Screening LncRNAs Of HCT-8 Cells Infected With Cryptosporidium Parvum And Its Effect On Proliferation Of Cryptosporidium

Cryptosporidium parvum,one of important enteric protozoa,is one of leading causes responsible for severe diarrhea in immunocompromised human,livestock and poultry.However,there are no effective drugs and vaccines used for controlling this parasite at present.It was found that the long non-coding RNA(lncRNA)played key roles in cancers,parasitic,virus and bacterial diseases through regulating the expression of host genes.However,the role of lncRNA in cryptosporidiosis was not clear.In the present study,the expression profiles of lncRNAs and mRNAs were analyzed in HCT-8 cells infected with C.parvum using the microarray,and the effect of significantly down-regulated lncRNA BACE1-AS on the proliferation of C.parvum was investigated by using siRNA and overexpression techniques.1.The expression profiles of mRNA in HCT-8 cells infected with C.parvum were obtained.A total of 20663 mRNAs were observed in HCT-8 cell infected with C.parvum at 24 h post infection,and 1349 mRNAs were significantly differentially expressed(fold change ? 1.2,P < 0.05),including 535 up-regulated and 814 down-regulated mRNAs.The qRT-PCR analysis of ten mRNAs(including 5 up-regulated and 5 down-regulated mRNAs)randomly selected verified the microarray results.The biological functional prediction indicated that these mRNAs were enriched in several pathways involved in the interaction between host and C.parvum,e.g.,Wnt signaling pathway,tight junction and hedgehog signaling pathway.2.The expression profiles of lncRNA in HCT-8 cells infected with C.parvum were obtained.A total of 16203 lncRNAs were detected in HCT-8 cell infected with C.parvum at 24 h post infection.Among them,821 were significantly differentially expressed(fold change ? 1.2,P < 0.05),including 557 up-regulated and 264 down-regulated lncRNAs.Further analysis indicated that these differentially expressed lncRNAs belonged to five types of lncRNAs,including 22 sense,280 antisense,312 intergenic,44 divergent,and 33 intronic lncRNAs.Furthermore,the qRT-PCR analysis of 11 significantly differentially expressed lncRNAs(including 5 up-regulated and 6 down-regulated lncRNAs)randomly selected confirmed the microarray results.The co-expression and target prediction analysis indicated that 27 mRNAs were cis-regulated by 29 lncRNAs and 109 were trans-regulated by 114 lncRNAs.The functional prediction showed that these targets of lncRNAs were enriched in hedgehog signaling pathway,tight junction and Wnt signaling pathway.3.The effect of lncRNA BACE1-AS on proliferation of C.parvum was preliminary analyzed.The lncRNA BACE1-AS was significantly down-regulated in HCT-8 cells infected C.parvum,and its expression was significantly lower than the control group at different infection doses(oocysts : cells = 1.125 : 1,2.25 : 1 and 4.5 : 1)and different times(8 h,12 h,24 h and 48 h)post infection(p.i.).To determined the effect of BACE1-AS on proliferation of C.parvum,HCT-8 cells were transfected with the recombinant vector of BACE1-AS and empty vector(pcDNA 3.1),respectively,and then infected with C.parvum at 24 h post transfection.Compared with cells transfected with pcDNA 3.1,qRT-PCR showed lower mRNA level of the HSP70 gene of C.parvum in HCT-8 cells transfected with the recombinant vector.Additionally,the expression of HSP70 gene of C.parvum was significantly higher after knockdown of BACE1-AS with siRNA(si-BACE1-AS)than the irrelevant siRNA(si-NC)group.Collectively,infection of C.parvum induced different expression of the lncRNAs and mRNAs in HCT-8 cells,and the significantly down-regulatrd lncRNA BACE1-AS inhibited the proliferation of C.parvum in HCT-8 cells.These findings preliminary revealed the role of host lncRNA in Cryptosporidium infection and provided basic data for developing vaccines and drugs of the prevention and control of cryptosporidiosis.