Whole Transcriptome Analysis Of HCT-8 Cells Induced By Cryptosporidium Parvum Infection

Cryptosporidium,a globally distributed diarrheal pathogen,can infect ?a variety of vertebrate,including humans as well as domestic and wild animals.Studies have shown that non-codingRNAs（ncRNAs）of host epithelial cells can defend against Cryptosporidium parvum infection by regulating the innate immune response of the host.In particular,long non-codingRNAs,microRNAs and circRNAs have attracted extensive attention from researchers.In this project,human colorectal adenocarcinoma cells（HCT-8）infected with C.parvum were used as a model to identify the whole transcriptome expression profile of HCT-8 cells associated with C.parvum infection by transcriptome sequencing（RNA-Seq）,and analyze the possible biological functions of these differentially expressedRNAs molecules and the possible signaling pathways involved by bioinformatics means such as GO annotation and KEGG enrichment.Based on the target relationships,a regulatory network map was predicted and constructed to further explore in depth the potential mechanisms of interactions between non-codingRNAs,thus providing basic data to subsequently reveal the mechanisms of the role of host epithelial cell ncRNAs in the innate immune response in defense against Cryptosporidium infection.1.Transcriptome analysis of miRNA in HCT-8 cells induced by C.parvum infection.UsingRNA-Seq sequencing to resolve the transcriptome sequencing data of miRNAs in HCT-8 cells at 3 h and 12 h after C.parvum infection,2076 miRNAs were identified,including 1665 known miRNAs and 411 newly identified miRNAs.69 miRNAs（21miRNAs）were screened at 3 h after C.parvum infection using Fold Change（FC）≥ 2 and Q-value ≤ 0.05 as the significant difference criteria.69 miRNAs（21 up-regulated and 48down-regulated,including 20 newly identified）were screened at 3 h after C.parvum infection as significant difference criteria;129 miRNAs（48 up-regulated and 81down-regulated,including 33 newly identified）were screened at 12 h after infection;24miRNAs with the same differential expression were screened at two time points.The relative expression levels of 7 randomly selected miRNAs were detected by qPCR,and the results showed that the expression levels of these 7 miRNAs were consistent with the results ofRNA-Seq sequencing,which proved that the sequencing results were effective and credible.The functional enrichment results showed that the differentially expressed miRNAs were mainly enriched in apoptosis and apoptosis-related pathways（such as PI3K-Akt signaling pathway and MAPK signaling pathway）.hsa-mi R-4722-5p、hsa-mi R-1915-3p、hsa-mi R-324-3p、hsa-mi R-6721-5p、hsa-mi R-4676-5p、hsa-mi R-33b-3p、mi R-103a-3p和 mi R-34c-5p,and several other miRNAs could be used as candidate miRNAs associated with apoptosis and apoptotic pathways.2.Transcriptome analysis of lncRNA,mRNA and circRNA in HCT-8 cells induced by C.parvum infection.Transcriptome sequencing data of lncRNA,mRNA and circRNA in HCT-8 cells at 3 h and 12 h after C.parvum infection were analyzed byRNA-Seq sequencing.A total of23,912 lncRNAs（including 10,687 known and 13,225 newly identified）,17,839 mRNAs and 51,358 circRNAs were identified.Using Fold Change（FC）≥2 and Q-value≤0.05 as the significant difference criteria,393 lncRNAs（218 up-regulated and 175 down-regulated,including 117 newly identified）and 115 mRNAs（61 up-regulated and 54 down-regulated）were screened 3 h after C.parvum infection.No circRNA with significant difference was screened;450 lncRNAs（284 up-regulated and 166 down-regulated,including 214 newly identified）,117 mRNAs（78 up-regulated and 39 down-regulated）and 1 circRNA with up-regulated expression（has_circ₀019973）were screened at 12 h post-infection.94 identical differentially expressed lncRNAs and 22 identical differentially expressed mRNAs were screened at these two time points.Eleven lncRNAs,eight mRNAs and one circRNA were randomly selected for qPCR validation,and the results showed that the qPCR results matched with theRNA-Seq sequencing results,which proved that the sequencing results were effective and reliable.The functional enrichment results showed that the differentially expressed lncRNAs were mainly enriched in C.parvum infection and pathogenicity-related pathways（e.g.tight junctions）and immune-related pathways（e.g.cell adhesion molecules）,and 7 candidate lncRNAs related to tight junctions and 23 candidate lncRNAs related to cell adhesion molecules were further discovered.The differentially expressed mRNAs were mainly enriched in nutrient absorption（e.g.protein digestion and absorption）,metabolic processes（tyrosine and retinol）and metabolism-related pathways（e.g.apelin signaling pathway and adipocytokine signaling pathway）,and 10 candidate genes related to nutrient metabolism were mined;while the only significantly up-regulated circRNA has_circ₀019973 was mainly enriched in MAPK signaling pathway and AMPK signaling pathway.3.Competitive endogenousRNA regulatory network construction.In order to explore the potential interaction mechanism of non-codingRNAs in HCT-8 cells after C.parvum infection,this study predicted and analyzed miRNA-mRNA and miRNA-lncRNA/circRNA relationship pairs from a bioinformatics perspective based on miRNA-target target relationships.Combined co-expression information,lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA network diagrams were constructed using Cytoscape v3.5.1,and the complex networks were integrated.The results showed that a total of 201RNAs interaction relationship pairs were obtained,which were composed of 1circRNA,129 lncRNAs,68 mRNAs and 3 miRNAs nodes,indicating that lncRNA/circRNA could adsorb the competitive binding mRNA of miRNA molecules through sponging;hsa-mi R-324-3p,hsa mi R-3127-5p could negatively regulate circRNA,lncRNAs and mRNAs simultaneously;data mining analysis revealed that lncRNAs（SEPTIN4-AS1,PPM1F-AS1,ASMTL-AS1,CASC11,etc.）/circRNA（has-circ-0019973）/hsa-mi R-324-3p/RPS6KA6 axis regulated MAPK signaling pathway;lncRNAs（PPM1F-AS1,SEPTIN4-AS1,LINC00158,etc.）/circRNA（has-circ-0019973）/hsa-mi R-3127-5p/BLNK axis regulated NF-κB signaling pathway.In conclusion,C.parvum infection induced differential expression of miRNAs,lncRNAs,circRNAs and mRNAs in HCT-8 cells at 3 h and 12 h post-infection;qPCR successfully verified the differentialRNAs obtained byRNA-Seq sequencing;Functional enrichment analysis revealed that differentialRNAs may play important biological functions,and candidate differentialRNAs associated with significant enrichment signaling pathways were discovered.hsa-mi R-324-3p and hsa-mi R-3127-5p are key miRNAs that can negatively regulate circRNA,lncRNAs and mRNAs simultaneously,and mined lncRNAs（SEPTIN4-AS1,PPM1F-AS1,ASMTL-AS1,CASC11,etc.）/circRNA（has-circ-0019973）/hsa-mi R-324-3p/ RPS6KA6 and lncRNAs（PPM1F-AS1,SEPTIN4-AS1,LINC00158,etc.）/circRNA（has-circ-0019973）/hsa-mi R-3127-5p/BLNK regulate two ceRNAs regulatory axis of MAPK and NF-κB signaling pathways,respectively.These findings lay the foundation for deeper insight into the molecular mechanisms of epithelial ncRNA regulation of microscopic C.parvum infection.