.parvum infection can benefit the prevention and treatment of cryptosporidiosis.Autophagy is a lysosomal dependent degradation pathway widely existing in eukaryotic cells,and plays an important role in the clearance of intracellular pathogens.Zhang et al.(2018)in our laboratory has showed that autophagy can be induced by C.parvum in the early stage of infection,and play a role in clearing the intracellular sporozoites.Reports show that autophagy can be regulated by many factors,including microRNA and MAPK signaling pathway.To elucidate the pathogenesis of C.parvum,and provide the potential target and basis for the prevention and control of the cryptosporidiosis,based on previous work in our group,this study aimed at exploring changes of microRNA and MAPK signaling pathway in HCT-8 after C.parvum infection,investigating their roles in regulating autophagy and controlling intracellular parasitism,The main research contents are as follows:1.Autophagy in C.parvum-infected HCT-8 cellsHCT-8 cells were infected with sporozoites at different time.expression of autophagy protein was detected by western blot,the location of EGFP-LC3 in the cells was observed by immunofluorescence.Results showed that the expression of Beclin1 was increased gradually from 0 to 24 h post infection.The accumulation of LC3-II protein was increased gradually from 0 to 8 h,slightly decreased at 12 h,and continued to increase from 18 to 24 h.The expression of p62 protein was low from 0 to 8 h,but increased gradually from 12 h.These indicated that C.parvum could induce autophagy in HCT-8 cells within 8 hours,but inhibit autophagy from 12 h to 24 h.Meanwhile,EGFP-LC3 can aggregate near the sporozoite.2.The role of miR-30 a and miR-26 a in C.parvum-regulated autophagy2.1 Screening microRNA of autophagy in C.parvum-infected HCT-8 cells.HCT-8 cells were infected with sporozoites at different times and the samples were collected.Total RNA was extracted and cDNA was synthesized.The dynamic expressions of miR-23 a,miR-375,miR-26 a and miR-30 a were detected by RT-PCR.The results showed that the expression of miR-30 a decreased in 0-8 h,but increased in 12 h-24 h;the expression of miR-26 a was low from 0 to 8 h,and increased from 12 h to 24 h.The changes of miR-26 a and miR-30 a expression were consistent with that of autophagy proteins regulated by C.parvum.The expression of miR-23 a and miR-375 was also different in 0-24 h,but there was no significant relationship with the expression of autophagy proteins.2.2 The role of miR-26 a and miR-30 a in autophagy regulated by C.parvum.After the expressions of miR-26 a and miR-30 a in HCT-8 were interfered,HCT-8 cells were infected with sporozoites and the autophagy proteins were detected by Western blot.When compared with N.C+C.parvum group,inhibition of miR-30 a led to significant up-regulation of Beclin1 and LC3-II but obvious p62 inhibition.However,up-regulation of miR-30 a showed contrary results.Up-regulation of miR-26 a did not change Beclin1 expression significantly,but increased the accumulation of LC3-II,and inhibited the p62 expression.Inhibition of miR-26 a did not change the expression of Beclin1 and LC3-II,but significantly increased p62 expression.These indicate that autophagy could be inhibited by C.parvum via up-regulating miR-30 a and inhibiting miR-26 a.2.3 The C.parvum proliferation affected by miR-26 a and miR-30 a.The expression of miR-26 a and miR-30 a in HCT-8 was interfered,then HCT-8 cells were infected with sporozoite,the number of parasites was detected by RT-PCR;the distribution of EGFP-LC3 in the cells was observed by immunofluorescence.The results showed that when compared with C.parvum group miR-30 a inhibition significantly decreased the number of parasites,while miR-30 a up-regulation showed an opposite role.On the other hand,miR-26 a up-regulation significantly decreased the number of parasites,when miR-26 a was inhibited,the number didn't change significantly;miR-30a-mimic and miR-26a-inhibitor could promote the aggregation of EGFP-LC3 near C.parvum.These indicate that C.parvum intended to survive by upregulating miR-30 a and down-regulating miR-26 a.3.The role of MAPK signaling pathway in autophagy regulated by C.parvum3.1 C.parvum activated MAPK signaling pathway.Western blot was used to detect the phosphorylation of MAPK signaling pathway in HCT-8 cells.The results showed that ERK phosphorylation increased continuously from 0 to 8h and decreased after 12 h,and phosphorylated p38 MAPK can be observed in varying degrees from 2 to 24 h after infection,but no change was found in JNK phosphorylation.Then miR-26 a in HCT-8 was interfered,and the phosphorylation of ERK and p38 MAPK was detected by Western blot after the sporozoite infected cells.The results showed that the phosphorylation of ERK and p38 MAPK was up-regulated when miR-26 a was inhibited,but the reverse result was observed when miR-26 a was up-regulated.It is suggested that C.parvum could promote the phosphorylation of ERK and p38 MAPK by inhibiting the miR-26 a.3.2 Effects of inhibiting ERK and p38 MAPK on C.parvum-regulated autophagy.The autophagy proteins were detected by Western blot.The results showed that after inhibiting the phosphorylation of ERK and p38 MAPK pathway,the expressions of Beclin1 and LC3-II were up-regulated,and the p62 was down-regulated compared with C.parvum group.It is suggested that C.parvum could inhibit autophagy by phosphorylating the ERK and p38 MAPK pathway.3.3 The C.parvum proliferation affected by ERK and p38 MAPK.The ERK and p38 MAPK pathways of the cells were inhibited,and the intracellular proliferation of C.parvum was detected by RT-PCR;the distribution of EGFP-LC3 in the sporozoites infected cells was observed by immunofluorescence.The results showed that when the phosphorylation of ERK and p38 MAPK pathway was inhibited,the number of parasites was decreased significantly,and the aggregation of EGFP-LC3 near C.parvum was increased.These indicate that C.parvum could promote its survival by activate the ERK and p38 MAPK pathway.In conclusion,after HCT-8 cells were infected by C.parvum,autophagy could be induced in HCT-8 cells within 8 hours,but inhibit autophagy from 12 h to 24 h;C.parvum can inhibit the autophagy to promote the survival in HCT-8 cells by upregulating the expression of miR-30 a,and also by enhancing the phosphorylation of ERK and p38 MAPK which was caused by the inhibition of miR-26 a.This study elucidated that the C.parvum can regulate autophagy through microRNA-MAPK signaling pathway,and found a new intracellular survival mechanism of C.parvum,this is of great significance for revealing the pathogenic mechanism of C.parvum and the prevention and treatment of the cryptosporidiosis. |
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