ABSTRACT
Objective To investigate the inhibitory effects of silencing expression ofEZH2 gene on the cell proliferation of human QBC939 cells and its mechanisms. Methods The targeting siRNA was designed and trans-fected into QBC939cells. The expressions of EZH2 mRNA and protein were detected by real-time qPCR and west-ern blotting,respectively. The ability of cellproliferationwas analyzed by MTT assay and plate clone formation assay. Cell apoptosis and cycle percentage weremeasured by flow cytometry. Cell senescence was assessed byβ-galactosi-dase dyeing.The expressions of H3K27me3,P14ARF,P16INK4a,P53,P21 and E2F1 proteinwere determined by West-ern blotting.Results Compared with the control group ,the expressions of mRNAand protein were significantly elevat-ed in experimental group. The ability of cellproliferation in the experiment group was significantly down regulated , which could also cause a rise of G1/S phase ,but not a marked variation of apoptosis rate. Silencing EZH2 would induce a obvious senescence phenotype in QBC939 cells. EZH2-siRNA transferredcould also down-regulate the expressions of H3K27me3 and E2F1 protein,while up-regulating the expressions of P14ARF,P16INK4a,P53 and P21 protein in QBC939 cells.Conclusions Silencing EZH2 could induce a significant inhibition on cell proliferation of QBC939 cells,the mechanism of which may be associated with the senescence pathway regulation.
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of silencing Bmi-1 expression in reversing cisplatin resistance in human lung cancer cells and explore the possible mechanisms.</p><p><b>METHODS</b>Cisplatin-resistant A549/DDP cells with small interference RNA (siRNA)-mediated Bmi-1 expression silencing were examined for cisplatin sensitivity using MTT assay and alterations in cell cycle distribution and apoptosis with flow cytometry, and the changes in cell senescence was assessed using β-galactosidase staining. The protein expressions of Bmi-1, P14(ARF), P16(INK4a), P53, P21, Rb and ubi-H2AK119 in the cells were determined with Western blotting.</p><p><b>RESULTS</b>A549/DDP cells showed significantly higher Bmi-1 expression than A549 cells. After siRNA-mediated Bmi-1 silencing, A549/DDP cells showed significantly enhanced cisplatin sensitivity with an increased IC50 from 40.3±4.1 µmol/L to 18.3±2.8 µmol/L (P<0.01) and increased cell percentage in G0/G1 phase from (48.9±2.3)% to (78.7±7.6)% (P<0.01). Silencing Bmi-1 did not cause significant changes in the cell apoptosis rate but induced obvious senescence phenotype in A549/DDP cells with down-regulated expression of ubi-H2AK119 and up-regulated expressions of P14(ARF), P16(INK4a), P53, P21 and Rb.</p><p><b>CONCLUSION</b>Silencing Bmi-1 by RNA interference can induce cell senescence and resensitize A549/DDP cells to cisplatin possibly by regulating INK4a/ARF/Rb senescence pathway.</p>