| Objectives:The aims of this study were to investigate the cellular and molecular biology mechanismsin intestinal mucosa injury of HIV/AIDS patients.Through analyzing differentially expressed IncRNA,mRNA and microRNA(miRNA)in intestinal mucosabetween HIV/AIDS patients and healthy individuals,we systematically illuminated the molecular mechanism in intestinal mucosa injury of HIV/AIDS patients,further expand the width and depth on the study of molecular mechanism in intestinal mucosa injury of HIV/AIDS patients and found the potential target for the treatment of HIV/AIDS.Methods:First,by IncRNA and miRNA assay,differentiall expressed IncRNA and its associated mRNA,and miRNA were detected.Then,the target genes of differentially expressed IncRNA and mRNA,GO and relative pathway of the target genes were predicted through GO and KEGG pathway analysis.Finally,the key IncRNA,mRNA and miRNA were found by intersecting the target genes of lncRNA,mRNA and miRNA.Results:The results of IncRNA assay showed that there were 6656 lncRNAs and 4823 mRNAs with fold change>2.Among 6656 lncRNAs,3917 lncRNAs were upregulated and 2516 lncRNAs were downregulated with P<0.05 and FDR<0.05.Among 4823mRNAs,1477mRNAs were upregulated and 3280mRNAs were downregulated with P<0.05 and FDR<0.05.The results of IncRNA assay showed that 2079 miRNAs were detected,among them,there were 295 differentially expressed miRNAs(203 upregulated and 92 downregulated)with fold change>2 and P<0.05.According to enrichment socre,mRNAs associated with upregulated lncRNAs and target genes of downregulated miRNAs were mainly involved in lymphocyte activation(BP),T cell receptor complex(CC)and signaling adaptor activity(MF),while mRNAs associated with downregulated IncRNAs and target genes of upregulated miRNAs were mainly engaged in anatomical structure morphogenesis(BP),extracellular matrix(CC)and protein binding(MF).The KEGG pathway analysis showed that mRNAs associated with upregulated lncRNAs and target genes of downregulated miRNAs were mainly involved in T cell receptor signaling pathway,while mRNAs associated with downregulated lncRNAs and target genes of upregulated miRNAs were mainly involved in ECM-receptor interaction,PI3K-Akt signaling pathway,MAPK signaling pathway and dilated cardiomyopathy.In addition,two genes,RUSC2 and ELN,were obtained through intersecting the target genes of downregulated miRNAs,upregulated mRNAs and targeted genes of all lncRNAs,while 52 genes were got by intersecting the target genes of upregulated miRNAs,downregulated mRNAs and targeted genes of all lncRNAs.Finally,by integrating the validation of q-PCR,lncRNA and miRNA assay,and bioninformatics analysis,we found that the expression of ITGA5 and IncRNA RP11-753H16.3 were obviously decreased,hsa-miR-32-5p were increased in intestinal mucosa of AIDS patients compared with health individuals.Moreover,ITGA5 was the common target of IncRNA RP11-753H16.3 and hsa-miR-32-5p.Conclusions:First,compared to intestinal mucosa of health individuals,various lncRNAs,mRNAs and miRNAs were altered in AIDS patients.Then,mRNAs associated with these lncRNAs and target genes of miRNAs were mainly involved in GO including lymphocyte activation,T cell receptor complex,signaling adaptor activity,anatomical structure morphogenesis,extracellular matrix and protein binding.Moreover,mRNAs associated with these IncRNAs and target genes of miRNAs were mainly involved in pathways including T cell receptor signaling pathway,ECM-receptor interaction,PI3K-Akt signaling pathway,MAPK signaling pathway and dilated cardiomyopathy.In addition,we found that ITGA5 was the common target of IncRNA RP11-753H16.3 and hsa-miR-32-5p.These suggested that IncRNA RP11-753H16.3 and hsa-miR-32-5p might involve the mechanism in intestinal mucosa injury of HIV/AIDS patients which need to further study.Objectives:The aims of this study were to investigate the role of lncRNA RP11-753H16.3 in intestinal epithelial cell subjected to HIV/AIDS.Methods:HIV/AIDS intestinal epithelial cell model was built using Caco-2 cell line and Tat protein.Recombinant lentivirus plasmids of lncRNA RP11-753H16.3interference(LV-lnc shl,LV-lnc sh2)were established and packed with lentivirus.Cell viability assay,cell matrigel invasion assay and permeability experiment were used to investigated the effects of interferinglncRNA RP11-753H16.3 on cell growth,migration and permeability after transfecting LV-lnc sh1,LV-lnc sh2 into HIV/AIDS Caco-2 cells,respectively.Western blot was used to detect the effects ofinterfering lncRNA RP11-753H16.3 on the expression of ITGA5,p-p38,p-ERK,p-JNK,p-cMET,p-beta-catenin,p53,?H2AX,acH4.Results:Quantitative analysis of Caco-2 cells growth in two-dimension showed that chemiluminescence values were no difference in groups on day 0(D0).While,on D3 and D7,the chemiluminescence values of Caco-2 cells in LV-sc group were both higher than LV-lnc sh-1 and LV-lnc sh-2.Quantitative analysis of Caco-2 cells growth in three-dimension showed that chemiluminescence values were no difference in groups on day 0(D0).While,on D3 and D7,the chemiluminescence values of Caco-2 cells in LV-sc group were both higher than LV-lnc sh-1 and LV-lnc sh-2 groups.These results showed interfering RP11-753H16.3 could reduce the viability of Caco-2 cells which indicated that interfering RP11-753H16.3 might inhibit the growth of HIV/AIDS intestinal epithelial cell.On D4,D12,D18,compared to LV-sc group,primmorph of Caco-2 cells were smaller inLV-lnc sh-1 and LV-lnc sh-2 groups.These results suggest that interfering RP11-753H16.3 might inhibit the migration of Caco-2 cells.At 30min,60min,90minand 120min,there were no differences in concentration of penetrativefluoresceinin Caco-2 cells between Naive and LV-sc groups.The concentration of penetrativefluoresceinin Caco-2 cells in LV-lnc sh-1 and LV-lnc sh-2 groups were both higher than Naive and LV-sc groups,and the concentration of penetrativefluorescein in Caco-2 cells in LV-lnc sh-1 group was higher than LV-lnc sh-2 group.Papp was calculated according to Papp=?Q/(?t·A·C0)(cm·s-1).The results showed that Papp in Naive and LV-sc groups was no difference.Papp in Caco-2 cells in LV-lnc sh-1 and LV-lnc sh-2 groups were both higher than Naive and LV-sc groups,and Papp in Caco-2 cells in LV-lnc sh-1 group was higher than.LV-lnc sh-2 group.These results indicated that interfering RP11-753H16.3 increase thepermeabilityof Caco-2 cells.Interfering RP11-753H16.3 could inhibit the protein expressions of ITGA5.p-p38,p-JNK,p-? catenin and p-53,promote the protein expressions ofp-cMET,yH2AXand acH4,and had little effects on the protein expression of p-ERK.Conclusions:Interfering RP11-753H16.3 might inhibit the cell growth and migration of HIV/AIDS intestinal epithelial cell,promote its permeability.These functions of RP11-753H16.3 might be associated and act through inhibiting the activation of ITGA5,p-p38 and/or promoting p-cMET.Objectives:The aims of this study were to investigate the role of ITGA5 and has-miR-32-5p in intestinal epithelial cell subjected to HIV/AIDS.Methods:Recombinant lentivirus plasmids of ITGA5 interference(LV-sh1,LV-sh2)and has-miR-32-5p overexpression were established and packed with lentivirus.Cell viability assay,cell matrigel invasion assay and permeability experiment were also used to investigated the effects of interferingITGA5 and ovexpression has-miR-32-5p on cell growth,migration and permeability after transfecting LV-sh1,LV-sh2and LV-premir into HIV/AIDS Caco-2 cells,respectively.Western blot was also used to detect the effects ofinterfering ITGA5 and overexpressing has-miR-32-5p on the expression of ITGA5,p-p38,p-ERK,p-JNK,p-cMET,p-beta-catenin,p53,?H2AX,acH4.Results:Quantitative analysis of Caco-2 cells growth showed that chemiluminescence values were no difference in groups on day 0(DO).While,on D3 and D7,the chemiluminescence values of Caco-2 cells in LV-sc group were both higher than LV-premir,LV-shl and LV-sh2 groups.These results showed interfering ITGA5 and overexpression of miR-32 could reduce the viability of Caco-2 cells which indicated that interfering ITGA5and overexpression of miR-32 might inhibit the growth of HIV/AIDS intestinal epithelial cell.On D2,D12,D18,there were no differences in primmorph of Caco-2 cells between Naive,LV-sc and LV-premir groups.Compared to LV-sc and Naive groups,primmorph of Caco-2 cells were smaller inLV-lnc sh-1 and LV-lnc sh-2 groups.These results suggest that interfering ITGA5 and overexpression of miR-32 might inhibit the migration of Caco-2 cells.At 30min,60min,90minand 120min,there were no differences in concentration of penetrativefluorescein in Caco-2 cells between Naive and LV-sc groups.The concentration of penetrativefluorescein in Caco-2 cells in LV-premir,LV-sh1 and LV-sh2 groups were bothhigher than Naive and LV-sc groups,whlie the concentration of penetrativefluorescein in Caco-2 cells in LV-sh2 group was higher than LV-premir and LV-shl groups.The concentration of penetrativefluorescein in Caco-2 cells in LV-shl group was higher than LV-premir group.These results indicated that interfering ITGA5 and overexpression of miR-32increase thepermeabilityof Caco-2 cells.The protein expression ITGA5 was decresed after transfecting LV-sh1 and LV-sh2.Interfering ITGA5 could inhibit the protein expressions of p-p38,p-JNKand ?H2AX,promote the protein expressions ofp-cMETand p-? catenin,and had little effects on the protein expression of p53 and acH4.Overexpression of miR-32 could inhibit the protein expressions of p-p38,p-? catenin,p53,yH2AX and acH4,promote the protein expressions ofp-cMET,and had little effects on the protein expression of ITGA5,p-ERK and p-JNK.Conclusions:Interfering ITGA5 and overexpression of miR-32 might inhibit the cell growth and migration of HIV/AIDS intestinal epithelial cell,promote its permeability.These functions of ITGA5 and miR-32 might be associated and act through inhibiting the activation of p-p38 and/or promoting p-cMET. |
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