The Expression And Significance Of Cxcr4, Sdf-1 And Vegf In Nephroblastoma

Nephroblastoma is one of the most commom abdominal maligant tumor in children. It consists of 8% in all pediatic solid tumors.Max wilms descripted its characteristics in 1899. so it was named after his name.The tumor usually generated in the first seven years of childhood.Its main prognosis factors are the histological stucture,age and metastasis.but the most important prognosis factor is local infiltration and instant metastasis of the tumor. During the past fourty years ,the mortality rate of nephroblastoma has decreased dramatically owing to the proper use of operation,chemotherapy and radiotherapy.But the children which have instant metastasis is still below the mark .There is no specific tumor diagnostic and prognostic marker been found in nephroblastoma now. So the researchers attempt to find the bio-marker to predict the ability of invasion and metastasis by using the molecular biology techniques. In order to evalute whether they can be used as a reliable predict for diagnosis and whether a new treatment pattern can be carried out.CXCR4 is a metastasis-associated gene which was detached in recent years.It was proved that its over-expression was closely correlated with invasion and metastasis of some tumors. SDF-1 is his ligand.There's combination plays an important role in tumor immigration,sticking,proliferation,infiltration,anti-apoptosis,vascularization and metastasis .VEGF is one of the strongest promoting angiogenesis factors,which plays a critical role in the process of tumor angiogenesis. The expression of VEGF is closely correlated with tumor's growth, invasion,metastasis and clinical prognosis.Our experiment detected the CXCR4mRNA expression by RT-polymerase chain reaction and used Immunohistochemitry to detect CXCR4,SDF-1 and VEGF protein in nephroblastoma and normal renal tissue near the tumor. The correlation of three protein was studied.The aim of experiment is to find new bio-marker to predict or diagnose the invasion and metastasis of nephroblastoma and to provide more experimental evidence for clinical therapy of nephroblastoma.Methods1. Patients and samples: This study analyzed 12 normal renal tissue near the tumor and primary cancer tissues from the 30 nephroblastoma patients treated at the department of the Pediatric Surgery of The First and The Third Affiliated Hospital of ZhengZhou University ,from 2007 to 2008 after their informed consents. We collected the patients clinical features such as genders,age,clinical stage and pathological type for statistical and study analysis.2. Total RNA was extracted from nephroblastoma specimens by using Trizol reagent according to the standard protocol.The quality of the RNA samples was determined by electrophoresis through agerosegels and staining with ethidium bromide,3. Total RNA were progressed directly to cDNA by reverse transcription using AMV reverse transcriptase produced by Promega company. We also quantified transcription of 6-actin to test the effect of reverse transcription.4. Design the primers of CXCR4 and 6-actin, We carried on quatative analysis of the expression of CXCR4 in nephroblastom comparedto their normal counterparts by RT-PCR.5. Immunohistochemical technique was used to detect the expression of CXCR4,SDF-1,VEGF proteins among these samples. Positive cytoplasmic staining, according to the staining intensity points respectively. Computer-aided image analysis for the optical density (OD) value on average.6 Statistical analysis : independent-samples T test,chi-square test and Fisher's exact probabilities were adopted to analysis the experimental data at SPSS15.0. Results were considerable statistically significant at the 0.05 level(2-tailed).Results1. Reverse transcrintion-PCR results indicated: Up-regulation OD value; (optical density value) of CXCR4 was observed in nephroblastoma compared with t -he normal renal tissue near the tumor (P<0.05).The expression level of CXCR4m RNA in lymph metastatic,clinical grade III or IV, Unfavorable histological groups is higher than that in non-metastatic,clinical grade I or II ,Favorable histological stage groups. There is a coincident result between Immunohistochemistry and RT- PCR about CXCR4.2. The positive ratio of SDF-1 protein in nephroblastoma is higher than normal renal tissue near the tumor (P<0.05).The average optical density value of SDF-1 in lymph metastatic,clinical grade III or IV , Unfavorable histological groups is higher than that in non-metastatic,clinical grade I or II, Favorable histological groups(P<0.05).3. The positive ratio of VEGF protein in nephroblastoma is significantly higher than normal renal tissue near the tumor (P<0.05). The average optical density of VEGF in lymph metastatic,clinical grade III or IV, Unfavorable histological groups is higher than that in non-metastatic,clinical grade I or II, Favorable histological groups (P<0.05).4. Analysis of correlation indicated:There was a significant correlation in CXCR4/SDF-1, CXCR4/VEGF, or SDF-1/VEGF expression(r=0.427,r=0.439 r=0.369,P<0.05) .Conclusion1. Immunohistochemical technique and RT- PCR was used to detect the expression of CXCR4mRNA and protein in nephroblastoma.The result indicated that its transcription and translation are corresponding.2. The highly express CXCR4,SDF-land VEGF in nephroblastoma associated with their lymph metastasis, clinical grade and histological type.3. There is dependablity with CXCR4,SDF-1 and VEGF. The high exression of CXCR4, VEGF and SDF-1, alone and in combination, promote the invasion and meastasis of nephroblasoma.4. The united detection of CXCR4,VEGF and SDF-1 expression can predict the ability of invasion and metastasis of nephroblastoma and supply the evidence for clinical therapy and judgement of prognosis.