Expression Of LncRNAs Associated With Cell Invasion Of Haemophilus Parasuis And Its Regulatory Mechanism

Haemophilus parasuis(H.parasuis)is a Gram-negative extracellular bacterium commonly found in the upper respiratory tract of pigs and can cause Glasserr’s disease in pigs under stress conditions.The current pathogenisis study on H.parasuis mainly focuses on the virulence factors,and there are few studies on the interaction between H.parasuis and the host.However,a better understanding of the interaction between H.parasuis and the host will be helpful to develop vaccines and drugs.lncRNAs are defined as transcripts lacking protein coding potential of greater than 200 nt in length.Although lncRNAs are expressed in most eukaryotic cells,specific lncRNAs clusters are found in different cells and tissues,and their levels are lower compared to mRNAs.lncRNAs regulate the expression of coding genes at the transcriptional,post-transcriptional and epigenetic levels and play an important role in the differentiation and embryonic development of eukaryotic cells.Studies have found that lncRNAs are involved in virus invasion process.The relationship between H.parasuis and lncRNAs has not been reported.Therefore,we hope to explore the role of lncRNAs played in the pathogenicity of H.parasuis by exploring the variations of lncRNAs and mRNA expression in H.parasuis infection in 3D4/21 cells.1.RNA-seq analysis of porcine alveolar macrophage cell line 3D4/21 infected by H.parasuisThis study was to investigate the changes in lncRNAs and mRNAs expression by infecting H.parasuis to pig alveolar macrophage cell line 3D4/21,thereby revealing the role of lncRNAs played in the pathogenicity of H.parasuis.Porcine alveolar macrophage cell line(3D4/21)was infected with standard serovar type 5H.parasuis(Hps5).RNA isolation,library preparation and sequencing,sequence data quality control and transcriptome analysis were used to identify differentially expressed genes.The potential lncRNAs were screened and target gene prediction and functional enrichment were analyzed.Real-time PCR was used to confirme differential expression of lncRNAs.The results showed that 2598 lncRNAs and 25512 mRNAs were identified in this study.2598 lncRNAs contain 2439 lincRNAs and 159 antisense lncRNAs.Compared with the control group,110 lncRNAs were up-regulated and 216 lncRNAs were down-regulated in infected cells.The 10 up-regulated lncRNAs identified by RNA-seq were stably up-regulated in Hps5-infected 3D4/21 cells by SYBR-Green real-time quantitative PCR.Moreover,compared with the control group,the expression levels of lncRNAs 165,3304,3472 and 1125 were significantly increased at 12 h,24 h and 36 h.These results indicated that our experiments initially screened four lncRNAs that were significantly up-regulated in Hps5-infected 3D4/21 cells.The four lncRNAs were targeted to investigate the interaction mechanism between H.parasuis and host.2.Association between differentially expressed lncRNAs and pathogenicity invasion of of H.parasuisTwo differentially expressed lncRNAs based on gene silencing and over-expression for subsequent in-depth studies to determine the relationship between lncRNAs expression and bacterial pathogenicity.The SYBR-Green real-time PCR was used to study the effect of lncRNAs expression on the adhesion and invasion of bacteria and the relationship between lncRNAs expression and bacterial virulence.By PCR amplification,the ATG start codon was added to the N-terminus of lncRNAs 165 and 3304 genes,the FLAG-tagged protein gene was added to the C-terminus,and then cloned into the pcDNA3.1 vector to construct recombinant over-expressing plasmids.The over-expression of lncRNA 165 and 3304 was identified by real-time PCR and western blot was used to detect the expression of FLAG-tagged protein.The results showed that the expression of lncRNA 165 and 3304 was up-regulated with the presence of viable bacteria in the cells,regardless of the adhesion of the inactivated bacteria.Expression of lncRNA 165 and 3304 was also associated with strain virulence at 24 and 36 hours post infection.We concluded that we successfully constructed eukaryotic expression plasmids to express non-coding 165 and 3304.The above results indicated that we have screened two lncRNAs with high expression levels during Hps5 invasion to 3D4/21 cells,which can be used as targets for studying the relationship between H.parasuis and host and provide the basis for studying the role of lncRNAs played in the pathogenicity of H.parasuis.3.LncRNA 165 and 3304 regulate TGF-β1 signal pathway to affect invasion of H.parasuisThe TGF-β1 pathway regulated by lncRNAs 165 and 3304 to affect H.parasuis invasion to cells was studied.The relationship between lncRNAs and bacterial invading to cells was investigated by interfering with the expression of lncRNA 165 and 3304 in 3D4/21 cells with specific small interfering RNA(siRNA)fragments.TGF-β1 expression in cells was studied by RNA interference assay and pretreatment cells with recombinant TGF-β1.The effects of lncRNAs 165 and 3304 on the expression of Fn and α5 were confirmed by RNA interference assay and flow cytometry.The over-expression lncRNA 165 and 3304 was performed to reveal bacterial invading to cells.The high homology encoding genes with lncRNA 165 and 3304 were analyzed by Nucleotide BLAST and the structures of lncRNAs 165 and 3304 and PTGS2 were analyzed by the website RNAfold web server.The results showed that the number of bacteria in the 3D4/21 cells infected with Hps5 was decreased after transfecting the cells with the siRNA fragments of lncRNA 165 and 3304.This supports the hypothesis that lncRNA 165 and 3304 are involved in the regulation of H.parasuis invasion.The expression of TGF-β1 in RNA interference-treated cells was significantly decreased,and the expression of fibronectin Fn and integrin α5 was also decreased.The levels of lncRNAs 165 and 3304 in 3D4/21 cells pretreated with TGF-β1 were also up-regulated.These results indicate that the expression of lncRNA 165 and 3304 is associated with TGF-β1 expression.Cells treated with LncRNA165-FLAG had more intracellular bacteria compared to the control group,while cells treated with LncRNA3 304-FLAG had fewer bacteria.We found that lncRNA 3304 and PTGS2 are highly homologous.Then,the structures of lncRNA 165,3304 and PTGS2 showed that they have different conformations.These results indicate that lncRNAs play an important role in H.parasuis infection,while lncRNAs 165 and 3304 affect the invasion of H.parasuis by regulating the TGF-β1 signaling pathway.