Study On The Function And Regulatory Mechanism Of Gln Transporter ASCT2 In Intestinal Barrier Injury And Repair

Objectives:Intestinal barrier injury plays an important role in the occurrence,development and prognosis of many diseases,especially in the progress of Acquired immune deficiency syndrome(AIDS).When HIV infects humans,it first attacks the gastrointestinal tract,causing damage to the intestinal barrier,which in turn leads to microbial displacement.Immune activation caused by microbial translocation is the main reason to increase the incidence of non-aids-related complications,reduce the quality of life of patients with chronic infection,and is an important factor to promote the progression of AIDS disease and accelerate the death of patients.Therefore,the research on the molecular mechanism of intestinal barrier injury and the search for therapeutic targets for the repair of intestinal barrier injury are of far-reaching significance for improving the quality of life of patients,delaying the disease process and prolonging the life of patients.Glutamine(Gln),as an important immune nutrient,plays a critical role in maintaining the function of intestinal mucosal barrier.In the absence of glutamine,intestinal barrier function is severely impaired.However,little is known about the abnormal glutamine metabolism in the damaged intestinal mucosa,especially in the hiv-infection-related damaged intestinal mucosa.Alanine,serine,cysteine transporter 2(Alanine,serine,cysteine-preferring from 2,ASCT2)is the most important glutamine transport carrier,widely distributed in the metabolism of organizations,such as intestinal,fat and tumor tissue.Available data show that ASCT2 can promote tumor deterioration by affecting the proliferation and apoptosis of tumor cells,but the exact role of ASCT2 in intestinal barrier damage and repair is still unknown.Our previous work found that low expression of ASCT2 was found in the damaged intestinal mucosa of HIV/AIDS patients,suggesting that ASCT2 may be related to the damage and repair of intestinal barrier in patients.Bacterial product Lipopolysaccharide(LPS)entered the intestinal lamella propria and induced inflammatory reaction,resulting in massive abnormal apoptosis of intestinal epithelial cells,which is the main reason for the damage of intestinal barrier associated with HIV infection.This study will establish a model of intestinal epithelial cells damaged by LPS,and then we will take ASCT2 as the center to study its biological function and related regulatory mechanism in the repair of lps-induced intestinal barrier injury,so as to provide new ideas for gene targeted therapy of intestinal barrier injury.Methods:The intestinal epithelial cell model was established after LPS injury,and the effects of LPS on the proliferation and apoptosis rate of intestinal epithelial cells were detected by MTT and flow cytometry,respectively.Real-time reverse transcription PCR was used to confirm the abnormal expression of ASCT2 in the intestinal epithelial cell model after LPS injury.Overexpression/interference of ASCT2 to explore the phenotypic effects of ASCT2 on lps-induced intestinal epithelial cell injury;High-throughput gene chip technology was used to analyze the influence of abnormal expression of ASCT2 on the whole transcriptome,and to find the signal pathway that may be regulated by ASCT2 for subsequent verification.Glutamine deletion and replacement experiment was used to study the repair effect of Gln on LPS damaged cells and the regulation effect of Gln on ASCT2.Bioinformatics predicted the miRNAs that might regulate ASCT2,and the miRNA with the most significant expression differences in the intestinal mucosa of HIV/AIDS patients were selected as subjects for further study according to the chip results at the early stage.miMiRNA mimics and inhibitors were used to investigate the regulatory effect of the miRNA on ASCT2 and its phenotypic effect on LPS injured intestinal epithelial cells.Luciferase reporter gene assay verified the target regulation effect of miRNA on ASCT2.Results:1.Compared with the control group,100 ng/ml LPS could significantly decrease the proliferation capacity of ccc-hie-2 cells(P<0.05)and significantly increase the apoptosis rate(P<0.05).100 ng/ml LPS was used as the modeling condition.2.Compared with the control group,the expression of ASCT2 in ccc-hie-2 with 100 ng/ml LPS injury was significantly down-regulated(P<0.05).3.ASCT2 silencing inhibited the proliferation of lps-damaged cells and promoted apoptosis(P<0.05).On the contrary,overexpression of ASCT2 can increase cell proliferation,reduce apoptosis and repair damage caused by LPS(P<0.05).4.By mRNA Chips analysis of ASCT2 overexpressed LPS injury model cells,20705 differentially expressed mRNAs were detected.Among them,there were 1066 mRNAs whose variation multiples were greater than 2 times.Of these 1066 mRNAs,732(P<0.05 and FDR<0.05)were up-regulated and 33(P<0.05 and FDR<0.05)were down-regulated.GO function enrichment analysis suggests that abnormal expression of ASCT2 may affect the development of a multicellular organism,cell proliferation,B cell-mediated immune response,immune globulin mediated immune response and so on a variety of cellular functions,KEGG signaling pathway analysis suggests that abnormal expression of ASCT2 may interact with cells factor-factor receptor,PI3K-Akt signaling pathways,fat protein digestion,absorption,digestion,absorption and glycerol phospholipid metabolism related pathways,etc.5.Verification of the PI3K-Akt-mTOR signaling pathway showed that,compared with the model group,the expressions of Akt,mTOR and P70S6K were significantly up-regulated after the overexpression of ASCT2(P<0.05),but the expressions of PI3K were not changed(P>0.05),while the expressions of Akt,mTOR and P70S6K were significantly down-regulated after the silencing of ASCT2(P<0.05),and the expressions of PI3K were not changed(P>0.05).6.With the increase of glutamine concentration in the culture medium,the expression of ASCT2 was gradually up-regulated,while the apoptosis of CCC-HIE-2 cells damaged by LPS was significantly reduced,but Gln at too high concentration could induce apoptosis.7.Three databases,miRTarBase,TarBase and TargetScan,were used to predict the miRNAs that might regulate ASCT2.Then,the intersection was taken to obtain three mimas,which are hsa-miR-125b-5p,hsa-miR-137 and hsa-miR-331-3p.Among them,hsa-miR-125b-5p has the most significant difference in miRNAs gene chips of damaged intestinal mucosa in HIV/AIDS patients,which is the object for further study.8.The target genes of hsa-miR-125b-5p were predicted,and 35 possible target genes(ASCT2 was one of them)were obtained by the intersection of them.The GO analysis showed that the biological process of the target gene set was mainly enriched in cell growth and/or maintenance,apoptosis,cell maturation and proliferation,etc.Analysis of KEGG signaling pathway suggested that its target genes were mainly involved in PI3K-Akt-mTOR signaling pathway,p53 pathway,SIP pathway,etc.It also involves cell proliferation,apoptosis and the PI3K-Akt-mTOR signaling pathway.9.Compared with the control group,the mimics of hsa-miR-125b-5p can lead to down-regulation of ASCT2 protein and mRNA level(P<0.05),while the inhibitor of hsa-miR-125b-5p can cause up-regulation of ASCT2 protein and mRNA level(P<0.05),indicating that hsa-miR-125b-5p has a negative regulatory effect on the expression of ASCT2.Dual-luciferin gene reporting assays demonstrated that hsa-miR-125b-5p performs its regulatory function by targeting the 3' UTR region of ASCT2.10.Compared with the control group,the mimics of hsa-miR-125b-5p inhibited cell proliferation and induced apoptosis(P<0.05),while the inhibitor of hsa-miR-125b-5p promoted cell proliferation and reduced apoptosis(P<0.05).The overexpression of hsa-miR-125b-5p inhibited the expression of important protein kinases(Akt,mTOR and P70S6K)in the AKt-mTOR signaling pathway(P<0.05),while the PI3K expression remained unchanged(P>0.05),while the hsa-miR-125b-5p inhibitor promoted the expression of Akt,mTOR and P70S6K(P<0.05),and PI3K expression remained unchanged(P>0.05).Conclusion:Our results suggest that hsa-miR-125b-5p regulates the expression of ASCT2 by binding to the 3'UTR region of ASCT2,and mediates the Akt-mTOR pathway to affect the proliferation and apoptosis of LPS-injured intestinal epithelial cells,which is one of the important causes of LPS-induced intestinal barrier injury.Down-regulating hsa-miR-125b-5p or up-regulating the expression of ASCT2 can repair the damage of CCC-HIE-2 cells caused by LPS to some extent.hsa-miR-125b-5p and ASCT2 are expected to be important targets for intestinal barrier injury repair.