Association Between Ebp1 And The Formation, Development And Prognosis Of Esophageal Cancer And Cardiac Cancer

In the world wide,high incidence areas(expressed as crude incidence per100,000) of esophageal cancer include:China(21 per 100,000),South America(13 per 100,000),Western Europe(11 per 100,000),South Africa(10 per 100,000),Japan(9 per 100,000) and the former Soviet Union(8 per 100,000).From the 90's,due to the efficient intervention to the risk factors of esophageal cancer,the incidence and mortality of this cancer decreased gradually in Henan Province,but still higher than the average level of the whole country. Esophageal cancer and cardiac cancer were a long process of evolution,understanding that will benefit for early diagnosis and proper treatment time,and also improve the treatment of esophageal cancer and cardiac cancer rate and survival rate.So increasing the 5-year survival rate of this cancer by early diagnosis and treating is an important topic.With the development of molecular biology and the experiment technology,the methods such as PCR,western blotting and immunohistochemistry are mature,all provide us a good condition to study the esophageal cancer on the molecular level.Ebp1 gene encodes an RNA-binding protein that is involved in growth regulation.This protein is present in pre-ribosomal ribonucleoprotein complexes and may be involved in ribosome assembly and the regulation of intermediate and late steps of rRNA processing.This protein can interact with the cytoplasmic domain of the ErbB3 receptor and may contribute to transducing growth regulatory signals.This protein is also a transcriptional co-repressor of androgen receptor-regulated genes and other cell cycle regulatory genes through its interactions with histone deacetylases.This protein has been implicated in growth inhibition and the induction of differentiation of human cancer cells.ObjectiveThe purpose of this study is to measure the mRNA levels of Ebp1 in paired primary esophageal cancers and their matched normal esophageal tissues.Furthermore,we determined whether the levels of expression of this gene was correlated with clinical and pathological parameters of esophageal cancers by using several statistical analysis models. We also took a follow-up to the patients from whom the specimens were gotten after operation resection to research the impact of Ebp1 on the survival.Materials and maethods1.Subjects and samplesThe subjiects are esophageal cancer and cardiac cancer patients treated at The First Affiliated Hospital of ZhengZhou University,Linxian Tumor Hospital,Henan Province, China,from 2003 to 2006.According to pathological analysis the cancer tissues and matched normal esophageal and cardiac tissues of the subjects were taken after operation.Most samples are stored at -80℃within one hour.The patients clinical data such as gender,age, family history,metastasis,Pathological type,tumor position,tumor size,tumor stage, Infiltrating depth and TNM stage were collected and the patients were also follow-uped.These data will be statistically analyzed.2.Determination of Ebp 1 mRNA in esophageal cancer and cardiac cancerReal-time PCR was used to determine the mRNA expression of Ebpl in esophageal cancer and cardiac cancer and matched normal esophageal and cardiac tissues.Ct valve represent the amouts of mRNA. 3.Protein determination of Ebp 1 in esophageal cancer and cardiac cancerWe also detected the protein expression of Ebp1 in esophageal cancer and cardiac cancer respectively,in 220 pairs of esophagus cancer and 120 pairs of cardiac cancer and corresponding non-cancer esophagus and camdiac tissues.It was observed 3 images at×200 magnification for each sample and calculate number of cell expressing target protein.The percentage of cells expressing target protein was estimated by dividing the number of positively stained cells by the number of total cells per high-power field area.It was established the index of protein expression level that the ratio was of percentage of positively immunostaining cells in esophagus cancer and cardiac cancer to that in matched normal esophagus and cardiac sample.4.We collected samples of esophageal cancer and cardiac cancer patients from 2003 to 2006 in the First Affiliated Hospital of Zhengzhou University and the Cancer Hospital in Linzhou city and.In March to April in 2008,we follow up the patients.Time of treatment of patients as the moment Start,and follow-up time as the termination of time of observation,lapse between the end of the incident and start time of the incident is defined as the survival time for patients.5.Statistical analysisFor each sample,aâ–³Ct(Ct_Ebp1—Ct_β-actin) value respresents the difference between the experimental gene and the internal reference gene,β-actin.â–³(â–³Ct)value was obtained by subtraction betweenâ–³Ct_normal andâ–³Ct_cancer.Paired-samples t-test was used forâ–³(â–³Ct) and protein expression.x²-test was adopted to study the expression of 220 pairs of esophagus cancer and120 pairs of cardiac cancer and matched normal tissues for Ebp1 gene and their association with single clinical factors,non-factor logistic regression analysis model was used for multi-factor analysis, For Ebp1 gene,Kaplan-Meier survival curves for patients with positive versus negative gene expression were plotted and log-rank test was used for comparing the equality of the two survival curves.Multivariable analyses with the Cox proportional hazards model were used to estimate the effects of the clinical characteristics and the expression of the Ebp1 gene on survival Results were considered statistically significant atα=0.05.All statistical analyses were performed using the program SPSS 12.0 for Windows(SPSS,Chicago,IL).Results1 Real time PCR results1.1 Expression of Ebp1 genes in esophageal cancer and cardiac cancerUp-regulation of Ebp1 mRNA expression was found in 150 of 220(68.18%) of esophagus tissues compared with the paired normal esophagus tissues.Down- regulation of Ebpl mRNA expression were found in 73 of 120(60.83%) of cardiac cancer compared with the paired normal tissues.Univariate analysis showed that mRNA level of Ebp1 gene in esophageal cancer and cardiac cancer have significant difference between cancer samples and paired normal ones(t=11.065,P=0.000:t=-3.534,P=0.001).1.2 Multi-factor analysis of Ebp1 mRNA expression in esophageal cancer and cardiac cancer with the individual clinical and pathological characteristicsIntroduce all the factors to non-factor Logistic regression analysis model for multi-factor analysis,results showed that:In esophageal cancer,family history,tumor size,tumor stage, infiltrating depth and TNM stage were the risk factors of Ebp1 mRNA expression,and pathological type was the protective factors of Ebp1 mRNA expression.Age.In cardiac cancer,age, family history,tumor size,tumor stage and TNM stage were the protective factors of Ebp1 mRNA expression,and gender was the risk factor of Ebp1 mRNA expression.2 Immuonhistochemistry result2.1 Ebp1 protein expression in esophageal cancer and cardiac cancerPaired-samples t-test indicated that,In esophageal cancer,the expression of Ebp1 (t=23.140,P=0.000) was really up-regulated at protein level in cancer tissues compared with normal counterparts.In cardiac cancer,the expression of Ebp1(t=32.588,P=0.000) was really down-regulated at protein level in cancer tissues compared with normal counterparts,which were similar to the results of real-time PCR.2.2 Multi-factor analysis of protein expression in esophageal cancer and cardiac cancer with the individual clinical and pathological characteristicsIntroduce all the factors to non-factor logistic regression analysis model for multi-factor analysis,results showed that:in esophageal cancer,family history,matastasis,tumor size, infiltrating depth and tumor stage were the risk factors of Ebp1 protein expression.In cardiac cancer,matastasis and tumor stage were the protective factors of Ebp1 protein expression.3 Survival analysisThe Univariate survival analysis showed that the expression of Ebp1 mRNA and its protein,metastasis,family history have significant association with esophageal cancer survival,and the relative risk are 2.717(confidence interval,1.190 to 6.200),2.526 (confidence interval,1.266 to 5.040) and 1.808(confidence interval,1.184 to 2.760) respectively.The Univariate survival analysis showed that Ebp1 mRNA,age and clinical stage remained a significant independent risk factors in cardiac cancer,and the relative risk are 0.198(confidence interval,0.085 to 0.461),1.429(confidence interval,1.081 to 1.890) and 1.950(confidence interval,1.289 to 2.950),respectively.Conclusion1.Up-regulation of Ebp1 mRNA expression was found in esophagus cancer tissues compared with the paired normal esophagus tissues.Down-regulation of Ebp1 mRNA expression was found in cardiac cancer compared with the paired normal tissues.In esophageal cancer, family history,tumor size,tumor stage,infiltrating depth and TNM stage were the risk factors of Ebp1 mRNA expression,and pathological type was the protective factors of Ebp1 mRNA expression.Age.In cardiac cancer,age,family history,tumor size,tumor stage and TNM stage were the protective factors of Ebp1 mRNA expression,and gender was the risk factor of Ebp1 mRNA expression.2.In esophageal cancer,the expression of Ebp1 was really up-regulated in protein level in cancer tissues compared with normal counterparts.In cardiac cancer,the expression of Ebp1 was really down-regulated in protein level in cancer tissues compared with normal counterparts,which were similar to the results of real-time PCR.In esophageal cancer,family history,matastasis,tumor size,infiltrating depth and tumor stage were the risk factors of Ebp1 protein expression.In cardiac cancer,matastasis and tumor stage were the protective factors of Ebp1 protein expression.3.The Univariate survival analysis showed that the expression of Ebp1 mRNA and its protein,metastasis,family history have significant association with esophageal cancer survival and Ebp1 mRNA,age and clinical stage remained a significant independent risk factors in cardiac cancer.4.Ebp1 was highly expressed in the esophageal cancer tissue compared with the normal and low expressed in cardiac cancer.It might play an important role in tumor formation and work on tumor advancement.However,they may be identified as a novel prognostic indicator and might be a potential therapeutic target.