Mesangial Proliferation Inhibition Factor Screening And Preliminary Function Study

Objective:Gene chip and bioinformatics methods have been used to explore the change law of gene expression in anti-Thy1 model of experimental mesangial proliferative glomerulonephritis to screen out possible new gene for inhibiting mesangial proliferation,and to verify its effects on mesangial proliferation via in vitro experiments.Methods:1) OX7 hybridoma cells were propagated in vivo,then ascites was collected and purified via Protein A affinity chromatography to prepare anti-Thy1 antibody. Anti-Thy1 model of experimental mesangial proliferative glomerulonephritis was induced by injection of the antibody(2.5mg/kg).2) Affymetrix Rat U230 2.0 gene chip was used to identify kidney gene expression profiles in anti-Thy1 model at different time points(day 3,5,7,10 and 14) and control rats before screening the differential expression genes by SAM analysis(Ratio≥1.50 or≤0.67,and Score≥2 or≤-2,FDR＜5%).Analysis of the main functions of these genes was performed with GOMiner software to select genes related to cell proliferation.KEGG was used to find out the main pathway involved by these genes.The mRNA expression levels were detected by real time PCR.Candidate gene which may be a novel inhibitor of mesangial proliferation was then selected.3) siRNA of the candidate gene was synthesized.Rat mesangial cells were divided into four groups:①serum free group (0%FCS group);②fetal calf serum group(10%FCS group);③control transfection group(10%FCS+siCon group);④candidate gene siRNA transfection group(10%FCS+siRNA group).Inhibition effects of siRNA were examined by RT-PCR and Western blot,the mesangial cell proliferative ability was detected by MTT,cell cycle profiles were determined by flow cytometry analysis,and the expression changes of cell cycle regulatory proteins(cyclin D1,CDK2,p21) were examined by Western blot. Results:1) After anti-Thyl antibody injection,mesangial dissolution was obvious at day 3,and the mesangial proliferation was significant at day 5 and day 7(abbreviation "proliferative period"),which recovered gradually at day 10 and day 14(abbreviation "recovery period").2) Of the 9033 gene sequences expressed on the microarray,1001 genes displayed differential expression at least at one time point compared to control group.Functional analysis software showed these genes had various molecular functions such as binding,catalytic activity,molecular transducer activity,transporter activity,transcription regulator activity,enzyme regulator activity,structural molecule activity,which were related to multiple processes including metabolism, biological regulation,development,localization,response to stimulus,immune system process,growth,biological adhesion,and reproduction,etc.During the proliferative period,genes which could stimulate proliferation,positively regulate cell cycle or inhibit apoptosis were predominant,while during the recovery period,genes that could inhibit proliferation,negatively regulate cell cycle or promote apoptosis were predominant.MAPK signal pathway which was closely related to proliferation was activated in proliferative period and was then inhibited partially in recovery period.Real time PCR showed that the tendency of mRNA expression of genes that were related to cell proliferation(Mxi1,TIMP-1,CCL2,S 100A4) were in accordance with the results of gene chip.Transcriptional repressor Mxi1 was down-regulated significantly in proliferative period compared to control group,and then the expression level was gradually elevated.So we selected Mxi1 as a novel potential inhibitor of mesangial proliferation.The effect of Mxi1 on mesangial proliferation was observed by RNAi technology.Compared to 10%FCS group and 10%FCS+siCon group,siMxi1 decreased mRNA and protein expression level of Mxil in mesangial cell(P＜0.05).MTT showed that proliferative ability was elevated after siMxi1 interference,with the absorbance of 24h and 48 h increased(P＜0.05). Cell cycle analysis showed that the percentage of the cells in S stage and proliferation index were markedly increased in 10%FCS+siMxi1 group(P＜0.05).siMxi1 also elevated the expression levels of cell cycle regulatory proteins including cycline D1, CDK2,and p21(P＜0.05). Conclusion:Mxi1 may inhibit mesangial cell proliferation via regulating the expression of cell cycle regulatory proteins,which maybe a new target of mesangial proliferative glomerulonephritis therapy.