Effect Of RNAi-mediated Inhibition Of EZH2 Gene Expression On Proliferation Of Human Bladder Cancer EJ Cell Line

Bladder transitional cell carcinoma (BTCC) is a very common malignancy inurogenital system in China. The etiology of bladder cancer is still unknown. Despiteadvances in early detection and treatment of BTCC, many patients with BTCC diedfrom recurrence and metastasis after operation. Some biomarkers may have definiteeffect on prognosis of BTCC. The patients with bladder cancer may benefit fromapplication of therapy designed according to expression of specific biomarker. Thus,there is a need for novel molecular predictors of tumor behavior at the time ofdiagnosis that will help guide clinical therapy decision and supervision of tumordevelopment.Recent studies have found that, from biological behavior to pathologicalcharacteristics, BTCC actually has two phenotypes: low malignant and aggressive, theso-called concept of two type bladder transitional cell carcinoma. The low malignantusually originate the atypical hyperplasia that is associated with high risk ofrecurrence and easily develop to the aggressive after recurrence. The aggressivegenerally arise from dysplasia which is characterized by invasiveness and metastasis.Under the guidance of the concept, we identified the different expression genesbetween the low malignant BTCC and normal transitional epithelium using lasercapture microdissection and gene chip analysis, indicting that high expression ofEZH2, a member of transcriptional repressors, is involved in early development ofBTCC.The EZH2 is a homolog of the Drosophila Polycomb group (PcG) gene enhancerof zest, a crucial regulator of homologous gene expression. EZH2 is thought to beinvolved in the early embryonic development and the control of cellular cycle.Overexpression of EZH2 can promote the cell entry to S phase and proliferation as transcriptional repressor. EZH2 was shown to be characteristic by inhibiting theability of anti-oncogene.Several studies have shown that EZH2 is highly expressed invarious tumors and linked to progression of human tumor cells and has beensuggested as a novel oncogen. In theory, the suppression of EZH2 expression mayinhibit the tumorigeness and metastasis. Mentioned above, the low malignant has atendency to develop the aggressive. Then, it is unclear for EZH2 how to be involvedin the progress of the development and what role to play. So, we evaluate the role andmechanism of EZH2 in bladder tumorigeness.Partâ… : Expression of EZH2 gene in BTCC and cell lines and itsclinical significanceEZH2 gene fimctions as a transcriptional repressor involved in gene silencing.Amplification of EZH2 has been reported in several malignancies, including prostate,breast and liver. In the study, we evaluated 49 BTCC specimens for EZH2 proteinexpression using immunohistochemistry and EZH2 mRNA expression was assessedby RT-PCR in 36 BTCC specimens. In addition, human bladder cancer cell lines EJand T24 were analyzed by RT-PCR and immunohistochemistry for EZH2 mRNA andprotein expression. The results indicated that three of ten normal urothelium tissuesversus 27 of 36 BTCC tissues had EZH2 mRNA expression (71% vs 20%, p=0.008).All of the high grade tumors had detectable EZH2 mRNA expression compared with10 of 19 low grade tumors (100% vs 53%, p=0.0038). In addition, EZH2 mRNAexpression was noted in 50% (7/14) superficial tumors verse 91% (20/22) invasivetumors (p=0.006). EZH2 protein expression, meanwhile, was increased in bladdercancer tissue compared with normal urothelium (70% vs 20%, p=0.0068). Thedifference was found between superficial tumors and invasive tumors (53% vs 83%p=0.02). High-grade tumors had increased EZH2 expression compared withlow-grade tumors (100% vs 60%,p=0.02). EJ and T24 expressed EZH2 mRNA andprotein. These data suggest that EZH2 gene may correlate with oncogenesis andprogression of BTCC. The expression level of EZH2 may predict the prognosis indefinite degree and help the early diagnosis, supervision of tumor progression and evaluation of prognosisPartâ…¡: Design and construction of siRNA expression plasmidtargetted EZH2 gene and its effect on the cell proliferationRNA interference (RNAi) is a novel approach for suppression of gene expression,which is characteristic by a gene-specific, posttranscriptional gene silencingmechanism. Small interference RNA (siRNA) homolog to silenced gene in sequenceis indued into cell to clearage the mRNA of target gene. Many studies havedemonstrated that RNAi is an effective tool for gene diagnosis and gene therapy.According to literature report, we selected the region containing the 461-481 basesof EZH2 complementation DNA as target and constructed EZH2 siRNA expressionplasmid and analyzed its effect on the proliferation of bladder cancer EJ cell line. Wedesigned the DNA model-strand containing the small hairpin of EZH2siRNA. Thenthe model strand was annealed and cloned into pRNAT plasmid to build therecombinatant plasmid pRNAT-EZH2-shRNA, which was confirmed by sequencing.We used lipofectamine to transfect the pRNAT-EZH2-shRNA into EJ cell. TheRT-PCR and Western-blot were used to detect the EZH2 expression. The proliferationof the transfected EJ cell was assessed with MTT, and flow cytometry. The resultsshowed that EZH2 expression was significantly suppressed by pRNAT-EZH2-shRNA,which led to cell decrease in S phase and blockage of cell proliferation. These dataindicate that EZH2 gene may,be involved in regulation of cell cycle and cellproliferation and be a potential target for gene therapy and application of RNAi mayan effective approach for gene therapyPartâ…¢: Analysis of gene expression profile by gene chip on humnanbladder cancer EJ cell line with knockdown of EZH2 geneTo elucidate the EZH2 functional mechanism in oncogenesis, we applied thegene chip of 22K Human Genome Array for analyzing the gen expressionprofile after EZH2 konckdown mediated by siRNA. The gene chip contains21522 Oligo DNA, each representative of human gene transcript. The samples were hybridised with gene chip after labelled by fluorescence. The resultsshowed 436 differented expression genes, among which 179 genes wereupregulated and 257 genes were downregulated. These genes were uploadedthe pathway miner and 152 genes were found in known biological pathways,including EGF signal pathway, p53 pathway, cell proliferation regulationpathway, cell cycle regulation pathway,Notch, Wnt pathway and so on. Thisimplied that EZH2 may regulate the cell proliferation by multiplebiological pathways. In the study, we found that various EZH2 target genesuch as MYT1, CNR1, WNT1 were upregulated, most of which are involved inthe embryonic development and cell differentiation, implying that EZH2is possible to be a potential biological marker of tumor stem cell. Inaddition, some protein relative to cell cycle regulation such as CDKN1A,CDKN1C and CCNE1 were showed to be downregulated, which suggested thatEZH2 may contribute to the oncogenesis by regulation of cell cycle.