Differential Expression And Function Of Long-chain Non-coding RNA(lncRNA) In The Brain Of Mice Chronically Infected With Toxoplasma Gondii

Toxoplasma gondii is an obligate intracellular parasitic protozoa that is distributed worldwide and is widely parasitic in human and animal nucleated cells.T.gondii can infect all warm-blooded animals,including humans,causing zoonotic toxoplasmosis.According to serological surveys,approximately 30%of people worldwide are infected with T.gondii.As an opportunistic pathogenic protozoan,it has been thought in the past that it is in the state of asymptomatic recessive infection in a host with normal immune function.However,in recent years,as far as studies have found that the recessive infection of T.gondii is not an asymptomatic worm,but it invades the host's brain cells in a very subtle way,causing the host to change intelligence and behave abnormally,even the occurrence of mental illness.Now,there is growing evidence that chronic infections can induce behavioral changes and participate in neurological diseases.Recent studies have found that Non-coding RNAs(ncRNAs)are important non-coding RNA in genomes.It does not have protein coding function,but plays an important regulatory function in life.Long non-coding RNA(IncRNA)of more than 200 nucleotides in length,as an important non-coding RNA of the nervous system,regulates post-transcriptional expression of genes by binding to target gene mRNA,it plays an important regulatory role in nerves related diseases.In this study,we used the PRU strain to establish a chronically infected mouse model.We used microarray chips to detect the expression levels of IncRNA and mRNA in the normal and infected groups.The differentially expressed lncRNA and mRNA were analyzed by heat map and scatter plot.The target gene was predicted by differential cis/trans target gene and the GO and KEGG enrichment analysis was performed to classify the genes according to different functions.Enrich the well-significant pathways,annotate and classify genes,find biological regulatory pathways for significant differences in experimental conditions,and identify lncRNAs and corresponding target genes.The IncRNA was localized and analyzed by bioinformatics methods to determine the location of the nucleoplasm.The expression of IncRNA and target genes was verified by real-time quantitative PCR amplification of the sample.The changes of the corresponding proteins in the infected group were detected by immunohistochemistry and immunofluorescence.The function of lncRNA was studied at the cellular level using CCK-8,flow cytometry,colony formation,TUNEL.By constructing shRNA and overexpression system,the effect of lncRNA expression changes on target gene expression was investigated,and protein changes were detected by Western blot.In addition,we pulled down the lncRNA-related proteins by RNA pulldown and identified the types of proteins by mass spectrometry to further reveal the mechanism by which lncRNA regulates downstream target genes.Microarray chips showed that the expression of IncRNA and mRNA in the brain tissue of mice infected with T.gondii changed greatly by heat map and scatter plot analysis.The expression of lncRNA in the brain tissue of mice infected with T.gondii was analyzed by GO,KEGG enrichment analysis.It was found that the difference of lncRNA11496 expression was the greatest in infection group.The target gene and mRNA of lncRNA were annotated and classified with GOand KEGG enrichment analysis.It was found that lncRNA11496 was closely related to the change of host's mental behavior.The target genes of lncRNA11496 were predicted by cis/trans and bioinformatics method.We confirmed that lncRNA11496 is located in the nucleus.The target gene of lncRNA11496 was determined to be Mef2c.We further verified by qPCR that both lncRNA11496 and Mef2c are down-regulated.After the interference and over-expression of lncRNA11496 were detected by real-time quantitative PCR(qPCR),the expression of the corresponding target gene Mef2c was detected.The results showed that lncRNA11496 positively regulated the expression of Mef2c.At the same time,we detected the protein-level expression of Mef2c by western blot,and further verified this result.We further test the function of IncRNA11496 in the culture of nerve cells in vitro.After transfected shRNA to interfere with the level of IncRNA11496 and overexpressed IncRNA11496,we found that cell proliferation became slow after interference with IncRNA11496,and cell proliferation became faster after overexpression of IncRNA11496 by CCK-8 and clone formation experiments.Furthermore,we detected cell cycle changes by flow cytometry and found that changes in IncRNA11496 affected the proportion of cells G1,G2 and S,which in turn affected cell proliferation.In addition,we used the TUNEL method to detect the effect of IncRNAl 1496 on apoptosis.It was found that decreased expression of lncRNAl1496 promoted apoptosis,while increased expression of IncRNAl 1496 inhibited apoptosis.Through RNA pulldown and mass spectrometry,we found that lncRNA11496 could interact with HDAC2 and affect the expression of Mef2c.In summary,the IncRNA and mRNA in the brain of mice infected with T.gondii were differently changed by microarray chip.LncRNA11496 was screened and it was detected that lncRNA11496 regulates the expression of Mef2c by binding to HDAC2,thereby affecting the proliferation,cycle and apoptosis of microglia.This study elucidates the role and regulation mechanism of IncRNA11496 in the brain of chronically infected mice of T.gondii,and point out a new direction for the prevention and treatment of T.gondi against host nervous system.