| Research backgroundThe upregulated expression of epithelial sodium channels(ENa C)can promote Na+ transport,which drives alveolar fluid clearance(AFC).Therefore,ENAC upregulation may benefit patients with acute respiratory distress syndrome(ARDS)by reducing lung edema.ENaC expression is mainly regulated by the mTORC2/SGK-1 signaling pathway.MicroRNAs(miRNAs)are small non-coding RNAs that regulate the translation of their target mRNAs.MiRNA-7-5p is involved in the regulation of mTOR in tumor cells.However,the mechanism of miRNA-7-5p regulation of ENa C in human alveolar epithelial cells is unclear,and whether it can affect the expression of ENaC in ARDS has not been reported.PurposeThe aim of this study was to investigate the effect of miRNA-7-5p on human alveolar epithelial sodium channels and clarify the pathway in which miRNA-7-5p regulates the expression of ENa C in ARDS.It can enrich the molecular biology theory of ARDS pathogenesis and provide new clues for the clinical treatment of the disease.MethodsThis study consists of three partsPart 1: Dual-luciferase assay reporter system to verify miRNA-7-5p target gene.First,the recombinant DNA plasmid was used to construct psiCHECK2-mTOR?psiCHECK2-mTOR-mut?psiCHECK2-SGK-1 and psiCHECK2-SGK-1-mut four groups of mRNA 3 '-UTR recombinant gene that containing miRNA-7-5p target sequence.Then,we confirmed that mTOR and SGK-1 are the target genes of miRNA-7-5p by Dual-luciferase assay reporter system respectively.Part 2: Study on the signaling pathway which miRNA-7-5p regulates the ENa C in human alveolar epithelial cells.Upregulation of miRNA-7-5p expression in alveolar epithelial cells by transfected with miRNA-7-5p mimics.Using qRT-PCR to detect the experimental group of miRNA-7-5p,mRNA and mTOR SKG-1 mRNA expression level.Western blotting was used to detect the mTOR?Total AKT?p-AKT-ser473 and SGK-1 protein expression.Finally,the effection of miRNA-7-5p on the expression of ENaC subunits was verified.Part 3: The study of miRNA-7-5p regulates the ENaC in human alveolar epithelial cells after LPS affection.LPS at a concentration of 10 ng/mL was used to stimulate the cells.The expressions of miRNA-7-5p,mTOR,SGK-1,p-Akt-Ser473,?-,?-,and ?-ENaC were detected by quantitative real-time polymerase chain reaction?western blotting and Immunofluorescence microscopy.ResultsPart 1: Using TargetScan,we found that the 3'-UTRs of mTOR and SGK-1 contained binding sequences for miRNA-7-5p,indicating that the two genes were the targets of miRNA-7-5p.To confirm this finding,a dual luciferase target gene assaywas conducted.Wild-type and mutant reporter gene vectors for mTOR and SGK-1 were constructed.A549 cells were transfected with the vectors and miRNA-7-5p mimics.After 48 h,the firefly luciferase activity was measured.Firefly luciferase units were normalized with Renilla luciferase units.We found that the expression of psiCHECK-2-mTOR relative luciferase activity was reduced to 57%±5%(7.1±0.6 versus 12.5±1.0)by miRNA-7-5p(p < 0.05),and the expression of psiCHECK-2-mTOR-mut was not significantly suppressed by miRNA-7-5p(p>0.05).Furthermore,it was found that the expression of psiCHECK-2-SGK-1 relative luciferase activity was reduced to 45% ±4%(5.1±0.4 versus 11.5±1.5)by miRNA-7-5p(p < 0.05),and the expression of psiCHECK-2-SGK-1-mut was not significantly downregulated by miRNA-7-5p(p > 0.05).Our data suggest that mTOR and SGK-1 are the target genes of miRNA-7-5p.Part 2: Using qRT-PCR,we found that the mRNA expression levels of both mTOR and SGK-1 were downregulated to 0.54-and 0.3-fold,respectively,in the miRNA-7-5p mimic groups than the blank controls(p < 0.01).Furthermore,it was found that the protein levels of all molecules described above showed the same results(p < 0.01).In addition,the expression of the mTORC2 downstream phosphorylation product p-Akt-Ser473 was reduced to 0.29-fold than the blank control(p < 0.01).Our data indicate that miRNA-7-5p may suppress the mTORC2/SGK-1 signaling pathway.We next used western blot analysis to examine the expression levels of ENaC subunits,and found that all three subunits,?,?,and ?,were downregulated compared to the controls(p<0.01).This suggests that miRNA-7-5p can suppress the expression of ENaC in alveolar epithelial cells.Part 3: we used 100ng/mL LPS to stimulate A549 cells for the simulation of ARDS.Afterwards,the expression of miRNA-7-5p was detected by qRT-PCR at 0,3,6,12,and 24 h.We found that the expression of miRNA-7-5p started to increase at 6 h and continued to gradually increase with the passage of time.The transcription of miRNA-7-5p was upregulated by 1.4-,1.9-,and 2.8-fold,respectively.This shows that after ARDS,the expression of miRNA-7-5p is significantly increased in the alveolar epithelial cells.A549 cells were transfected with miRNA-7-5p inhibitors or negative controls,and then stimulated with LPS.After 24 h,we found that the expression levels of mTOR and SGK-1 were higher significantly in the LPS+miRNA-7-5p inhibitor group than in the pure LPS group(p < 0.01).Moreover,the protein level of mTOR,SGK-1,and p-Akt-Ser473 was also increased(p < 0.01).These results indicate that reduced expression of miRNA-7-5p can result in elevated activity of the mTORC2/SGK-1 signaling pathway after ARDS.Western blotting was used to analyze the expression of ENa C.We found that after transfection with the miRNA-7-5p inhibitor,the expression of ENaC ?,?,and ? was significantly upregulated compared to the expression in the LPS-only group(p < 0.01).This indicates that after ARDS,the reduced expression of miRNA-7-5p can leave ENaC more stable,which may promote AFC.Conclusion 1.mTOR and SGK-1 are the target genes of miRNA-7-5p.2.miRNA-7-5p suppresses the expression of ENaC through Mtorc2/SGK1 signaling pathway.3.Expression of miRNA-7-5p increases with time in A549 cells stimulated by LPS.Decreased miRNA-7-5p expression may stimulate the mTORC2/SGK-1 signaling pathway.Reduced miRNA-7-5p expression increased ENaC expression in alveolar epithelial cells stimulated with LPS.Therefore,miRNA-7-5p is a potential therapeutic target worthy of further investigation. |
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