| ObjectiveRenal clear cell carcinoma(RCCC)is the most common malignant tumor in genitourinary system.This malignancy is one of the dominant diseases in urological practice.The researches in recent years indicate that renal cancer is related to multiple genes with unclear complicated pathogenesis.Chemokine receptor is a category of G-protein-coupled transmembrane receptor(GPCR)by which mediated chemotaxis function.Chemokines and their receptors are important actors in the tumorigenesis, development,progression and metastasis of human tumors.Chemokine receptors are interacted or regulated with many cytokines.Chemokine CXC receptor 4(CXCR4)is one of the hot point in chemokines.Recent researches show that CXCR4 is closely related to tumorigenesis,development,infiltration,and metastasis.There are high expressions of CXCR4 in many tumors,which may be regulated by such cytokines as tumor necrosis factor-α(TNF-α).There are few studies about the relationship between chemokine CC receptor 3(CCR3)and tumorigenesis,development,infiltration,and metastasis.It is not clear if CCR3 is regulated by TNF-α.Some studies reported that CXCR4 and CCR3 participated in tumorigenesis of clear cell renal cell cancer,but the pathogenesis was unclear.The interaction between TNF-αand CXCR4,CCR3 in renal cancer was nuclear,too.The aim of this study is to investigate the expression of CXCR4 and CCR3 in clear cell renal cell cancer,and the regulation of CXCR4 and CCR3 expression by TNF-αin clear cell renal cell cancer cell lines,and analyze the relation between TNF-αand CXCR4,CCR3 in the pathogenesis,development and infiltration of clear cell renal clear cell carcinoma.The results may provide a new clue to prevention and cure for the renal clear cell carcinoma. Methohs1.51 cases of clear cell renal cell cancer specimens(resected during 2001-2007) were selected from the patients in the urology department of the first affiliated hospital of China Medical University.18 cases of normal renal tissues close to the tumor were taken as normal controls.All specimens were fixed by formaldehyde solution,and embedded by paraffin.The embedded specimens were cut into sections with 4μm in thickness.S-P method of Immunohistochemistry was employed to detect the expression of CXCR4 and CCR3 in clear cell renal carcinoma and normal renal tissues.The relation between the expression and the clinical features,stage of the patients with renal cancer was also analyzed.Histochemistry results assessment:CXCR4 and CCR3 expressed within the cells with brown color was defined positive expression.5 High Power Field in each section was randomly selected.100 cells were analyzed in each High Power Field.The percentage of the positive expressed cells was calculated.We defined:"-",no stain;+,stained cells less than 1%;++, stained cells between 1%and 10%;+++,stained cells from 10%to 50%;and ++++, stained cells over 50%.2.RT-PCR method was used to detect the mRNA expression of chemokine receptor CXCR4 and CCR3 in the renal clear cell carcinoma cell lines A498 and 786-0. Western blot method was employed to investigate chemokine receptor CXCR4 and CCR3 expression in protein level in the renal clear cell carcinoma cell lines A498 and 786-0.3.Detection of endogenous TNF-αin renal cell carcinoma cell lines:Real time PCR method was employed to investigate the mRNA of TNF-αin the cell lines A498 and 786-0.Elisa method was used to detect the content of TNF-αin the supernatant of cell lines A498 and 786-0.4.The interaction between the endogenous TNF-αand CXCR4,CCR3:The mRNA expressions of TNF-α,CXCR4 and CCR3 in the cell line A498 and 786-0 were detected by real time PCR method.We investigated the mRNA expressions of TNF-α, CXCR4 and CCR3 in the cell line A498 at 3,6,12,24,and 48 hour after the cell lines were treated with anti-TNF-αneutral antibody,while the untreated cell lines as control.5.The inducement effect of exogenous TNF-αon the mRNA expressions of CXCR4 and CCR3 in renal cell cancer cell lines:The renal cell cancer cell line 786-0 was treated with TNF-α(1ng/ml,10ng/ml and 100ng/ml,respectively).The mRNA expressions of CXCR4 and CCR3 were detected by RT-PCR at 1,3,6,12,and 24 hour after the treatment,while the untreated cell lines as controls.5.Statistics analysis:All data were analyzed by SPSS software.P＜0.05 was as statistic significance level.Results1.The immunohistochemistry results showed that:There were high expression of CXCR4 and CCR3 in the renal clear cell carcinoma,while no expression of CXCR4 and CCR3 in the normal renal tissues.The expressions of CXCR4 and CCR3 were positively related to the TNM stage of the renal clear cell cancers,but not related to the patients age,sex and the tumor size..2.RT-PCR results:RT-PCR method was employed to investigate the mRNA expression of the chemokine receptor CXCR4 and CCR3 in the renal clear cell carcinoma cell lines.There were different mRNA expressions between the cell line A498 and 786-0.There were significantly higher expressions of CXCR4 and CCR3 in cell line A498 than in the cell line 786-0.3.Western blot results:We used the human renal clear cell carcinoma cell line A498 and 786-0 as the material.Western blot method was employed to investigate chemokine receptor CXCR4 and CCR3 expression at protein level in the cell lines. There were different expression between the cell line A498 and 786-0.There were significantly higher expressions of CXCR4 and CCR3 in cell line A498 than in the cell line 786-0.4.The detection of endogenous TNF-α:Real time PCR method was employed to investigate the mRNA of TNF-αin the cell lines A498 and 786-0.The results show that there was high expression of TNF-αin the cell line A498,while there was low expression of TNF-αin the cell line 786-0.The TNF-αmRNA expression in the cell line A498 was 9.3 times higher than that in the cell line 786-0(P＜0.01).Elisa method was used to detect the content of TNF-αprotein in the supernatant of cell lines A498 and 786-0.The TNF-αwas significantly higher in cell line A498 than in the cell line 786-0(P＜0.01).The TNF-αmRNA expressions paralleled the TNF-αprotein contents.5.The interaction between the endogenous TNF-αand CXCR4,CCR3 in the renal clear cell cancer cell lines:We investigated the mRNA expressions of CXCR4 and CCR3 in the cell line A498 at 3,6,12,24,and 48 hour after the cell lines were treated with anti-TNF-αneutral antibody,while the untreated cell line A498 as control.The results indicated that the expression of CXCR4 and CCR3 mRNA was reduced after the treatment。Compared to the control,the expressions at 6,12 and 24 hour was significantly reduced.The expressions at 12 hour after the treatment were reduced to 30.4%and 41.7%respectively.The expressions at 12 hour after the treatment were the lowest of all.6.The regulation of exogenous TNF-αto the expression of CXCR4 and CCR3 mRNA:The renal cell cancer cell line 786-0 was treated with TNF-α(1ng/ml,10ng/ml and 100ng/ml,respectively).The mRNA expressions of CXCR4 and CCR3 were detected by Real Time PCR method at 1,3,6,12,and 24 hour after the treatment,while the untreated cell line was as control.The results showed that the expression of CXCR4和CCR3 mRNA in the renal cancer cell line were up-regulated with different levels after the treatment of different concentrations of exogenous TNF-αdescribed previously(P＜0.05).The expressions of CXCR4 and CCR3 mRNA improved significantly after the treatment of TNF-αat the 10ng/ml concentration,and at 6 hour after the treatment.Compared to the control the expressions of CXCR4 and CCR3 mRNA improved 21.7 times and 16.4 times respectively at 6 hour after the treatment of 10ng/ml TNF-α(P＜0.01). Conclusion1.There are over-expressions of CXCR4 and CCR3 in the renal clear cell carcinoma and renal clear cell cancer cell lines.The result showed that CXCR4 and CCR3 participated to the morbidity of the renal clear cell cancers.The expressions of CXCR4 and CCR3 were positively related to the TNM stage of the renal clear cell cancers.2.There were significantly over-expressions of CXCR4 and CCR3 in renal clear cell cancer cell lines.The expressions in renal clear cell cancer cell line A498 were higher than that in the cell line 786-0.These imply that the invasive ability of A498 is higher than that of cell line 786-0.The expressions of CXCR4 and CCR3 may be positively related to the invasiveness of renal cancer cell lines.3.Renal clear cell cancer cell lines can produce endogenous TNF-α.The ability of producing endogenous TNF-αin cell line A498 is higher than in cell line 786-0.This suggests that the invasive ability of A498 may be higher than that of cell line 786-0.4.TNF-αcan up-regulate the expression of CXCR4 and CCR3 in renal clear cell cancer cell lines,by which can improve the invasiveness.
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