LncRNACNN3-206 Selected By High-throughput Sequencing Promoted The Apoptosis And Invasion Of Intestinal Epithelial Cells,Mediated The Occurrence Of Crohn’s Disease Through Activating Of Caspase-10

Background :Although the incidence of Crohn’s disease(CD)in China is not as high as in European and American countries,there has been a clear increasing trend in recent years.Little is known about its pathogenesis,cause of deferment and the range of complications associated with the disease.Local and international scholars have presented many hypotheses of CD pathogenesis based on experimental and clinical studies,including genetic susceptibility,immune function defects,intestinal microflora disorders,delayed hypersensitivity and food antigen stimulation.But the specific mechanism leading to this immune imbalance,which causes persistent intestinal mucosal damage,and the source of the inflammatory cascade reaction are still unclear.So far,the results of research studies differ locally and internationally.The most current research in immune correlation is presented in this summary.However,these results are not only the same,but there are no well-recognized and highly reproducible studies to fully demonstrate the pathogenesis of Crohn’s disease.At present,the tendency of Crohn’s disease is the result of the combination of genetic and environmental factors.Most of the changes in the environment lead to mutations,which increase or decrease the production of non-codingRNAs such as lncRNA,miRNA,circleRNA,siRNA,and these non-coding genes.In particular,the most abundant expression of lncRNA,which is widely involved in epigenetic regulation,may result in a sustained inflammatory response in intestinal epithelial cells.There are few reports at home and abroad on whether lncRNA is involved in the pathogenesis of CD.ObjectivesBy using High-throughput Sequencing Technology,the abnormal intestinal mucosal tissue at terminal ileum and the difference of lncRNA and mRNA expression in the normal tissues at terminal ileum in healthy controls are screened by High-throughput Sequencing Technology;the co-expressed mRNA of lncRNA differential expression is predicted by using bioinformation analytical technique CNC,which lays a foundation for exploring the role of lncRNA in intestinal mucosa.To determine which cell and cell substructure lncrna is located in intestinal mucosa.To predict possible regulatory mechanisms such as Ce mechanisms or Cis/Trans based on the location of lncRNA in cells.According to the regulatory mechanism,to construct lncRNA,mRNA co-expression network by using correlation analysis.To predict intermediate regulatory factor miRNA by using miRBase/Target Scan,and finally lncRNA-miRNA-mRNA gene regulatory pathway may get involve in the regulation mechanism of inflammatory responses in CD will be obtained.According to the predicted regulatory pathway of lncRNA-miRNA-mRNA gene,the possible mechanism involved in the regulation is verified at the clinical and cellular levels.At the animal level,by intervening the level of lncRNA,we observed whether the physiological state,the degree of pathological damage of colon and the proportion of related immune cells of the model animals were significantly improved.MethodsThe totalRNA was extracted from the diseased intestinal mucosa of 40 CD patients and40 normal intestinal mucosa of 40 healthy subjects.The totalRNA of 3 purified samples from each group was randomly selected for high-throughput sequencing and bioinformatics analysis.The lncRNA and mRNA expressed differently in the abnormal ileal mucosa of CD patients and the normal ileal tissues of healthy people were screened out.Screening criteria:lncRNA and mRNA molecules were screened out in pathological intestinal mucosa and normal intestinal mucosa according to the criteria of difference multiple greater than or equal to 10 and P less than or equal to 0.05.Using the method of Pearson correlation coefficient(CNC analysis)to predict the co-expressed mRNA of differentially expressed lncRNA;aiming at the target genes of differentially expressed lncRNA,using the method of bio-informatics to predict and analyze whether there are regulatory factors in the mRNA of CO expressed target genes of differentially expressed lncRNA.To enlarge the sample size,use the totalRNA of CD pathological intestinal mucosal and normal intestinal mucosal tissues(40: 40)to validate the representative differential expression of lncRNA,co-expression gene mRNA and whether they are the really differential expression in intestinal mucosal tissues byRT-PCR Use statistical methods of bio-information analytical technique to find the correlation between the significantly differential expression of lncRNA and significantly differential expression of mRNA.According to mRNA,perform GO analysis and Pathway analysis to predict the function of its regulators,which lay a foundation to explore the function of lncRNA in intestinal mucosa.In this study,the lncRNACNN3-206 with the most significant difference and the co-expression gene Caspase10 are selected as research objectives.FISH experiment determined the distribution of lncRNACNN3-206 mainly in intestinal mucosal epithelial cells,lymphocytes,fibroblasts and other cells.The electron microscope was used to observe whether it was mainly distributed in cytoplasm or nucleus.Because the target gene is mainly regulated by Ce mechanism if it located in cytoplasm and the target gene is mainly regulated by Cis/Trans mechanism if it located in nucleus.Construct the co-expression network of lncRNACNN3-206,mRNA(Caspase10)based on correlation analysis,use miRBase/Target Scan to predict intermediate regulatory factor miRNA(miR-212).Preliminarily establish lncRNACNN3-206-miR-212-Caspase10 gene expression regulatory network.At the cell level,clarify the specific molecular mechanism of lncRNACNN3-206 on regulating the target gene Caspase 10 and the biologic functions on specific cells of intestinal mucosa(cell proliferation,apoptosis,cell migration,autophagy,etc.).Finally,at the animal level,by intervening the level of lncRNACNN3-206 in intestinal epithelial cells,to observe whether the physiological state of CD model animals,the degree of pathological damage of colonic mucosa,the proportion of immune cells promoting the progress of CD in blood circulation etc.show the significant improvements.Results:A total of 51388 lncRNAs were detected in overallRNA sequencing and screening of6 tissue specimens,and a total of 400 lncRNAs experienced significant difference in comparison of pathological tissues in patients with CD and normal tissues,in which the expression up-regulation of 243 lncRNAs in pathological tissues was obviously superior to the normal mucosa in the control group,and expression down-regulation of 157 lncRNAs in the positive tissues was significantly more than that in the control group(p < 0.01).The top 10 lncRNAs with the most prominent up-regulation and down-regulation predicted in the biological information analysis were subjected to screening and cluster analysis,and Top5 lncRNAs in differential expression subjected to expanded clinical specimen verification.CD terminal ileum lesions and terminal ileum mucosa of normal subjects(40 cases: 40 cases)were used forRT-PCR verification,and a t-test comparison analysis was performed to prove the differential expression in the tissues and to confirm whether it was consistent with the gene sequencing results.It was found that 6 lncRNAs were consistent with the results of chip inspection,wherein the difference was most significant in lncRNA ZNF292 and lncRNACNN3-206.Since lncRNAZNF293 data standard deviation is greater than lncRNACNN3-206,lncRNACNN3-206 with greater data reliability was selected as the object of study.FISH experiments determined that lncRNACNN3-206 was mainly distributed in the cytoplasm of intestinal epithelial cells,and starting from Ce mechanism of lncRNA inside the cytoplasm,miRBase/Target Scan was used for prediction of intermediate adjustment factor miRNAs(miR212)of lncRNACNN3-206 and Caspase10.The lncRNACNN3-206-miR-212-Caspase10 gene expression regulation network was established initially.In intestinal epithelial cells,regulation of Caspase10 by lncRNACNN3-206 was achieved by adsorption of miR212,and by adsorption of large amount of miR212,lncRNACNN3-206 reduced the concentration of intracellular free miR212,thereby suppressing the silencing of miR212 on Caspase10.The ultimate result is that lncRNACNN3-206 promotes the generation of a large number of apoptotic Caspase10 in the cell.At the cellular level,it expounded that by adsorption of miR-212 regulation target gene Caspase10,lncRNA promoted intestinal epithelial cell apoptosis and migration,and the molecular mechanism of autophagy,and fundamentally explained that intestinal inflammation of patients with CD is different from normal intestinal inflammation and ulcerative colitis manifestation,the intestinal mucosa layer quickly infiltrates into the mucosal muscle layer,submucosa layer,and serosa layer,easily forming transmural intestinal inflammation and even intestinal fistula.It revealed why intestinal fistula is the root cause of major complications in patients with CD.Finally,in the intestinal tract of Crohn’s disease model mice,the level of intestinal mucosa lncRNACNN3-206 was intervened.After treatment,the physiological condition of the model mice was observed to be significantly improved,the degree of colonic pathological damage was significantly reduced,and the proportion of relevant immune cells was close to normal.Conclusions:The type and content of lncRNA in the diseased intestinal mucosal tissue of patients with CD and normal tissues of healthy subjects were significantly different,indicating that the differentially expressed lncRNA in the diseased tissue plays an important role in the progress of intestinal mucosal inflammation.lncRNACNN3-206 stimulates the biological behaviors of intestinal epithelial cell apoptosis,migration,and autophagy through lncRNACNN3-206-miR-212-Caspase10 gene regulation network,and plays a key role in inducing the onset of CD and maintaining the continued development of the disease.Therefore,lncRNACNN3-206 can be used as a new target for the study of CD treatment protocols,providing new ideas and research directions for human gene therapy of CD.