Study On The Association Between Ctgf Expression And Prognosis Of Esophageal Cancer

Esophageal cancer is one of the most common malignancies throughout the world, the incidence of the tumor accounts for the eight position, the mortality rate of it accounts for the six. China's mortality rate of esophageal cancer ranks the first position in the world, In recent years, the mortality rate of esophageal cancer although had decreased, but the decline was not apparent. In some high-incidence area of esophageal cancer, the incidence and mortality remain high. Because the early symptom wasn't manifest in most of the patients with esophageal cancer, so they were found in the late stage. The five-year survival rate was only about 25% after surgical operation. But if operation in early stage, the five-year survival rate probably elevates to 90%, and the ten-year survival rate is about 73%. At present approaching the pathogenesis of esophageal cancer and searching for the biomarker for early detection and diagnosis become one of the main directions of the scientific research. But the etiology and pathogenesis is still in nubilous up to now. So it would be a gigantic mission to continue the research on esophageal cancer. The methodology of molecular biology and other experimental technology has been well developed, such as PCR and immuno-histochemistry methods, which can be applied at the molecular level for the investigation on esophageal cancer. Connective tissue growth factor (CTGF) is a member of the CCN family, encoded by immediate early genes that play various roles in angiogenesis and tumor growth. Recently, CTGF expression has been shown to be associated with progression, tumorigenesis, diversion, angiogenesis, invasion potentiality and prognosis of various cancers. According to the reports, CTGF expression was different in different cancers, as in laryngeal squamous carcinoma, thyroid carcinoma, breast cancer, colon carcinoma, hepatocellular carcinoma and so on (some overexpression, some lowexpression). The relationship between CTGF and tumor was not accepted argument. In the present study, we applied real-time PCR and immunohistochemistry techniques to measure the mRNA levels of CTGF genes in esophageal cancers and their matched normal esophageal tissues. The correlation of the gene expression and the factors was also analyzed by using statistical analysis models. The patients were followed up to study the relationship between the gene expression levels of CTGF and the survival time.Materials and methods1. Subjects and sample collectionThe subjects were esophageal cancer patients hospitalized in The First Affiliated Hospital of ZhengZhou University, Cancer hospital in LinZhou city, Henan Province, from year 2004 to 2007. The cancer tissues and matched normal esophageal tissues from the subjects were taken during surgery. The identification of normal and tumor tissues was based on the pathological analysis. The samples were snap frozen in liquid nitrogen and then stored at -80℃for future processing. The data such as gender, age, family history, tumor location, size, tumor differentiation and metastasis were collected.2. Total RNA was extracted from esophageal cancer tissues by using TRIzol reagent. The equality of the RNA samples were determined through OD visualized underUV lihgt.3. Total RNA was reversed directly to cDNA by using AMV reverse transcriptase kit (Promega company). We also quantified transcription ofβ-actin to test the effect of reverse transcription.4. Determination of CTGF mRNA in samplesThe mRNA level of CTGF in esophageal cancer tissues and matched normal esophageal tissues was determined by Real-time PCR technique. Ct value represents the quantity of mRNA.5. Protein determination of CTGF in samplesCTGF protein expression were determined in 186 pairs of esophageal cancer tissues and matched normal esophageal tissues by immunohistochemistry. Three images at×200 magnification were observed for each sample and the number of cells expressing of CTGF protein was calculated. 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 esophageal cancer sample to that in matched normal esophagus sample.6. survival analysisWe collected samples of esophageal cancer patients from 2004 to 2007 in the Cancer Hospital in Linzhou city. 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 asthe survival time for patients.7. Statistical analysisProgram SPSS12.0 for Windows (SPSS, Chicago, IL) was used to analyze all the statistical analyses performed. For each sample, aΔCt (Ct_CTGF-Ct_β-actin) value represents the difference between the CTGF gene and the internal control gene,β-actin. Paired-sample Mest was used forΔCt analysis and the protein expression. The associations of CTGF gene mRNA and protein expression with the factors were analyzed by x²-test. Kaplan-Meier survival curve for sample with positive versus negative gene expression were plotted and log-rank test were used to compare the equality of the two survival curves. Multivariable analyses with the Cox proportional hazards model were used to estimate the effects of the factors and the CTGF expression on survival time. The statistically significant level was set atα=0.05.Results1 The Relationship between CTGF mRNA and Esophageal Cancer 1.1 CTGF genes expression in esophageal tumor tissue and the matched normal esophagealtissuesCTGF mRNA expression were found up-regulation in 154of 186 (82.8%) esophageal tissues compared with the paired normal esophageal tissues. CTGF mRNA level had significant difference between cancer and normal tissues (P = 0.000).1.2 Relationships between mRNA levels of CTGF in esophageal cancer and the factorsCTGF mRNA expression level was significantly higher in lymph node metastasis group and family history group compared with non-metastasis group and non-family history group (P=0.000, P=0.001). There were significant differences between different tumor size, infiltrating depth and tumor differentiation (P=0.000, P=0.000, P=0.000). However there were no relationships between CTGF mRNA expression level and sex, age, pthological type and tumor position.2 The Relationship between CTGF protein and Esophageal Cancer2.1 CTGF protein expression in esophageal cancer tissues and the paired normal tissuesThe expression of CTGF was up-regulated at protein level in cancer tissues compared with the paired normal esophageal tissues (P=0.000), which was similar to the results ofreal-time PCR.2.2 Relationship between protein expression of CTGF and the factorsUnivariate analysis demonstrated that CTGF protein expression was significantly associated with family history, metastasis, tumor size, tumor infiltrating depth and tumor differentiation. CTGF protein expression level was higher in lymph node group and family history group compared with non-metastasis group and non-family history group (P=0.000, P=0.000). There were significant differences between different tumor size, infiltrating depth and tumor differentiation (P=0.000, P=0.000, P=0.000). However, there were no relationships between CTGF protein expression level and sex, age, pthological type and tumor position.3 Survival analysis3.1 Univariate survival analysisThe Univariate survival analysis showed that CTGF mRNA expression, protein expression, sex, family history, infiltrating depth, tumor size, tumor metastasis, and tumor differentiation were significantly associated with patient survival time. Patients with the higher expression of CTGF showed a significantly decreased average survival time (P=0.000, P=0.000). Similarly, Patients with cancer family history and lymph node metastasis showed a significantly decreased survival time in comparison with those without family history and without metastasis (P=0.0017, P=0.000). Infiltrating depth was also significantly associated with average survival time (P=0.0012). Moreover, tumor differentiation, sex and tumor size were also associated with average survival time (P=0.001, P=0.0104, P=0.0381), poorly differentiated had decreased survival time than well-differentiated, women had decreased survival time than men, patients with bigger tumor showed also a decreased mean survival time comparing with those with small.3.2 Cox regression analysisThe multivariate (Cox regression) survival analysis showed that CTGF mRNA expression, protein expression, family history, tumor metastasis and tumor differentiation were significantly associated with patient survival time. The relative risk respectively were 5.008 (confidence interval, 1.562～16.057), 1.567 (confidence interval, 1.17～2.681), 1.65 (confidence interval, 1.037～2.641), 2.247 (confidence interval 1.309～3.860), 1.507 (confidence interval 1.104～2.056).Conclusion1. CTGF mRNA expression level was significantly higher in cancer tissues compared with the normal tissues. CTGF mRNA expression level was correlated with lymph node metastasis, family history, tumor size, infiltrating depth and tumor differentiation.2. The expression of CTGF was up-regulated at protein level in cancer tissues compared with the normal tissues. CTGF protein expression level was correlated with lymph node metastasis, family history, tumor size, infiltrating depth and tumor differentiation.3. Patients with the higher expression of CTGF showed a significantly decreased average survival time. The multivariate (Cox regression) survival analysis showed that the expression of CTGF, family history, tumor differentiation and tumor metastasis were independent risk factors for esophageal cancer patient survival time.