Differential Expression And Functional Study Of Non-coding RNA Of Trichinella Spiralis Invading Intestinal Epithelial Cells

Backgrounds and AimsTrichinosis is a serious zoonotic parasitic disease mainly caused by raw or semi-raw meat products containing Trichinella larva sacs.The muscle larvae（ML）of Trichinella spiralis are released from the capsular under the action of digestive fluids in the stomach of the host,exposed to intestinal contents,bile and other small intestinal environment,and then activated as intestinal infective larvae（IIL）.After the invasion of small intestinal mucosa in molting,development.Therefore,whether Trichinella spiralis can invade host intestinal epithelial cells（IECs）is a key step for successful infection and development.Since Trichinella spiralis does not have spiny or spear like structures in its orifices,it may invade intestinal epithelial cells through chemical or mechanical damage.Found in a previous study Trichinella spiralis larvae invade host IECs changed significantly before and after the insect body protein,including a variety of protein hydrolysis enzyme activity,has for some scholars both at home and abroad of Trichinella spiralis protease（such as serine protease,glutathione s transferase,etc）gene cloning expression,found that the recombinant protein antibody of larvae invade IECs have partially block effects.Previous studies have shown that during invasion of host IECs,some genes of Trichinella spiralis are expressed and regulated,and the proteins encoded by them may be related to injury and invasion of host IECs.However,the key factors and molecular mechanism of invasion of host IECs by Trichinella spiralis are still unclear.In this study,high-throughput sequencing was used to identify non-coding RNA（ncRNA）differentially expressed before and after intestinal infectious larvae of Trichinella spiralis invaded host IECs.Bioinformatics techniques were used to analyze the GO function and KEGG signal enrichment pathway of differentially expressed IncRNA,miRNA and circRNA.Furthermore,the regulatory network of circRNA,miRNA and mRNA was further analyzed,which provided a new idea for comprehensively understanding the molecular regulatory mechanism and gene function of trichinosis invasion host IECs,and searching for specific and efficient protective vaccine and therapeutic target of trichinosis.Materials and methods1.Screening of differential non-coding RNA before and after intestinal infective larvae invaded mice IECsML were collected and incubated with mouse bile for 3 h.After repeated washing with sterile normal saline,intestinal infectious larvae（pre-invasion larvae）were obtained.It was mixed with DMEM semi-solid medium and inoculated into the monolayer of mouse primary culture IECs.At 6 h of culture,the larvae had invaded the IECs but had not yet started molting,and the larvae were collected at this time（post-invasion larvae）.Intestinal infectious larvae before and after invading IECs were collected,and total RNA of larvae was extracted,and the purity and concentration of extracted RNA were detected for high-throughput sequencing and RT-qPCR verification.The non-coding RNA library was constructed,Illumina sequencing was performed,and the sequencing data were filtered to remove the fragments with a length less than 18 nt to obtain clean data.Data quality assessment of clean data,identification and prediction of different types of ncRNAs（lncRNA,miRNA,circRNA）through UCSC,Ensembl,and GENCODE databases,RNA that did not conform to the characteristics of lncRNA,miRNA and circRNA were filtered out,and the number and distribution locations of the three non-coding RNA were counted.Common and specific nucleotide sequences among samples were analyzed,and reference genome comparisons with Trichinella spiralis were compared and annotated.The differential expression of ncRNA before and after intestinal infection larvae invaded IECs was obtained.The expression levels of differentially expressed lnCRNA,miRNA and circRNA obtained by high-throughput sequencing were verified by RT-qPCR.2.Function and regulatory network analysis of differentially expressed ncRNAsTarget genes of ncRNA were predicted by Bedtools,Blast and Pearson correlation coefficient.Rsem software was used to calculate the gene expression levels of different types of ncRNA.The software used FPKM method to calculate gene expression,and the threshold value of FPKM was greater than 0.1 to determine whether the gene was expressed.RT-qPCR was used to detect its expression level in intestinal infectious larvae before and after invasion of IECs.Bioinformatics techniques were used to analyze the GO function and KEGG pathway enrichment of ncRNA differentially expressed before and after invasion,so as to comprehensively understand the gene regulation function of Trichinella spiralis during invasion of host IECs.A co-expression network was constructed by using differential lncRNA and differential mRNA to analyze its gene expression regulation and function during the invasion of Trichinella spiralis to IECs.CircRNAs,as competitive endogenous RNAs（ceRNAs）,can competitively bind with miRNAs to regulate mRNA expression.In this study,known miRNA mature sequences of Trichinella spiralis were downloaded from miRBase database,and miRNA-3.3a software was used to predict miRNA binding sites of circRNA circular sequences and target gene mRNA of miRNA.The predicted miRNA was intersected with the differential miRNA obtained by high-throughput sequencing,and the target gene mRNA predicted by miRNA was intersected with the differential mRNA obtained by high-throughput sequencing.CircRNA-miRNA-mRNA was obtained,namely,the ceRNA regulatory network of differentially expressed ncRNA in the process of Trichinella spiralis invading host IECs.Results1.Differentially expressed lncRNA,miRNA,and circRNA after Trichinella spiralis invaded host IECs.（1）High-throughput sequencing results showed that 101 lncRNAs were differentially expressed in larvae,including 54 up-regulated genes and 47 down-regulated genes.The results of GO functional enrichment analysis showed that the molecular functions of differentially expressed IncRNA were mainly enriched in"binding","catalytic activity","nucleic acid binding transcription factor activity","molecular transcriptional activity",and "molecular function regulator".The results of KEGG pathway enrichment analysis show that 10 pathways may be involved in these differential IncRNA signaling pathways,among which "Wnt signaling pathway","Autophagy regulation","PI3K-Akt signaling pathway",and "phagocyte"are the most significant enrichment pathways.（2）Sequencing results showed that 40 miRNAs were differentially expressed in larvae,including 16 up-regulated miRNAs and 24 down-regulated miRNAs.The results of GO functional enrichment analysis showed that differential expression of miRNA mainly had the functions of "transporter activity" and "MAP kinase activity".The KEGG pathway is mainly concentrated in three pathways:"protein transport","gap junction",and "splicing".（3）Sequencing results showed that 161 circRNAs were differentially expressed,including 69 up-regulated circRNAs and 92 down-regulated circRNAs.The results of GO functional enrichment analysis showed that the differential expression of circRNA mainly had the molecular functions of "SMAD binding","Chymosin binding",and "1-phosphatidylinositol binding".The KEGG pathway is mainly enriched in three pathways:"cell adhesion molecule","regulation of actin cytoskeleton",and "calcium signaling pathway".The KEGG pathway is mainly enriched in three pathways:"cell adhesion molecule","regulation of actin cytoskeleton",and "calcium signaling pathway".（4）In addition,the differentially expressed mRNAs of intestinal infected larva before and after invading host IECs were also analyzed in this study.The results showed that 607 mRNA were differentially expressed in the larva,including 339 and 268 mRNA up-regulated.The results of GO functional enrichment analysis showed that the differential expression mRNA mainly had the molecular functions of"binding","catalytic activity","transport activity",and "signal transducer activity".The KEGG pathway is mainly enriched in "lysosomes","protein digestion and absorption",and "Hippo signaling".2.The lncRNAs,miRNAs,and circRNAs with significant differentially expressed expressions were selected for RT-qPCR verification.In the selected 7 lncRNAs,4（Tsp₀0004318,Tsp₀0032297,Tsp₀0004317,Tsp₀0015911）were up-regulated and 3（Tsp₀0017706,Tsp₀0023512,Tsp₀0018305）down-regulated;2（Tsp-miR-22,Tsp-miR-59）of 6 miRNAs were up-regulated and 4（Tsp-miR-68,Tsp-miR-29,Tsp-miR-98,Tsp-miR-101）down-regulated;One（Tsp_circ₇562755）of the 6 circRNAs was up-regulated and 5（Tsp_circ₁681119,Tsp_circ₁0433765,Tsp_circ₂352178,Tsp_circ₁0812042,Tsp_circ₁455）down-regulated.The results of RT-qPCR were consistent with those of high-throughput sequencing,which further verified the accuracy of the sequencing results and provided a target for the subsequent analysis of the molecular regulation pathway of the invasive host IECs.3.The co-expression network analysis of differentially expressed IncRNA and differentially expressed mRNA showed that there were 49 differentially expressed lncRNA and 43 mRNAs,of which 23 were up-regulated lncRNA and 26 were down-regulated lncRNA.The analysis of GO function and KEGG pathway of differential mRNA in co-expressing network showed that differential mRNA the functions of "catalytic activity","transferase activity" and "calcium ion binding",and KEGG pathway was mainly enriched in "GATA binding protein 4","PH domain and leucine-rich protein phosphatase".4.The differentially expressed circRNA-miRNA-mRNA regulatory networks before and after Trichinella spiralis invasion of mouse IECs include three circRNAs（Tsp_circ₈387433,Tsp_circ₄149397,and Tsp_circ₂957817）,four miRNAs（Tsp-miR-103,Tsp-miR-107,Tsp-miR-4,and Tsp-miR-59）and five mRNAs（Tsp₀6890,Tsp₀6880,Tsp₀8514,Tsp₁2418,and Tsp₀8514）.The results of GO function and KEGG pathway enrichment analysis of differential mRNA in regulatory network showed that differential mRNA mainly had the molecular functions of"receptor activity" and "transport activity",and KEGG pathway was mainly enriched in "axon-guided" signaling pathway.Conclusions1.Before and after the Trichinella spiralis invaded the IECs,the differentially expressed ncRNAs occurred in the insect body,including 101 lncRNAs,40 miRNAs,and 161 circRNAs.The differentially expressed ncRNAs had the molecular functions of "catalytic activity","transport activity","signal transducer activity" and participated in "PI3K-Akt signal pathway","autophagy regulation","lysosome","Wnt signal pathway","protein transport" and other signal pathways.The gene regulation and expression pattern may play an important role in the invasion of host IECs by Trichinella spiralis and the completion of intracellular molt development.2.There are three circRNAs,four miRNAs,and five mRNAs in the ceRNA regulatory network of Trichinella spiralis invasion IECs,which have the molecular functions of "receptor activity" and "transport activity".This regulatory network provides clues to reveal the molecular regulatory mechanism of Trichinella spiralis invasion IECs.