Role Of EZH2 In Mechanism Of Cisplatin Resistance In Ovarian Cancer

Part One EZH2 gene silencing in A2780/DDP cells by RNA interferenceObjectiveTo examine the difference of EZH2 expression between cisplatin-resistant ovarian cancer cell line A2780/DDP and its parental cell line A2780, and to deplete expression of EZH2 in A2780/DDP cells by RNA interference.Methods1. Expression of EZH2 mRNA and protein in cells were detected by Real-time quantitative PCR and Western blotting.2. Four shRNA sequences targeting EZH2 gene were synthesized and inserted into Supersilencing shRNA plasmid expression vector pGPU6/GFP/Neo.3. After 4 recombinant vectors were transfected into A2780/DDP cells by LipofectamineTM 2000 respectively, the best effective vector was chosen by Real-time quantitative PCR. The stable transfected cell clone was obtained after being screened with G418. EZH2 knockdown in stable transfected cells was validated by Real-time quantitative PCR and Western blotting.Results1. Expression of EZH2 mRNA and protein were overexpressed by (3.50±1.06) and (2.10±0.29) times in A2780/DDP cells compared with A2780 cells (P<0.01).2. Four shEZH2 plasmid vectors were constructed successfully, and the shEZH2-1583 vector was the most effective one to knock down expression of EZH2.3. After shEZH2-1583 plasmid was transfected stably into A2780/DDP cells, mRNA and protein expression of EZH2 were decreased by (83.66±5.65)% and (77.57±3.90)% respectively (P<0.001).ConclusionEZH2 was overexpressed in A2780/DDP cell line compared with parental cell line A2780. The EZH2 shRNA vector was constructed and the stable EZH2-knockdown A2780/DDP cell line was obtained successfully.Part Two Effect of EZH2 gene silencing on cisplatin resistance in A2780/DDP cellsObjectiveTo investigate the effect of EZH2 knockdown on cisplatin resistance in cisplatin-resistant A2780/DDP cells.MethodsAfter the stable EZH2-knockdown A2780/DDP cells was obtained, assessment of chemoresistance to cisplatin was determined by the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays, the expression of H3-(me)3-K27 was evaluated by Western blotting, cell apoptosis was analyzed by Flow cytometry.ResultsThe 50% inhibition concentration (IC50) was decreased by (61.33±11.40)% in EZH2-knockdown A2780/DDP cells compared with vector-transfected cells (P<0.05). Expression of H3-(me)3-K27 was decreased by (50.50±7.80)%(P<0.001). No difference in cell apoptosis rate was observed between EZH2-knockdown cells and control cells (P>0.05).ConclusionRNA interference targeting EZH2 gene in A2780/DDP cells could resensitize the drug resistant ovarian cancer cells to cisplatin, EZH2 may play an important role in platinum resistance in ovarian cancer via H3K27 methylation.Part Three Effect of EZH2 gene silencing on cell proliferation and migration in A2780/DDP cellsObjectiveTo investigate the effect of EZH2 knockdown on cell proliferation and cell migration in ovarian cancer cell line A2780/DDP.MethodsCell viability was assessed by MTT assays, cell cycle was analyzed by Flow Cytometry, and migration capability was examined by Transwell migration assay.ResultsEZH2 knockdown suppressed cell proliferation capability, caused G2/M cell cycle arrest, and also inhibited cell migration capability of A2780/DDP cells (P<.05).ConclusionRNA interference silencing EZH2 gene could significantly inhibit cell proliferation and migration of A2780/DDP cells, thus provide a novel target for epigenetic therapy of cisplatin resistance in ovarian cancer.Part Four Effect of EZH2 gene silencing on cisplatin sensitivity of ovarian tumor xenograftsObjectiveTo investigate the effect of EZH2 gene silencing on cisplatin sensitivity of ovarian tumor xenografts.MethodsFour-week old female BALB/c-nu nude mouse were randomized in 2 groups for experiments. ShEZH2 cells and shVector cells were injected subcutaneously into the right flank of nude mouse respectively. When the mean tumor diameter was at least 0.5 cm, the animals of each group were further randomized into cisplatin treated group and untreated group. Weight of mice and tumor volumes were measured everyday after cisplatin treatment, tumor growth inhibition rate was calculated as (Vuntreated group—Vcisplatin-treated group)/Vuntreated group. Expression of EZH2 and H3-(me)3-K27 of tumor were analyzed by immunohistochemistry, Hematoxylin-eosin staining was performed to investigate the morphological structure of liver and kidney.Results1. After the cisplatin treatment on day 0, the growth inhibition rate of shEZH2 tumor xenografts on day 1,3,5,7 were 39%,59%,71%,80%, while the inhibition rate of shVector tumor xenografts were 12%,29%,43%,43% respectively. The tumor inhibitory rate of shEZH2 group was much higher than that of shVector group (P<0.05). There was no difference between the body weight of cisplatin-treated mice and untreated mice (P>0.05). The histological structure of liver and kidney of nude mice were normal.2. Five weeks after tumor formation, the tumor growth of shEZH2 xenografts was significantly decreased compared with shVector tumor xenografts.3. Immunohistochemistry showed that the expression of EZH2 and H3-(me)3-K27 in shEZH2 tumor xenografts were decreased significantly than that in shVector tumor xenografts (P<0.05).ConclusionEZH2 gene silencing enhanced cisplatin sensitivity of ovarian tumor xenografts and inhibited the tumor growth in vivo. Gene therapy targeting EZH2 could reverse cisplatin resistance of ovarian tumor xenografts efficiently and safely.