Contribution Of PLCε/NF-κB Signal Pathway On Proliferation Of Tumor Cells In Renal Cell Carcinoma

PART ONE:Objective Investigation the expression of phospholipase C epsilon in Renal cell carcinoma and its relationship with clinicopathologyMethod To detect the expression of PLCεin renal cell carcinoma tissues and corresponding adjacent tissues and analyze the relationship between PLCεmRNA and the grade and stage of clinical pathology by RT-PCRResult The expression of PLCεmRNA was higher in in renal cell carcinoma tissues than corresponding adjacent tissues(P<0.001), the expression of PLCεmRNA was correlation with clinical stage(P<0.05),but not related with clinical grade(P>0.05).Conclusion The expression of PLCεmRNA is obviously creased in renal cell carcinoma tissues, and correlation with clinical stage,suspect that PLCεmaybe relationship with the development and proceed of renal cell carcinoma. PART TWO:Objective To detection the effect of shRNA PLCεwhich transfected 786-0 renal cell carcinoma cells and to analyze the possible molecular mechanism.Method To detection the expression change of PLCε,NF-κB,COX-2 and c-myc mRNA and protein by RT-PCR and Western blot. Then detection the expression change of the cytoplasm and the nucleus protein of NF-κB p65. Detection the effect of shRNA PLCεon the proliferation of 786-0 cells by MTT and clonogenic cell survival assay. Detection the status of nuclear translocation of NF-κB after transfected shRNA PLCεin 786-0 cells by immunofluorescence assay.Result The expression of PLCε,NF-κB,COX-2 and c-myc mRNA and protein were inhibited obviously by shRNA PLCε,and inhibited the expression of cytoplasm and the nucleus protein of NF-κB. The result of MTT and clonogenic cell survival assay show that ShRNA PLCεinhibited proliferation of 786-0 cells. ShRNA PLCεinhibited the exoression of NF-κB protein by immunofluorescence assay.Conclusion shRNA PLCεinhibited the proliferation of 786-0 cells maybe through down-regulation the expression of NF-κB,COX-2 and c-myc mRNA and protein.PART THERE:Objective Assessed the effects of NF-κB inhibitor BAY11-7082 on the proliferation of 786-0 cells and the expression of COX-2 and c-myc protein, and the expression of whole protein, cytoplasm and the nucleus protein of NF-κB, In order to further confirming that PLCεinhibited proliferation of 786-0 cells and down-regulation the expression of COX-2 and c-myc protein through NF-κB.Method Treated 786-0 cells with different concentrations of BAY11-7082, assessed the effects of BAY11-7082 on 786-0 cells proliferation by MTT and clonogenic cell survival assay. Examination the expression of NF-κB, c-myc and COX-2 protein before and after treated with BAY11-7082. Detection the effect of nuclear translocation of NF-κB after treated with BAY11-7082 by immunofluorescence assay.Result Proliferation of 786-0 cells were inhibited obviously by BAY11-7082. The expression of c-myc and COX-2 protein were down-regulated by BAY11-7082(P<0.01) , but there was no influence NF-κB protein(P>0.05), then, we examined the nuclear protein and cytoplasm protein of NF-κB, nuclear protein of NF-κB was reduced after treated with different dose of BAY11-7082.Conclusion BAY11-7082 inhibited the proliferation of 786-0 cells and the expression of COX-2 and c-myc protein maybe through down-regulation the expression of nucleus protein of NF-ΚB.