Mechanisms Of Hsa-microRNA-1297 Negatively Regulating PLC?1 Participating In Intestinal Barrier Injury

Background and Objectives:The intestinal barrier serves as the first line of defense against the harsh environment of the lumen.Destruction of the barrier can lead to serious pathological consequences such as inflammation or diarrhea;Acquired immunodeficiency syndrome(AIDS)is caused by humans.A chronic consumptive infectious disease caused by immunodeficiency virus(HIV)caused by a decrease in CD4+T cells in peripheral blood.The gut is the most important target organ of HIV attack.CD4+T cells in the early intestinal tract are depleted,intestinal mucosal immunity is impaired,intestinal mucosal barrier permeability is increased,allowing microbes and immunostimulatory biological products to first The intestinal lumen in the lamina priory passes through and then passes through the systemic circulation,thereby continue to promote local and systemic inflammatory responses.Therefore,the mechanism of intestinal barrier injury,to find the biomarkers repaired after intestinal barrier injury,is of great significance to the prognosis and progression of the patient.Changes in miRNAs in the small intestine of HIV-infected patients play a major role in intestinal lesions.Inflammation affects the regeneration of intestinal epithelial cells.Some miRNAs regulate intestinal epithelial membrane permeability and viral proliferation through target genes,possibly involving inflammation,peroxidation,and cells.Apoptosis,etc.Changes in miRNAs in the gut of HIV-infected individuals may be an important cause of intestinal lesions.However,how miRNA-mRNA affects the integrity of the downstream intestinal epithelial barrier,gastrointestinal physiology and function remains unclear.MiR-1297 has been widely studied in the function of the digestive tract,such as diagnostic biomarkers in esophageal squamous cell carcinoma;targeting cyclooxygenase-2 to regulate invasion of colorectal cancer cells;its down-regulation targeting CREB1 enhances gastric cancer cells Growth,etc.Our preliminary work has predicted that miR-1297 may be an important molecule regulating the growth and migration of intestinal mucosal injury cell model,further confirming that its target gene and its mechanism provide a better understanding of the regulation of intestinal mucosal injury repair process in AIDS patients.Our previous work also screened and signaled pathways to AIDS intestinal mucosal genomic expression(including miRNAs and mRNAs),and obtained target genes that may play important roles in key pathways such as tight junction-associated pathways and cell adhesion.PLC?1 and its downstream ZO-1 gene,and prediction of miR-1297 by genomics may be important factors regulating PLC?1 and its downstream pathway.The epithelial repair mechanism rapidly rebuilds continuous epithelial monolayers to prevent bacterial toxin absorption and reduce the ability of pathogens and their toxins to invade the mucosa.Cytoplasmic plaques bind to membrane proteins to form a tight junction that regulates cell-by-cell permeability.The protein ZO-1 of the granulate kinase(MAGUK)superfamily constitutes a major component of cytoplasmic plaques and binds to muscle movement towards/away mucosal repair.Protein rearranges the cytoskeleton.Therefore,PLC?1 and its downstream ZO-1 gene expression are associated with the repair of damaged intestinal mucosa.Lipopolysaccharide(LPS)in the cell wall of Gram-negative bacteria treats human colonic epithelial cell lines to mimic the damage to intestinal epithelial cells in the body,and the intestinal mucosa undergoes an inflammatory process.In this study,we constructed a human colon epithelial cell model of LPS-induced intestinal barrier injury,and focused on the key gene PLC?1 and the possible upstream regulator miR-1297.We explored the changes of these two molecules in the process of LPS-induced intestinal barrier injury repair,and preliminarily explored the important targets related to the early intestinal mucosal injury of HIV/AIDS,which will be better to understand and clarify the repair process of intestinal mucosal injury in AIDS patients provides.Methods:Establishment of LPS-damaged intestinal epithelial cell model,construction of microRNA-1297 overexpression and interference lentivirus system,transfection of LPS-damaged CCC-HIE-2 cells,detection of microRNA-1297 expression in cells by RT-q PCR,Western blot assay to detect the effect of microRNA-1297 on the expression of target gene PLC?1 in cell model.The expression of tight junction protein ZO-1 downstream of PLC?1 was detected by Western blot and double fluorescence assay.Cell viability and permeability were measured by MTT and fluorescence yellow,and the biological function of CCC-HIE-2 cells damaged by LPS was observed by over-expression or interference of PLC?1 and over-expression and interference of miR-1297.High-throughput sequencing is used to analyze the effect of over-expression of PLC?1 on transcriptome in cell models.Bioinformatics identifies the differentially expressed genes and their functional annotations after over-expression ofPLC?1,and explores the signaling pathways that differentially expressed genes may participate in in the cell for subsequent validation studies.Results:1.Overexpression of miR-1297 down-regulated the expression of PLC?1 protein,and miR-1297 inhibited the up-regulation of PLC?1 protein expression,demonstrating that miR-1297 negatively regulates gene PLC?1.2.Inhibition of miR-1297 enhanced the activity of CCC-HIE-2 cells damaged by LPS,indicating that inhibition of miR-1297 may promote the growth of intestinal mucosal epithelial cells injured by LPS.LPS injury after overexpression of PL?1 gene CCC-HIE-2 cell viability assay showed that overexpression of PLC?1 enhanced cell viability.3.The expression of miR-1297 was up-regulated by the expression of miR-1297 and the downstream ZO-1 gene was up-regulated by miR-1297ib transfect LCC-infected CCC-HIE-2 cells.Overexpression of PLC?1 gene can up-regulate the expression of downstream ZO-1 gene,and the expression of the two is positively correlated Overexpression of miR-1297 down-regulated the expression of PLC?1 protein,and down-regulated the expression of downstream ZO-1 protein.4.Inhibition of miR-1297 expression reduced cell permeability;overexpression of PLC?1 reduced LPS-damaged CCC-HIE-2 cell permeability.Overexpression miR-1297 enhanced the permeability of CCC-HIE-2 cells;knockdown of PLC?1 increased CCC-HIE-2 cell permeability.5.The transcriptome sequencing results showed that 2908 genes were up-regulated in the transcriptome expression profile of over-expressing PLCK?1 gene.The most enriched biological process(BP)is oxidation-reduction process,the highest concentration of cellular components(CC)is Cytoplasm,the most enriched molecular function(MF)is protein binding;the most significant signaling pathway is Ribosome.6.The transcriptome sequencing results showed that 1645 genes were down-regulated in the transcriptome expression profile of over-expressing PLC?1 gene.The most enriched biological process(BP)is regulation of transcription,the most enriched cellular component(CC)is cytoplasm,the most enriched molecular function(MF)is metal ion binding,the most significant signaling pathway is TGF-beta signaling pathway.7.In the CCC-HIE-2 cells,the transcriptome sequencing results showed that 181 genes were up-regulated in the transcriptome expression profile of knockout miR-1297,and the biological process(BP)with the highest degree of enrichment was:urea cycle;The most enriched cell component(CC)is:mitochondrial matrix;the most enriched molecular function(MF)is:calmodulin binding The most significant signaling pathway for downregulating mRNAs enrichment is:Arginine biosynthesis.8.In the CCC-HIE-2 cells,the transcriptome sequencing results showed that 2948 genes were down-regulated in the transcriptome expression profile of knockout miR-1297,the most enriched biological process(BP):chemical stimulation in olfactory perception Detection of chemical stimulus involved in sensory perception of smell;the highest enriched cell component(CC)is:keratin filament;the most enriched molecular function in down-regulated genes(The molecular function(MF)is:olfactory receptor activity;the most significant signaling pathway for down-regulating mRNAs is:Olfactory transduction.Conclusion:The results showed that miR-1297 negatively regulated PLC?1.The down-regulation of PLC?1 in intestinal barrier repair was caused by the over-expression of miR-1297 and the down-regulation of downstream tight junction protein ZO-1,which resulted in the blockage of cytoskeleton rearrangement in the process of epithelial cell repair and the decrease of viability and permeability of intestinal epithelial cell model damaged by LPS.Apoptosis.Correspondingly,up-regulation of the expression of PLC?1 or down-regulation of the expression of miR-1297 can repair the damage of CCC-HIE-2 cells caused by LPS,and leading to apoptosis ultimately.PLC?1 and miR-1297 may become important targets for the repair of intestinal barrier damage.