The Roles And Mechanisms Of EZH2and VEGF In The Development Of Clear Cell Renal Cell Carcinoma

Kidney cancer is the third most common urological malignancy and isresponsible for an estimated120,000deaths per year worldwide. The incidenceof kidney cancer has increased significantly in the past. Renal cell carcinoma(RCC) is the most common type of kidney cancer in adults, and accounts foraround90%of primary malignant renal tumors, and remains a major cause ofmorbidity and mortality.Enhancer of zeste homolog2(EZH2) is the core member of Polycombrepressive complex (PRC) and acts as histone methyl transferase and results insilencing of various genes, including tumor suppressor genes, through directmethylation of lysine-27of histone H3. Dysregulated expression of EZH2isassociated with many cancers, but little is known about the roles of EZH2inClear cell renal cell carcinoma (CCRCC). Vascular endothelial growth factor(VEGF) is a potent angiogenic factor and plays important roles in RCC, andanti-VEGF therapy has been widely used for treatment of RCC. Reports haveshown that regulatory interplays exist between EZH2and VEGF-A. Howeverresults differ in different kinds of cancer. Regulatory roles and mechanisms ofEZH2in renal cell carcinoma are still unknown. The relationship between EZH2and VEGF is contradictory in CCRCC. In order to explain the relationshipbetween EZH2and VEGF expression and investigate the function of EZH2andVEGF in CCRCC,185paraffin-embedded primary tumor samples and10normal kidney tissues,5cm to the tumor area were used for analysis.Immunohistochemistry (IHC) and tissue microarray were used to investigate theexpression of EZH2, and analyze the relationship between EZH2expression and clinical characteristics. The recombinant EZH2plasmid overexpressing EZH2was constructed and used to investigate the function of EZH2in786-O cell line,and siRNA was used in knockdown experiment. The results are shown below:(1) Detection of EZH2and VEGF expression in CCRCC tissuesIn order to detect the expression of EZH2and VEGF in CCRCC tissues,185paraffin-embedded primary tumor samples and10normal kidney tissueswere collected and used for analysis with IHC and tissue microarray. The resultsof IHC array showed that EZH2expression was negative in normal kidneytissues while positive in all tumor samples, with low expression level in97cases(97/185) and high expression level in88cases (88/185). The positive signal wasin the nuclei of cancer cells. VEGF expression was positive in both the normalkidney and tumor tissues. In tumor tissues, VEGF with low expression in82cases (82/185) and with high expression level in103cases (103/185). Thepositive signal was in the cytoplasm of cancer cells. Spearman’s rank correlationcoefficient analysis showed that the protein expression level of VEGF and EZH2was positively correlated (correlation coefficient=0.850, P<0.001).(2) The association analysis between the expression of EZH2and VEGFand clinical features and survivalTo analyze the association between the expression of EZH2and VEGF andclinical features as well as survival, we collected the clinical information ofCCRCC patients, including age, gender, grade, TNM, metastasis and survivaltime. The results showed that the expression level of EZH2and VEGFcorrelated with the TNM stage (P=0.013, P=0.001, respectively) and distantmetastasis (P=0.011, P=0.038, respectively) of CCRCC patients. In patientswith low EZH2expression,60cases (60/97) were in TNM stage Ⅰ-Ⅱ, while37cases (37/97) were in stage Ⅲ-Ⅳ. In patients with high EZH2expression,38cases (38/88) were in TNM stage Ⅰ-Ⅱ, while50cases (50/88) were in stageⅢ-Ⅳ. In patients with low VEGF expression,32cases (32/82) were in TNM stage Ⅰ-Ⅱ, while50cases (50/82) were in stageⅢ-Ⅳ. In patients with highVEGF expression,66cases (66/103) were in TNM stage Ⅰ-Ⅱ, while37cases(37/103) were in stage Ⅲ-Ⅳ. Cases with low EZH2expression were mainly lowgrade tumors (61%), while cases with high EZH2expression were high gradetumors (57%). Patients with high EZH2and VEGF expressions present withdistant metastasis with the metastatic rate of62%and67%, respectively.Kaplan–Meier survival analysis revealed that patients with high EZH2expression showed a shorter overall survival time compared to patients with lowEZH2expression (34.3vs.67.2, P<0.001).(3) Effects of EZH2silencing on cell cycle, apoptosis, proliferation andVEGF expressionIn order to understand the function of EZH2in CCRCC comprehensively,the expression of EZH2was downregulated using siRNA in786-O cell line. Cellcycle, apoptosis, proliferation and the expression of VEGF were detected. Theresults showed that knocking down EZH2significantly decreased the expressionof VEGF mRNA and protein, normalized with GAPDH. Immunofluorescencemicroscopy showed that the expression of EZH2and VEGF were significantlydecreased, and the proliferation was also decreased. The apoptosis assayshowed that EZH2silencing significantly increased apoptosis, as shown byHoechst staining of the tumor cell nuclei and flow cytometry analysis. In cellcycle analysis, decreased EZH2expression increased the number of G1cellsfrom46.65%to70.65%and68.29%, but the number of G2cells decreased from17.30%to4.31%and5.36%, and the number of S cells decreased from36.05%to25.04%and26.35%.(4) Effects of EZH2overexpression on cell cycle, apoptosis, proliferationand VEGF expressionIn order to investigate the roles of EZH2on cell cycle, apoptosis,proliferation and the expression of VEGF, we overexpressed EZH2in786-O cell line, and detected the cell cycle, apoptosis, proliferation and the expression ofVEGF. The results showed that overexpression of EZH2could increase theexpression of VEGF mRNA and protein, normalized with GAPDH.Immunofluorescence microscopy showed that EZH2was expressed in the nuclei,and VEGF was expressed in the cytoplasm, and proliferation rate wassignificantly increased. The apoptosis assay showed EZH2upregulationsignificantly decreased the apoptosis of786-O cells. The apoptosis rate wasdecreased for around50%detected by Hoechst33258staining, and similarresults were found in AnnexinV-PE assay. In the cell cycle analysis,overexpression of EZH2increased the number of G2cells from16.62%to33.28%, and increased the number of S cells from30.67%to35.26%, butdecreased the number of G1cells from52.71%to31.46%.In the present study, the functions of EZH2and VEGF were investigatedboth in CCRCC tissues and786-O cell line. The results showed that EZH2positively correlates with VEGF and associates with adverse clinicopathologiccharacteristics, such as TNM and distant metastasis, and shorter survival time inCCRCC patients. EZH2accelerates proliferation, antiapoptosis and cell cyclein786-O cells. This study revealed the functions of EZH2and VEGF in CCRCC,and pointed out the relationship between EZH2and VEGF, and provided a novelinsight into the development of CCRCC, and may have an important clue intreatment of CCRCC.