| BACKGROUND:Recent research shows that,the incidence and mortality of lung cancer increased gradually,to 2014,lung cancer is the malignant tumor in the first.Currently,the curative effect of comprehensive treatment of lung cancer has increased,but the 5 year survival rate is still less than 20%,tumor invasion and metastasis is the main cause of death in patients with lung cancer.The prognosis of patients with lung cancer are mostly bad,thus,early discovery,early diagnosis,early treatment is very important to improve the cure rate and survival rate.Further explore the molecular mechanism of distant metastasis lung cancer,to find effective molecular targeted therapy,it has the vital significance to develop a plan of clinical comprehensive treatment for lung cancer.EZH2(Enhancer of Zeste Homologue 2)is the human homolog of Drosophila zeste gene enhancer,it is a member of the Polycomb-group(PcG)family,locating in human chromosome 7q35,its coding protein contains highly conserved SET structure domain,the domain structure has methyltransferase activity,inhibition of expression the downstream gene by methylation.EZH2 is associated with cell growth regulation,regulation of cell proliferation by inhibiting target gene of chromatin,and its high expression can cause cells to enter S phase.In addition,it plays an important role in maintenance of cell characteristics,cell cycle regulation,cell aging and cell differentiation.A growing number of evidence indicates that EZH2 is high expressionin in many human malignant tumors,such as oral cancer,esophageal cancer,gastric cancer,liver cancer,colorectal cancer,bladder cancer,breast cancer,lymphoma,endocrine tumor and prostate cancer.It is suggest that the abnormal expression of EZH2 is closely related to the prognosis of patients and tumor malignant progression.E-cadherin,locating in human chromosome 16q22.1,is a member of the cadherin family,mainly distributed in various epithelial cells,it is an important medium for the regulation of cell and between cells and the extracellular matrix adhesion,plays an important role in maintaining the structural form,is considered to be an important tumor suppressor gene.A recent study found that:EZH2 can methylate the promoter H3K27 of the tumor suppressor gene E-cadherin gene,and downregulate the expression,promote the progression of gastric cancer.Does the tissue expression of two in non small cell lung cancer has relevance?In this study,we use immunohistochemistry and image analysis technology to investigate the expression of EZH2 protein in lung cancer at the quantitative level and semi-quantitative level and its relationship with proliferation and prognosis,at the same time detection E-Cadherin expression in lung cancer group,analysis the correlation of EZH2 and E-Cadherin in non-small cell lung cancer,then we investigated its expression in different human lung cancer cell lines both at the protein and mRNA levels,and down-regulate of EZH2 expression,the changes of E-cadherin expression level.Finally,in order to investigate the effect of EZH2 on the morphology,proliferation,apoptosis,cycle and metastasis of human lung adenocarcinoma cancer cell line A549,we apply RNA interference technique to suppress the expression of EZH2 in A549 cell.OBJECTIVE:1.To investigate the expression of EZH2 and its prognostic significance in non-small cell lung cancer;2.To investigate the correlation between EZH2 and E-Cadherin in lung cancer;3.To investigate the effect of EZH2 on the morphology,proliferation,apoptosis,cycle and metastasis of human lung cancer cell line A549 by downregulating EZH2 expression.METHOD:1.EZH2 expression in normal lung tissue,lung benign lesion,non-small cell primary lung cancer and lymph node metastases,and its prognostic significanceImmunohistochemical SP method was used to detect the expression of EZH2 in 32 cases of normal lung tissue,24 cases of lung benign lesion tissues,223 cases of lung cancer and 57cases of corresponding metastasis lymph node.ImageproPlus6.0(IPP)image analysis software was applyed to test protein expression of positive unit(PU value).Analysis the expression intensity variation of EZH2 protein in different types of lung tissues,and explore the relationship between its high or low levels and clinpathological characteristics in non-small cell lung cancer.Statistical analysis and postoperative survival analysis were analyzed together with clinical pathological characters and follow-up data.2.The expression of EZH2 and E-cadherin correlation analysis in non-small cell lung cancerInmunohistochemical SP method was used to detect the expression of EZH2 and E-cadherin in 78 cases of lung cancer,we adopted the method of semi-quantitative analysis for immunohistochemical results.Judgment the method of semi-quantitative results are as follows:according to the stain intensity and the percentage of stained cells were scored.Stain grade:no coloring is 0;Color is light yellow to 1;color is yellow to 2;Color is brown to 3.Counting the percentage of Staining positive cells,calculation the proportion of positive tumor cells to total tumor cells,which is divided into 5 grades:counting the staining positive cells<5%was 0(-),5%?25%was 1(+),>25%?50%was 2(++),>50%?75%was 3(+ + +),>75%was 4(+ + + +).The staining degree classification and dyeing percentage of cell multiplicated,multiplication of? 4 divided into positive expression(high expression),0-3 divided into negative expression(low expression).3.To detect the expression of EZH2 in different human lung cancer cells,and to detect the relationship between EZH2 and E-Cadherin expression in lung cancer cells.Western Blot was used to detect EZH2 protein expression in A549,GLC-82 and H358 cells,EZH2 mRNA expression was detected in A549,GLC-82 and H358 cells by qRT-PCR.We Select the highest expression of EZH2 of a cell to interfere.using qRT-PCR and Western Blot method to verify the interference efficiency,the expression of EZH2 and E-Cadherin were detected,we want to explore wether the EZH2 decline will affect the E-Cadherin expression.4.The effect of EZH2 down-regulation on the morphology,proliferation,apoptosis,cycle and metastasis in human lung cancer cell A549.EZH2 siRNA was transiently transfected into human lung cancer cell line A549 after design and synthesis to inhibit the expression of EZH2.The inhibition efficiency was detected by qRT-PCR and Western Blot.MTT assay was used to detect ability of cell proliferation changes,wound healing assay and transwell experiment were used to detect cell migration and movement changes,Annexin V-FITC/PI double staining cell apoptosis assay was used to detect apoptosis changes,cell cycle assay was used to detect cell cycle changes,5.Statistical analysisAll statistical analysis were carried out with the use of SPSS 19.0 statistical software package.One-way ANOVA test was used in the comparison of multiple sample averages.When the variances are heterogeneous,Dennett's T3 test were used for multiple comparison between groups.Otherwise,LSD test were used;Paired samples used paired samples't test;Two independent-sample t-tests were used between the two groups;Correlation of non-normol distribution between two variable used Spearman method;Five-year survival rate was checked with Pearson ?2 test.Kaplan-Meier method was used for univariate survival analysis,log-rank test was for the significance of differences in survival,and Cox proportional hazards regression model was for the identification of relevant prognostic factors.A 2-sided P<0.05 was considered statistically significant in all the analysis.RESULTS:1.EZH2 expression in normal lung tissue,lung benign lesion,non-small cell primary lung cancer and lymph node metastases,and its prognostic significanceThe expression of EZH2 in lung cancer tissue was mainly localized in lung cancer cell nucleus,which were clearly brown-yellow stained.It was almost no expression in the lung benign lesions and normal lung tissues,it showed a small amount expression of EZH2 in mucosal epithelium cells and wall glands epithelial cells in bronchietasis.The EZH2 PU value of lung cancer tissues significantly greater than that of normal lung tissues and benign lung disease(P=0.000,P=0.000).The EZH2 PU value in normal lung tissues similar to that in benign lung lesions(P=0.716).There was no significant difference amongs the EZH2 PU value of bronchiectasis,bullae and tuberculosis belonged to benign lung disease(F=1.574,P=0.231).The expression of EZH2 PU value in various histological types of lung cancer were compared with normal lung tissue and benign lung lesions respectively,the difference was statistically significant(F=35.063,P=0.000),In 226 cases of different histological types of lung cancer tissues,the PU value of EZH2 protein expression was different(F = 3.38,P = 0.01),the EZH2 PU value in squamous cell carcinoma were lower than that of adenocarcinoma,small cell carcinoma,adenosquamous carcinoma(P=0.010,P=0.017,P=0.048),the EZH2 PU value in large cell carcinoma were lower than that of small cell carcinoma(p=0.044),the rest EZH2 PU value in various histological types were pairwise comparison,there was no significant difference(P>0.05).The EZH2 PU value in primary lung tumors was smaller compared with that in corresponding lymph node metastases tissues(P=0.000).In 226 cases of lung cancer tissues with different degree of differentiation,the well differentiated lung cancer had smaller EZH2 PU value than moderately-poorly differentiated,the difference was statistically significant(t=-4.666,P=0.000).In the general types of lung cancer,the EZH2 PU value in peripheral lung cancer was higher than the central type of lung cancer,the difference was statistically significant(t=3.172,P=0.002).The PU value of maximum tumor diameter>3cm lung cancer is higher than the maximum tumor diameter ? 3 cm lung cancer(t=2.927,P=0.004).EZH2 PU value was not associated with the patients' gender,age,smoking status,pTNM stage and lymph node metastasis(P>0.05).2.The relationship between the semi quantitative EZH2 expression and prognosis in non small cell lung cancerThe prognostic value of EZH2 expression in 153 NSCLC patients with 5-year follow-up data was analyzed.5-year overall survival rate in NSCLC was 31.4%(48/153)in this study.The survival rate of EZH2 for low expression at the 5th year was 48.8%(21/43),and for high expression was 24.5%(27/110),difference in survival rates between the two groups was obvious(?2=10.826,P=0.001).Kaplan Meier analysis results showed that:the expression of EZH2 protein,lymph node metastasis,TNM stage,distant metastasis was associated with the prognosis of patients with non-small cell lung cancer.The median survival time in patients with low expression of EZH2 was significantly higher than that in patients with high expression(?2 = 10.826,P = 0.001);The median survival time in patients without lymph node metastasis was significantly higher than that in patients with lymph node metastasis(?2 = 16.119,P = 0.000);The median survival time in patients with TNM stage ?-? was significantly higher than that in patients with stage ?-?(?2=8.973,P=0.003);The median survival time in patients without distant metastases was significantly higher than that in patients with distant metastasis(?2=5.008,P=0.025);The prognosis of patients was not associated with the patients' gender,age,smoking status,histological type,degree of differentiation,gross type,tumor diameter.Patients with high expression of EZH2 also predicts poor prognosis in tumor stage ?-?(?2=7.391,P=0.007),whereas patients of stage ?-IV showed a poor prognosis both in low and high expression(?2=1.458,P=0.227).Patients with high expression of EZH2 also predicts poor prognosis in without lymph node metastasis(?2=4.879,P=0.027),patients with high expression of EZH2 also predicts poor prognosis in lymph node metastasis(?2=4.597,P=0.032);Patients with high expression of EZH2 also predicts poor prognosis in without distant metastasis(?2=8.215,P=0.004),whereas patients with distant metastasis showed a poor prognosis both in low and high expression(?2=1.719,P=0.190).3.Cox proportional hazards multivariate analysisFor all risk factors that may affect the prognosis:gender,age,smoking history,gross type,tumor size,histological type,degree of differentiation,lymph node metastasis,distant metastasis,pTNM stage,EZH2 expression were performed univariate Cox model analysis,the results showed that lymph node metastasis,distant metastasis,pTNM stage,EZH2 expression were independent prognostic factors affecting overall survival time(P=0.000,P=0.030,P=0.004,P=0.002),relative risk(HR)were 0.449,1.847,1.801,0.464.Further multivariate Cox model analysis for the above results show that the lymph node metastasis,distant metastasis,EZH2 expression were independent prognostic factors affecting overall survival time(P =0.004,P = 0.027,P = 0.002),the relative risk(HR)were 0.474,1.888,0.472,that is to say,lymph node metastasis and distant metastasis in patients with high risk,the high expression of EZH2 in patients with high risk.4.Correlation of EZH2 and E-cadherin expression in lung cancerThe expression of E-cadherin in lung cancer tissue was mainly localized in lung cancer cell membrane or cytoplasm,which were clearly brown-yellow stained.Expression of E-cadherin protein in squamous cell carcinoma and adenocarcinoma are differences,squamous cell carcinoma was higher than that of adenocarcinoma,the difference was statistically significant(P=0.035),the rest E-cadherin PU value in various histological types were pairwise comparison,there was no significant difference(P>0.05).The expression of E-cadherin protein is related to gender,the expression of E-cadherin in female was higher than that in males,the difference was statistically significant(P=0.003);The expression of E-cadherin in patients with smoking history was significantly higher than that in patients without smoking history,the difference was statistically significant(P=0.005);The PU value of maximum tumor diameter<3 cm lung cancer was higher than the maximum tumor diameter>3cm lung cancer,the difference was statistically significant(P=0.006);The expression of E-cadherin protein was not associated with the patients' age,gross type,differentiation degree,lymph node metastasis,distant metastasis and pTNM stage(P>0.05).The result of correlation analysis of expression of EZH2 and E-cadherin showed:EZH2 was correlated negatively with E-cadherin expression from the quantitative point of view(r =-0.224,P = 0.048);EZH2 was also correlated negatively with E-cadherin expression from the semi quantitative point of view(r=-0.232,P = 0.041),the two methods showed negative correlation with the expression of EZH2 and E-cadherin protein.5.The effect of EZH2 gene silence on human lung adenocarcinoma cell line A549 by RNA interferenceqRT-PCR was used to detect EZH2 mRNA expression in A549,GLC-82 and H358 cells,the results showed that:EZH2 mRNA in three lung cancer cell lines in the expression were significantly different(F=797.969,P=0.000);The expression of EZH2 mRNA in GLC-82 cells was highest,and which in H358 cells was lowest.Compared with the negative control group,qRT-PCR results showed that,EZH2 SiRNA interference after 48h,its related genes-E-Cadherin,mRNA expression levels increased(t =-21.931,P = 0.000).Western Blot was used to detect EZH2 protein expression in A549,GLC-82 and H358 cells,the results showed that:EZH2/p-actin protein ratio grayscale in three lung cancer cell lines were significantly different(F = 64.679,P = 0.000),the expression of EZH2 protein in A549 cells was highest,and which in H358 cells was lowest.We extracted the total cellular protein when SiRNA interference EZH2 after 72h,Western Blot results showed that:the expression of EZH2 protein after interference was significantly reduced,while the expression of EZH2 silence allows upregulation of E-Cadherin.The results of MTT assay in A549 cell showed that:the OD values in EZH2 inhibitor group at 48h,72h and 96h were lower compared with negative control group(P(0.05),the cell growth inhibition rate was 25.7%,20.7%and 18.8%respectively at every time point after transfection,the cell growth inhibition rate was most top at 48h.The results of wound healing assay in A549 cell showed that:Compared with negative control group,the cell migration speed in EZH2 inhibitor group at Oh,12h,24h,36h,48h and 72h after wound significantly slowed down.The results of transwell chamber assay in A549 cell showed that:the cells have crossed the membrane in EZH2 inhibitor group was significantly less than negative control group(t=4.895,P=0.000).Cell cycle assay show that:compared with the negative control group,the G1 phase cells in EZH2 inhibitor group increased,S phase cells decreased,the difference was statistically significant(t = 3.386,P = 0.028;t =-4.735,P = 0.009);while G2 phase cells were no difference between the two(t = 0.434,P = 0.687).The results show that SiRNA down regulated the expression of EZH2,it can lead to the number of cells in G1 phase increased,S phase cells decreased.Annexin V-FITC cell apoptosis assay show that:when SiRNA down regulated the expression of EZH2,it can lead to tumor cell apoptosis ratio increased,but the difference was not statistically significant(t = 0.511,P = 0.636).CONCLUSIONS:1.The expression level of EZH2 was associated with histological type,differentiation,gross type,tumor diameter,the higher expression levels,the poorer the prognosis.Multivariate Cox model analysis showed that lymph node metastasis,distant metastasis,the expression of EZH2 were independent prognostic factors affecting overall.survival time.2.The result of correlation analysis of expression of EZH2 and E-cadherin showed:from the point of view of quantitative and semi quantitative,there is a negative correlation between EZH2 and E-cadherin.3.Down regulation of EZH2,the expression of E-cadherin can recover both in the protein and mRNA levels.4.Expression of EZH2 can promote proliferation,migration and movement of lung adenocarcinoma cell line A549,the impact on cycle is:make the G1 phase cells increased,S phase cells decreased,and no effect on the G2 phase cells.It does not affect apoptosis. |
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