Sublytic C5b-9 Induces The Proliferation Of Glomerular Mesangial Cells In Thy-1 Nephritis Rats And Its Molecular Mechanism

Part I The effects of RGC32 up-regulation induced by sublytic C5b-9 on rat glomerular mesangial cells(GMCs)proliferation in the rats with Thy-1 nephritisObjective: To investigate the expression of response gene to complement 32(RGC32)both in the renal tissue of rat Thy-1 nephritis(Thy-1N)and rat glomerular mesangial cells(GMCs)stimulated by sublytic C5b-9 complexes.And to explore the role of RGC32 in sublytic C5b-9-mediated GMCs proliferation.Methods: Firstly,an animal model of rats Thy-1N was reproduced,and the cultured rats GMCs were stimulated with sublytic C5b-9.The protein levels of RGC32 in the renal tissue of Thy-1N rats(in vivo)and in the GMCs stimulated by sublytic C5b-9(in vitro)were measured using western blot at fixed time.Thereafter,RGC32 eukaryotic expression plasmid(p IRESII-EGFP/RGC32)and its short hairpin RNA(sh RNA)expression plasmid(sh RGC32)were well constructed,and then transfected into rat GMCs respectively for 48 h,followed by sublytic C5b-9 stimulation for another 10 h.Moreover,the protein levels of RGC32,cyclin D2,PCNA,FN and collagen IV were detected using western blot.Meanwhile,the proliferation of rat GMCs in the different groups was also determined by CCK8 test.Results: The protein level of RGC32 in the renal tissue of Thy-1N rats and GMCs stimulated by sublytic C5b-9 was significantly up-regulated and peaked at 10 h.Over-expression of RGC32 in rat GMCs could obviously increase the production of RGC32,cyclin D2,PCNA,FN and collagen IV,and promote the GMCs proliferation.Transfection of the GMCs with sh RGC32 treatment could effectively suppress the protein level of RGC32 and inhibit the proliferation of GMCs.Conclusion: In the renal tissue of the rat Thy-1N and rat GMCs,sublytic C5b-9 can promote GMCs proliferation and ECM production through up-regulating the expression of RGC32 gene.Part II The effects and mechanism of MZF1 on promoting RGC32 gene transcription and GMCs proliferation in the GMCs exposed to sublytic C5b-9Objective: To explore the effect of myeloid zinc finger gene1(MZF1)up-regulation both in the renal tissue of rat Thy-1 nephritis(Thy-1N)and rat glomerular mesangial cells(GMCs)stimulated by sublytic C5b-9 complexes on RGC32 transcription.And to evaluate the role of MZF1 in sublytic C5b-9-mediated GMCs proliferation.Methods: Firstly,Rat RGC32 promoter(-686~-1 nt)and different truncated XAF1 gene promotors(-486~-1 nt,-286~-1 nt and-86~-1 nt)were amplified by PCR and cloned into the luciferase reporter plasmid(p GL3-basic).The recombinant plasmids(p GL3-RGC32-FL,p GL3-RGC32-1,p GL3-RGC32-2 and p GL3-RGC32-3)were transfected into GMC accompanied with p RL-SV40 for 48 h,followed by sublytic C5b-9 stimulation for 10 h,and then detected the luciferase activity.Meantime,the possible transcription factor binding sites within RGC32 promoter were predicted by bioinformatics software(TF search).Thereafter,the protein levels of the possible transcription factor in the renal tissue of Thy-1N rats(in vivo)and in rat GMCs stimulated by sublytic C5b-9(in vitro)were measured using western blot at fixed time.The plasmid of possible transcription factor eukaryotic expression and its short hairpin RNA(sh RNA)expression plasmid were constructed successfully,and then transfected into rat GMCs accompanied with p GL3-RGC32-FL.Thereafter,according to the prediction of TF search,the corresponding primers were designed to determine the possible transcription factor binding sites by Ch IP.Finally,The plasmid of transcription factor expression and its sh RNA expression plasmid were transfected into rat GMCs respectively for 48 h,followed by sublytic C5b-9 stimulation for another 10 h.The proliferation of GMCs and production of ECM were determined using western blot and CCK8.Results: The GMCs treated with sublytic C5b-9 could significantly enhance the RGC32 promotor luciferase activity(p GL3-RGC32-FL,p GL3-RGC32-1 and p GL3-RGC32-2),whereas the luciferase activity of p GL3-RGC32-3 was obviously reduced,which indicated that the region of rat RGC32 promoter(-286~-86 nt)may contain a possible transcription factor binding element.Meanwhile,MZF1 was predicted to bond to the RGC32 promoter binding element by bioinformatics software(TF search).Thereafter,the binding element was further determined by Ch IP assay.The protein level of MZF1 in the renal tissue of Thy-1N rats and GMCs stimulated by sublytic C5b-9 was significantly up-regulated and peaked at 10 h.Over-expression of MZF1 in rat GMCs could markedly increase RGC32,cyclin D2,PCNA,FN and collagen IV production,and promote the GMCs proliferation.Knockdown of MZF1 by sh MZF1 could effectively decrease the protein level of RGC32 and inhibit the proliferation of GMCs and production of ECM.Conclusion: Sublytic C5b-9 can indeed up-regulate RGC32 gene transcription and GMCs proliferation via increasing MZF1 expression.