沉默EZH2表达逆转人非小细胞肺癌顺铂耐药性

Reverse effect of silencing EZH2 expression on human cisplatin-resistant non small cell lung cancer

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;β-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。

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.