Study On The Relationship Between Wisp3 And Prognosis Of Esophageal Cancer

In the world,there are high risk regions of esophageal cancer such as China (21/100,000),South America(13/100,000),Western Europe(11/100,000),South Africa (10/100,000),Japan(9/100,000) and the former Soviet Union(8/100,000).Esophageal cancer is the common malignant tumor in our country,especially in Henan,Shanxi and Hebei provinces,its incidence is very high,100/10,000.Due to the efficient intervention to the risk factors of esophageal cancer,the incidence and mortality of this cancer declined gradually in Henan Province,but still higher than the average level of the whole country.The 5-year survival rate is no more than 15/100;it is an important topic to improve the 5-year survival rate of this cancer by early diagnosis and treatment.Esophageal cancer had been studied for a long time and has had many achievements,It had been reported that environmental and genetic factor may contribute to the procees of esophageal cancer.Like other cancers,the progress of esophageacl cancer is involved in the activation of cancerogen,and the damage and repair of DNA,the proliferation and apoptosis of cell.It is reported that CCN is related to the turmor.CCN family consist of CTGF,Cyr61, NOV,WISP1,WISP2 and WISP3,which have complex function.They not only participate in the nomal organism,but they also have intimate connection with tumor.WISP3 is located in 6q21-22,encoding a cysteine-rich protein with 354 amio acid.The relationship between WISP3 expression level and esophagus cancer has been infrequently studied.ObjectivesTo know whether the expression level of WISP3 is related to the clinical and pathological characteristic,and whether the expression level of WISP3 is related to the prognosis of esophageal cancer.Materials and methods1.Subjects and samplesThe subjects were esophageal cancer patients treated in the First Affiliated Hospital of ZhengZhou University and Linxian Tumor Hospital from 2004 to 2007.The cancer tissues and matched normal esophageal tissues of the subjects were collected after operation,all depending on pathological analysis.All samples were stored in the icebox at -80℃within half an hour.The patients' clinical data such as gender,age,family history,tumor position, size,stage and metastasis were collected and the follow-up was conducted.These data were statistically analyzed.2.Expression of WISP3 mRNA in samplesReal time-PCR was used to determine the mRNA expression levels of WISP3 in the esophageal cancer tissues and matched normal esophageal tissues.Ct valve represents the amounts of mRNA.For each sample,ΔCt(Ct_WISP3--Ct_β-actin) value respresents the difference between WISP3 andβ-actin,which is the internal reference gene.Δ(ΔCt) value comes fromΔCt_normorl -ΔCtcancer.3.Expression of WISP3 protein in samplesWISP3 protein expression was determined in 186 pairs of esophageal cancer tissues and matched normal esophageal tissues,which is at 200 magnifications.The number and percentage of positive cells were calculated.The ratio is the index of WISP3 protein expression level. 4.Survial analysisFrom March to April in 2008,we followed up the patients.The date of the patients is the beginning of follow up.and the date of the follow-up is the end point,lapse between the end of the incident and the date of the incident is defined as the survival time for patients.5.Statistical analysisTo study the association between the WISP3 gene expressions to single clinical factors in 186 pairs of esophageal cancer and matched normal tissues,we used aχ²-test. Kaplan-Meier survival curves described as positive and negative WISP3 gene expression. The difference of the two survival curves was analyzed by Log-rank test.Cox proportional hazards model has used to estimate the effects of the clinical characteristics and the expression of the WISP3 gene on survival.The statisticl significance level was 0.05.All statistical analyses were performed using the program SPSS12.0 for Windows(SPSS, Chicago,IL).Results1 Real time-PCR results1.1 Expression level of WISP3 mRNA in esophagus cancer tissues and matched normal esophagus tissuesUp-regulation of WISP3 mRNA expression was found in 68%(128/186) of esophagus tissues compared with the paired normal esophagus tissues.The result by univariate analysis showed that there is significant difference of WISP3 mRNA level between cancer samples and matched normal ones(t=7.641,P = 0.001).1.2 Relationship between expression level of WISP3 mRNA in esophageal cancer and the individual clinical and pathological characteristicsThe result by univariate analysis showed that there was significant association between expression levels of WISP3 mRNA and smoke,metastasis,family history,Invasion depth, and tumor size and tumor stage.The difference of WISP3 expression level between the smokers and non-smokers was significant(P=0.007).Expression of WISP3 in metastatic esophagus tissues were significantly higher than in those non-metastatic ones(P=0.001). Level of WISP3 in the patients with family history was significantly higher than those without family history(P=0.031).Levels of WISP3 expression were increased significantly in poor differentiation tumors compared to high differentiation tumors(P=0.008).Moreover, Large size of the cancer patients had also significantly higher level of WISP3 than smaller ones(P=0.001).There is no association between expressions of WISP3 and sex,age and tumor type.2 Immuonhistochemistry result2.1 WISP3 proteins expression in esophageal cancer tissues and paired normal esophageal tissues.Up-regulation of WISP3 protein expression was found in 60%(120/186) of esophagus tissues compared with the paired normal esophagus tissues.The result by paired-samples t-test indicated that the expression of WISP3 was really up-regulated in protein level in cancer tissues compared with normal esophageal tissue(P=0.001),which was similar to the results of real-time PCR.2.2 Relationship between protein expression of WISP3 in esophageal cancer and the individual clinical and pathological characteristicsThe result by univariate analysis showed that there is a significant association existed between expression level of WISP3 protein and smoke,metastasis,family history,invasion depth,and tumor stage.The difference of WISP3 protein expression level between the smokers and non-smokers is significant(P=0.001).For metastasis,protein expression of WISP3 in metastatic esophagus tissues was significantly higher than in those non-metastatic ones(P=0.037).WISP3 protein expression level in the patients with family history was significantly higher than those without family histroy(P=0.002).WISP3 protein expression level was increased significantly in low stage tumors compared to high stage tumors (P=0.038).There were no association between expressions of WISP3 protein expression level and sex,age,tumor size,tumor position and tumor type.3 Survival analysisThe result by univariate survival analysis showed that the expression levels of WISP3 protein,metastasis,and family history had a significant association with esophageal cancer survival,and the relative risk were 1.277(95%confidence interval,1.080 to 1.509),1.692 (95%confidence interval,1.067 to 2.683) and 2.009(95%confidence interval,1.176 to 3.433) respectively.Conclusion1.Up-regulation of WISP3 mRNA expression was found in 68%(128/186) of esophagus tissues compared with the paired normal esophagus tissues.There is a significant association between expression levels of WISP3 mRNA and smoke,metastasis,family history,Invasion depth,and tumor size and tumor stage.There is no association between expressions of WISP3 and sex,age and tumor type.2.Up-regulation of WISP3 protein expression was found in 60%(120/186) of esophagus tissues compared with the paired normal esophagus tissues.There is significant association between expression level of WISP3 protein and smoke,metastasis,family history,Invasion depth,and tumor stage.There is no association between expressions of WISP3 protein expression level and sex,age,tumor size,tumor position and tumor type.3.The expression levels of WISP3 protein,metastasis,and family history have significant association with esophageal cancer survival.4.WISP3 was highly expressed in the esophageal cancer tissue compared with the normal esophageal tissue.It might play an important role in tumor formation and work on tumor advancement.However,it may be identified as a novel prognostic indicator and might be a potential therapeutic target.