DOT1L介导Notch1信号通路对喉鳞状细胞癌上皮间质转化的机制

The mechanism of DOT1L on epithelial mesenchymal transition in laryngeal squamous cell carcinoma via Notch1 signaling pathway

目的探讨类端粒沉默干扰体1(disruptor of telomeric silencing 1-like,DOT1L)介导Notch1信号通路对喉鳞状细胞癌(laryngeal squamous cell carcinoma,LSCC)上皮间质转化(epithelial mesenchymal transition,EMT)的作用机制。方法选择30例病理确诊LSCC患者的肿瘤组织和癌旁组织,免疫组织化学染色检测DOT1L及其催化产物H3K79me3的阳性表达率,qRT-PCR检测DOT1L和H3K79me3的mRNA相对表达量。选择人LSCC细胞系TU686分为空白对照组、过表达组和低表达组,培养48 h后MTT法检测细胞增殖率,流式细胞术检测细胞凋亡率,Western blot法检测Notch1、E-cadherin、N-cadherin和Vimentin蛋白相对表达量。结果(1)与癌旁组织相比,肿瘤组织中DOT1L和H3K79me3阳性表达率[(41.5±8.9)%vs.(18.3±3.6)%,t=56.963,P<0.001;(40.5±7.7)%vs.(10.2±2.3)%,t=96.635,P<0.001]以及mRNA表达量[(0.69±0.13)vs.(0.13±0.09),t=102.302,P<0.001;(0.42±0.19)vs.(0.09±0.03),t=85.659,P<0.001]均显著增加,差异比较有统计学意义。(2)与空白对照组相比,过表达组细胞增殖率明显升高(t=45.628,P<0.001),凋亡率降低(t=40.125,P<0.001),Notch1、N-cadherin和Vimentin蛋白表达升高,E-cadherin降低(t=22.524、30.263、45.629、27.859,P<0.001);低表达组细胞增殖率显著下降(t=33.427,P<0.001),凋亡率增加(t=78.529,P<0.001),Notch1、N-cadherin和Vimentin蛋白表达下降,E-cadherin增加(t=19.864、23.524、28.957和33.426,P<0.001)。结论DOT1L在LSCC中高表达,影响组蛋白的甲基化水平,进而调控细胞增殖、凋亡和EMT行为,DOT1L有望成为肿瘤靶向干预的新位点。

OBJECTIVE To investigate the mechanism of disruptor of telomeric silencing 1-like(DOT1L)on epithelial mesenchymal transition(EMT)in laryngeal s quamous cell carcinoma(LSCC)via N otch1 signal pathway.METHODS Fresh tumor tissues and adjacent normal tissues from 30 pathologically confirmed LSCC patients were selected.The positive expression rates of DOT1L and its catalytic product H3K79me3 were detected by immunohistochemistry staining,the relative mRNA expression levels of DOT1L and H3K79me3 were measured by qRT-PCR.The human LSCC cell line TU686 was selected and divided into blank control group,over-expression group,and low-expression group.After 48 hours of cultivation,cell proliferation rate was measured using MTT method,cell apoptosis rate was measured using flow cytometry,and the relative expression levels of Notch1,E-cadherin,N-cadherin,and Vimentin proteins were detected using Western blot.RESULTS 1.Compared with normal tissues adjacent to cancer,the positive expression rates[(41.5±8.9)%vs.(18.3±3.6)%,t=56.963,P<0.001;(40.5±7.7)%vs.(10.2±2.3)%,t=96.635,P<0.001]and mRNA expression levels[(0.69±0.13)vs.(0.13±0.09),t=102.302,P<0.001;(0.42±0.19)vs.(0.09±0.03),t=85.659,P<0.001]of DOT1L and H3K79me3 in tumor tissues were significantly increased.2.Compared with the blank control group,cell proliferation rate in the over-expression group was significantly higher(t=45.628,P<0.001),while apoptosis rate was lower(t=40.125,P<0.001),Notch1,N-cadherin,and Vimentin proteins expressions were increased,E-cadherin was decreased(t=22.524,30.263,45.629,27.859,P<0.001).The cell proliferation rate in the low expression group was significantly decreased(t=33.427,P<0.001),but apoptosis rate was increased(t=78.529,P<0.001),levels of Notch1,N-cadherin,and Vimentin proteins were lower,E-cadherin was higher,too(t=19.864,23.524,28.957,33.426,P<0.001).CONCLUSION DOT1L is highly expressed in LSCC,affecting the methylation level of histones,thereby regulating cell proliferation,apoptosis,and EMT behavior.DOT1L is expected to become a new site for tumor targeted intervention.