CAJ | 학술논문

目的探讨沉默EZH2表达逆转人肺癌顺铂耐药性的作用及机制。方法以实时荧光定量 PCR 和 Western blot法检测人非小细胞肺癌A549及其顺铂耐药株A549/DDP细胞中EZH2的表达；以siRNA沉默EZH2的表达, MTT法检测细胞增殖力；流式细胞术检测细胞周期分布与凋亡；?-半乳糖苷酶染色法检测细胞的衰老； Western blot法检测细胞衰老通路 P14ARF、P16INK4a、P53、P21、CDK1、CDK2、Rb、E2F1和H3K27me3的表达。结果 A549/DDP细胞的EZH2在mRNA和蛋白质水平上的表达均明显高于A549细胞。沉默EZH2的表达并未引起细胞发生明显的凋亡现象,但G0/G1期比值明显升高(从0.53±0.08升到0.84±0.09, P <0.01),明显增加耐药株对顺铂的敏感性[IC50从(41.2±4.3)μmol·L-1降低到(19.4±3.3)μmol·L-1,P<0.01]。细胞呈衰老特征,细胞衰老通路的 P14ARF、P16INK4a、P53、P21、Rb表达上调,CDK1、CDK2、H3K27me3、E2F1表达下调。结论沉默EZH2的表达能诱导A549/DDP细胞衰老,逆转其对顺铂的耐药性,其机制与调节INK4a/ARF/Rb衰老通路有关。
목적탐토침묵EZH2표체역전인폐암순박내약성적작용급궤제。방법이실시형광정량 PCR 화 Western blot법검측인비소세포폐암A549급기순박내약주A549/DDP세포중EZH2적표체；이siRNA침묵EZH2적표체, MTT법검측세포증식력；류식세포술검측세포주기분포여조망；?-반유당감매염색법검측세포적쇠로； Western blot법검측세포쇠로통로 P14ARF、P16INK4a、P53、P21、CDK1、CDK2、Rb、E2F1화H3K27me3적표체。결과 A549/DDP세포적EZH2재mRNA화단백질수평상적표체균명현고우A549세포。침묵EZH2적표체병미인기세포발생명현적조망현상,단G0/G1기비치명현승고(종0.53±0.08승도0.84±0.09, P <0.01),명현증가내약주대순박적민감성[IC50종(41.2±4.3)μmol·L-1강저도(19.4±3.3)μmol·L-1,P<0.01]。세포정쇠로특정,세포쇠로통로적 P14ARF、P16INK4a、P53、P21、Rb표체상조,CDK1、CDK2、H3K27me3、E2F1표체하조。결론침묵EZH2적표체능유도A549/DDP세포쇠로,역전기대순박적내약성,기궤제여조절INK4a/ARF/Rb쇠로통로유관。
Aim Toinvestigatethereverseeffectofsi-lencing EZH2 on the human cisplatin-resistant non-small cell lung cancer cell line A549/DDP and its mechanism.Methods TheexpressionofEZH2was detected by real time qPCR and Western blot. siRNA was used for silencing gene expression of EZH2 . As-sessment of proliferation and chemoresistance to cispla-tin in A549/DDP cells was evaluated by MTT assay. Cell cycle and apoptosis were analyzed by flow cytome-try. Cell senescence was assessed by ?- galactosidase dyeing. The expression of H3K27me3, P14ARF, P16INK4a, P53, P21, CDK1,CDK2, Rb and E2F1 was determinedbyWesternblot.Results Theexpressions of EZH2 mRNA and protein were significantly elevated in A549/DDP cells compared with A549 cells. Silen-cing EZH2 expression resensitized A549/DDP cells to cisplatin [ IC50 from ( 41. 2 ± 4. 3 ) μmol · L-1 to (19. 4 ± 3. 3)μmol·L-1, P<0. 01], and also caused a rise of G0/G1 phase from (0. 53 ± 0. 08) to (0. 84 ± 0. 09 ) ( P<0. 01 ) , but there was not a marked varia-tion of apoptotic rate. EZH2 knockdown induced obvi-ous senescence phenotype in A549/DDP cells. EZH2-siRNA could also down-regulate the expression of CDK1 , CDK2 , H3 K27 me3 and E2 F1 , while it up-regulated the expression of P14ARF, P16INK4a, P53, P21andRbinA549/DDPcells.Conclusions Silen-cing EZH2 by RNA interference could induce cell se-nescence and resensitize A549/DDP cells to cisplatin. The mechanism of resistance reversal is associated with INK4a/ARF/Rb senescence pathway.