EZH2在小细胞肺癌恶性表型中的作用及其潜在调控机制

Role of EZH2 in malignant phenotype of small cell lung cancer and its potential regulatory mechanism

目的探讨Zeste同源物2增强子(enhancer of zeste homologue 2,EZH2)对小细胞肺癌(small cell lung cancer,SCLC)细胞增殖、凋亡和化疗耐药性的影响,并初步阐述EZH2作为长链非编码RNA HOTTIP的下游效应分子的调控通路。方法采用RNA干扰技术和EZH2小分子抑制剂GSK-126处理SCLC细胞,并使用qRT-PCR、Western blot、CCK-8实验、流式细胞术及免疫组化法分析EZH2参与SCLC恶性表型的调控作用。结果与癌旁肺组织相比,EZH2在SCLC组织中的表达显著升高(P<0.05);与人支气管上皮细胞相比,EZH2在SCLC细胞株中的表达较高;与SCLC化疗敏感细胞相比,EZH2在SCLC化疗耐药株中的表达更高(P<0.05)。采用EZH2小分子抑制剂GSK-126和EZH2 shRNA干扰序列抑制或沉默EZH2表达后,CCK-8实验分析发现,沉默EZH2表达后,SCLC细胞对三种化疗药物的敏感性增强(P<0.001)、增殖能力下降(P<0.01)。流式细胞术分析发现,EZH2下调后两株细胞G_(2)期细胞比例减少(P<0.05)、细胞早凋率(P<0.01)及晚凋率(P<0.05)均呈增加趋势。生物信息学技术分析发现,EZH2启动子区甲基化水平在肺鳞状细胞癌及肺腺癌组织中均较低(P<0.05),采用GSK-126处理细胞,发现细胞中EZH2蛋白表达随GSK-126升高而下降(P<0.05),细胞中甲基转移酶H3K27me3的表达量亦同时呈梯度下降趋势(P<0.05)。HOTTIP下调的细胞和裸鼠瘤体组织中EZH2和H3K27me3蛋白的表达呈下降趋势(P<0.05)。结论EZH2是长链非编码RNA HOTTIP的效应分子之一,其可能在SCLC恶性进展中发挥重要作用。

Purpose To explore the effects of enhancer of zeste homologue 2(EZH2)on the proliferation,apoptosis and chemo-resistance of small cell lung cancer(SCLC)cells,and to preliminarily explain the molecular regulatory pathway of EZH2 as the downstream effector of long non-coding RNA HOTTIP.Methods The SCLC cells were treated with RNA interference technique and EZH2 small molecule inhibitor GSK-126,and then qRT-PCR,Western blot,CCK-8,flow cytometry and immunohistochemistry were used to analyze the role of EZH2 in the regulation of malignant phenotype in SCLC.Results It was found that the expression of EZH2 in cancer tissues was significantly increased in SCLC compared with adjacent lung tissues(P<0.05).Further analysis in cell lines showed that the expression of EZH2 in SCLC cell lines was higher than that in human bronchial epithelial cells.In comparison,the expression of EZH2 in SCLC chemoresistant cells was higher than that in SCLC chemosensitive cells(P<0.05).After EZH2 small molecule inhibitor GSK-126 and EZH2 shRNA interference sequence were used,CCK-8 analysis showed that SCLC cells were more sensitive to three chemotherapeutic drugs(P<0.01)and decreased proliferation was observed after EZH2 silencing(P<0.001).Flow cytometry analysis showed that the proportion of G_(2) cells decreased(P<0.05),the rate of early and late cell death(P<0.01),and the rate of late cell death(P<0.05)increased after EZH2 down-regulation.Bioinformatics analysis showed that the methylation level of the EZH2 promoter region was low in patients with lung squamous cell carcinoma and lung adenocarcinoma(P<0.05).Further treatment with GSK-126 showed that the expression of EZH2 protein decreased with the increase of GSK-126(P<0.05),and the expression of methyltransferase H3K27me3 also reduced in a gradient pattern(P<0.05).Further analysis of the correlation with HOTTIP found that the expression of EZH2 and H3K27me3 proteins in cells and nude mouse tumor tissues downregulated by HOTTIP showed a downward trend(P<0.05).Conclusion As one of the effector molecules of long non-coding RNA HOTTIP,the above studies suggest that EZH2 may play an important role in the malignant progression of SCLC.