Exprssion Of TFPI-2 In Renal Cell Carcinoma And The Effect Of EGCG On Renal Cell Carcinoma Cell Line

Exprssion of TFPI-2 in renal cell carcinoma and the effect of EGCG onrenal cell carcinoma cell lineObjective:To investigate the effect of TFPI-2 on invasiveness andapoptosis of renal cell carcinoma,to analysis the relationship betweenpromoter methylation and expression of TFPI-2.EGCG can inhibit activityof DNMT(DNA methyltransferase),cause CpG demethylation and reactivationof methylation-silenced genes.In the present study we investigated theeffect of EGCG on renal cell carcinoma cell line 786-0 and expressionregulation of TFPI-2 by EGCG.Methods:Immunohistochemistry,Western blot and real-time RT-PCR wasperformed to detect the expression of TFPI-2 in 37 renal cell carcinomacases and 11 normal kidney cases.TUNEL was used to study apoptosis statusof renal cell carcinoma.Real-time methylation specific PCR was performedto analysis the methylation status of TFPI-2 gene promoter.The humanrenal cell carcinoma cell line 786-0 was incubated with variousconcentrations of EGCG (10μM,20μM) or PBS buffer (as control) for 6days.Every group was made up of 5 samples.Cell cycle S phrase rate andcell line apoptosis index were evaluated by MTT assay and flow cytometryanalysis.Exprssion of TFPI-2 in all three groups was evaluated by Westernblot and real-time RT-PCR.Real-time methylation specific PCR was usedto detect methylation status of TFPI-2 gene promoter in all three groups.15 four-week-old nude mice were divided into three groups.Nude mice wereinjected with 5×10~6 786-0 cells into the right flank.After 6 weeks,subcutaneous tumor tissue,liver and lung tissue were taken to performHE staining.We evaluate the invasiveness of tumor cells among threegroups.Results:Immunohistochemically,high level expression rate of TFPI-2 inrenal cell carcinoma and normal kidney were:59.5%,100%,x~2=6.50,P＜0.05.The opticaldensity of Western-blot strip in renal cell carcinomaand normal kidney were:0.92±0.36,1.61±0.13,t=9.82,p＜0.05.Therelative expression of TFPI-2 mRNA in renal cell carcinoma and normalkidney were:0.0019±0.0011,0.0065±0.0008,t=12.51,p＜0.05.Comparedwith normal kidney,lower expression of TFPI-2 mRNA(r:-0.69931,p＜0.05) and protein(r:-0.93270,p＜0.05) was detected in renal cell carcinoma.The relationship between expression of TFPI-2 and stage of renal cellcarcinoma was negative correlation.Apoptosis index of renal cellcarcinoma specimens were:2.41%(TFPI-2 expression:grade 1),3.90%(TFPI-2expression:grade 2),6.78%(TFPI-2 expression:grade 3),9.57%(TFPI-2expression:grade 4),F=194.97,P＜0.05.The relationship betweenexpression of TFPI-2 and apoptosis index of renal cell carcinoma waspositive correlation,r:0.94948,p＜0.05.The relative expression of TFPI-2mRNA in methylated and unmethylated tumors were:0.0015±0.0011,0.0024±0.0009,t=-2.57,P＜0.05.The opticaldensity of Western-blot strip inmethylated and unmethylated tumors were:0.82±0.35,1.04±0.34,t=-2.03,P=0.05.Exprssion of TFPI-2 mRNA and protein were significantly lower inmethylated tumors than those in unmethylated tumors.In MTT assay,the mean OD value of 10μM EGCG group,20μM EGCG group andcontrol group were:0.526,0.441 and 0.577.The inhibition rate of 10μMand 20μM EGCG treatment groups were:8.8%,23.6%.S phrase rate in EGCGtreatment groups was lower than that in control group (F=15.023,p＜0.05).The mean S phrase rate of 10μM EGCG group,20μM EGCG group and controlgroup were:54.31%,60.50% and 71.32%.The mean apoptosis rate of 10μMEGCG group,20μM EGCG group and control group were:1.09%,2.27% and 0.94%.Apoptosis rate in EGCG treatment groups was higher than that in controlgroup (F=40.349,p＜0.05).In subcutaneous tumor tissue of nude mice,HEstaining revealed no invasiveness of tumor cell.We also didn't findmetastatic tumor in nude mice liver or lung tissue.The opticaldensityof Western-blot strip in 10μM EGCG group,20μM EGCG group and controlgroup were:0.85±0.23,1.47±0.12,1.73±0.15,F=34.391,P＜0.05.Real-timeRT-PCR demonstrated that the mean relative expression amount of TFPI-2mRNA was:2.33% (10μM EGCG),4.16% (20μM EGCG) and 0.35% (control group),F=-103.622,P＜0.05。Real-time methylation specific PCR detected nomethylation of TFPI-2 gene promoter in 10μM EGCG group,20μM EGCG groupand control group.Conclusions:The relationship between expression of TFPI-2 andinvasiveness of renal cell carcinoma was negative correlation.Overexpression of TFPI-2 may induce tumor cell apoptosis in renal cell carcinoma.Lower expression of TFPI-2 in renal cell carcinoma was partlydue to hypermethylation of gene promoter.EGCG can upregulate expressionof TFPI-2 in renal cell carcinoma cell line 786-0.The upregulation ofTFPI-2 by EGCG may have nothing to do with gene promoter methylation status.The exact pathogenesis of upregulation needs to be further investigated.EGCG inhibits growth and induces apoptosis in renal cell carcinoma throughTFPI-2 overexpression.