| Objective:Colorectal cancer(CRC)is one of the most common malignant tumors in the world.Its morbidity and mortality are third in both male and female patients.With the advancement of surgery techniques and the improvement of neoadjuvant and adjuvant therapy,the prognosis of locally advanced colorectal cancer has improved greatly.However,approximately50%of the CRC patients still develop local recurrence and distant metastasis.Therefore,finding a sensitive biomarkers to predict prognosis of CRC patients is the hot spot of current researches.Enhancer of zeste homologure 2(EZH2)is an important member of Polycomb protein family,which can inhibit the function of tumor suppressor genes by trimethylation of lysine 27 on histone H3(H3K27me3).Our study aimed at investigating the relation betwwen expression of EZH2 and H3K27me3 and chinicopathological features of colorectal cancer patients by using immunohistochemotherapy(IHC)in 55 paried tumor and normal colorectal tissues.Their expression relation and impact on prognosis of CRC patients were also analyzed.Then EZH2 knockdown colon cell lines were established in vivo to detect its regution of invasion ability of colon cells by using Transwell invasion assay.Methods:1.A total of 55 paried cancer tissue and corresponding normal tissue of CRC patiants were recruited from January 2012 to December 2012 at Department of Colorectal Surgery in Center Hospital of Cangzhou Province.The expression of EZH2 and H3K27m3 in colorectal cancer tissue and normal tissue were detected by IHC method.The expression level of their expression in cancer tissue and normal tissue were compared and their expression relation was evaluated.2.The clinicopathological information of 55 eligible CRC patients were collected and the relation of expression level of EZH2 and H3K27m3 and clinicopathological characteristics were assessed.3.The predictive value of EZH2 and H3K27m3 for overall survival(OS)and disease-free survival(DFS)of CRC patients was evaluated by using follow-up information and the expression patterns of the two biomarkers.4.The expressioin level of EZH2 in four different colon cancer cell lines,such as RKO,HCT-116,LoVo and DLD-1,was detected by real-time quantitative PCR and Western blot.The two colon cancer cell lines highly expressing EZH2 were used to establish knockdown cell lines of EZH2 by transfecting siRNA,and real-time quantitative PCR and Western blog were used to verify knockdown effect.5.The effect of EZH2 knockdown on the invasion ability of colon cancer cells was detected by Transwell invasion assay using EZH2-knockdown cells and the control cells.6.Statistical analyses were performed by SPSS 22.0 software.The results of IHC stain were analyzed using Chi-square test and Spearman correlated examination.The survival curves of OS and DFS were drawn by Kaplan-Meire method.Univariate and multivariate survival analyses were used to evaluate the association between various clinicopathological factors with OS and DFS by Cox proportional hazards model.Differences of paired groups were assessed by paired-sample t test and differences among multiple groups were evaluated by one-way ANOVA method with multiple compari-sons made by Tukey test.Results were expressed as mean±SD.For each protocol,at least three independent experiments were performed.All statistical tests were considered significant at P<0.05.All confidence intervals(CIs)were stated at the 95%confidence level.Results:1.In the 55 cases of CRC specimens,58.2%cases displayed high expression of EZH2,while 45.5%cases displayed high expression of H3K27me3.In the paired normal tissues,14.5%cases presented high expression of EZH2,while 18.2%cases presented high expression of H3K27me3.Comparing the immunal reaction score of EZH2 and H3K27me3expressioin in cancer and normal tissue,the expression differences of EZH2and H3K27me3 between cancer and normal tissue was of statistical significance(P<0.001).2.The expression level of EZH2 and H3K27me3 in cancer tissue were positively relavated(r~2=0.2396,P=0.0001).3.χ~2 test was used to analyze the relation between clinicopatholgical features and expression level of EZH2 and H3K27me3.We found that high expression level of EZH2 and H3K27me3 were correlated with more lymph node metastases(EZH2,P=0.008;H3K27me3,P=0.021),and distant metastases(EZH2,P=0.003;H3K27me3,P=0.002).Besides,the high expression level of EZH2 was also correlated with venous invasion(P=0.009).4.Univaraite analysis indicated that high levels of EZH2 and H3K27m3were correlated with worse OS EZH2:HR 4.56,95%CI 1.31-15.78,P=0.008,H3K27me3:HR 3.86,95%CI 1.46-10.19,P=0.003)and DFS(EZH2:3.89,95%CI 1.24-12.17,P=0.019;H3K27me3:2.85,HR 95%CI 1.69-6.98,P=0.011).5.Adjusted by multiple clincopathological factors,multivariate analyses indicated only TNM clinical stage was an independent factor for predicting OS(P=0.024)and DFS(P=0.012),while the expression level of EZH2 and H3K27me3 were not.6.Real time quantatitave PCR and Western blot were used to detect the relative expression level of EZH2 in RKO,HCT-116,LoVo and DLD-1 and the results showed that EZH2 was highly expressed in RKO and LoVo,with statistical significance(P<0.05).7.The knockdown cell lines of EZH2 in RKO and LoVo was established by transfecting siRNA.Real time quantatitave PCR and Western blot were used to verify the knockdown effect and found the expression level of EZH2was decreased with statistical significance in RKO-siEZH2-1,RKO-siEZH2-2and LoVo-siEZH2-1,LoVo-siEZH2-2,compared with RKO-scramble and LoVo-scramble(P<0.01).8.Transwell invasion assay indicated that the invasion ability of cells in RKO-siEZH2 and LoVo-siEZH2 declined significantly(P<0.001),compared with control groups(RKO-scamble and LoVo-srcamble).Conclusions:1.The expression of EZH2 and H3K27me3 increases in colorectal cancer tissue compared with normal tissue.The expression of EZH2 and H3K27me3were positively relavated.2.In colorectal cancer tissue,the expression of EZH2 and H3K27me3 are correlated with late stage,more lymph node metastasis and distant metastasis;EZH2 is also highly expressed in patients with venous invasion.3.High levels of EZH2 and H3K27me3 are correlated with worse OS and DFS,but they are not independent factors for predicting OS and DFS of CRC patients.4.Knockdown of EZH2 cell lines in RKO and LoVo are successfully established and RKO-siEZH2 and LoVo-siEZH2 cell lines show decreased invasion ability. |
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