Sublyticc5b-9 Stimulate Gmcs Induced Egr-1 And Atf3 Expression Relationship Study

Part I The expression of Egr-1 and ATF3 in GMCs induced by sublytic C5b-9stimulationandintherenaltissueofratswithThy-1nephritisTo investigate the expression of early growth response gene-1(Egr-1)and activating transcription factor 3 (ATF3) in rat glomerular mesangial cells (GMCs) inducedbysublyticC5b-9(sC5b-9)stimulationandinrenaltissue ofratswithThy-1nephritis (Thy-1N).Methods: The rat GMCs were cultured in vitro and stimulated with or without sC5b-9. And ananimal model of rat Thy-1N by injection of anti-thymocyte serum (ATS) to rats was induced.The level of Egr-1 and ATF3 expression was measured by reverse transcription PCR (RT-PCR)and Western blot in the cultured GMCs following sC5b-9 attack and in the renal tissue of ratswith Thy-1N at fixed time. Results: Both in vitro and in vivo, Egr-1 and ATF3 expressioninduced by sC5b-9 attack were found to be significantly up-regulated at 3h compared to controlgroups. Moreover, Egr-1 and ATF3 had the same expression changes. Conclusion: Egr-1 andATF3 expression in sC5b-9-treated rat GMCs and renal tissue of rats with Thy-1N weremarkedlyelevatedat3h,andATF3hadthesameexpressionchangesasEgr-1.Partâ…¡ConstructionandidentificationofEgr-1geneanditsshRNAeukaryoticexpressionvectorTo construct early growth response gene-1 (Egr-1) and its shRNAexpression vectors, and to assess their function on rat glomerular mesangial cell (GMCs). Methods: The CDS area of Egr-1 and three reverse repeated motifs targeting of Egr-1 gene weresynthesized and cloned into eukaryotic expression plasmid pcDNA3.1-myc-his-A andpGCsi.U6.neo.GFP. After screened and confirmed, the recombinant plasmids were transfectedinto GMCs, thenthe levels of Egr-1 protein in ratGMCswere measured byWestern blot to proveits expression and find out the optimal shRNA. Results: It was verified by partial nucleotidesequencing and restriction endonuclease digestion that the constructed eukaryotic vectors werecorrect. The results by Western blot showed that the constructed pcDNA3.1/Egr-1 plasmid couldexpress correctly and the optimal shRNA, which could effectively silence the target gene, wasEgr-1 shRNA-2. Conclusion: The pcDNA3.1/ Egr-1 plasmid and its shRNA were constructedsuccessfully. These data provide experimental tools for studying biological functions of Egr-1geneinthefuture.Part III The relationship between Egr-1 and ATF3 expression in GMCsstimulatedwithsC5b-9To explore the effects on ATF3 expression and GMCs apoptosisinduced by sC5b-9 stimulation via Egr-1 overexpression or knockdown. In addition, todemonstrate that Egr-1 can bind to ATF3 promoter and regulate its transcription directly.Methods: Firstly, the cultured rat GMCs were transfected with pcDNA3.1/ Egr-1 and Egr-1shRNA-2 respectively, then the cells were divided into different groups with or without sC5b-9attack. The apoptosis of GMCs was evaluated by Flow cytometry after AV-PI staining, and themRNAandproteinlevelsofEgr-1andATF3indifferentgroupsweremeasuredbyRT-PCRandWestern blot. Moreover, dual luciferase reporter system was used to verify the regulation ofATF3promoteractivitybyEgr-1.Finally,chromatinimmunoprecipitation(ChIP)assaywasdone to identify whether Egr-1 binds to the ATF3 promoter in GMCs. Results: (1) Upregulation ofATF3 by Egr-1 overexpression can facilitate sC5b-9-induced GMCs apoptosis. In contrast,downregulation of ATF3 by Egr-1 knockdown had the opposite effect. (2) Egr-1 increased theactivity of ATF3 promoter using luciferase reporter assay, and Egr-1 can bind to the ATF3promoter using ChIP experiment. Conclusion: Egr-1 can bind to specific elememt on ATF3promoter and facilitate its expression. The GMCs apoptosis induced by sC5b-9 stimulation isregulatedthroughupregulationofATF3expression.