苯丁酸钠通过下调CLDN4基因DNA甲基化对喉鳞状细胞癌增殖与凋亡的作用研究  

Effect of Sodium Phenylbutyrate on Proliferation and Apoptosis of Laryngeal Squamous Cell Carcinoma by Down-regulating DNA Methylation of CLDN4 Gene

作　　者：卡衣赛尔·卡哈尔 艾力根·阿不都热依木[1] 谭元元[1] Kayisaier·Kahaer;Ailigen·Abudureyimu;Tan Yuanyuan(Department of Otolaryngology,People′s Hospital of Xinjiang Uygur Autonomous Region,Xinjiang 830001,China)

机构地区：[1]新疆维吾尔自治区人民医院耳鼻喉科,乌鲁木齐830001

出　　处：《医学研究杂志》2020年第7期62-68,共7页Journal of Medical Research

基　　金：国家自然科学基金资助项目(81760023)。

摘　　要：目的观察苯丁酸钠对喉鳞状细胞癌(laryngeal squamous cell carcinoma,LSCC)细胞株Hep-2增殖及凋亡的作用,探讨其作用机制与紧密连接蛋白4(claudin4,CLDN4)基因甲基化的相关性。方法使用0.5、1.0、2.0、4.0、8.0mmol/L的苯丁酸钠分别处理Hep-2细胞24h,利用四甲基偶氮唑蓝(MTT)比色法、流式细胞术检测Hep-2细胞增殖、细胞周期与细胞凋亡,细胞免疫荧光法检测Hep-2细胞中caspase-3表达,实时荧光定量PCR(qRT-PCR)检测Hep-2细胞中DNA甲基转移酶(DNMT1、DNMT3a)mRNA表达水平,免疫印迹试验(Western blot)法检测Hep-2细胞CLDN4蛋白表达水平,利用甲基化特异性PCR法(MSP)分析苯丁酸钠对Hep-2细胞CLDN4基因甲基化的影响。结果苯丁酸钠呈剂量依赖地抑制Hep-2细胞增殖,使细胞周期阻滞在G 0/G 1期,并诱导细胞凋亡;4.0mmol/L苯丁酸钠处理Hep-2细胞后,caspase-3蛋白表达明显增加;苯丁酸钠处理Hep-2细胞后,CLDN4蛋白表达水平随苯丁酸钠浓度增加而升高,而DNMT1、DNMT3a mRNA表达水平随苯丁酸钠浓度增加而下降;苯丁酸钠作用后CLDN4基因的高甲基化被逆转,并呈剂量依赖性。结论苯丁酸钠可通过抑制甲基转移酶DNMT1、DNMT3a的表达使CLDN4基因去甲基化,从而抑制Hep-2细胞的增殖并诱导其凋亡。Objective To observe the effects of sodium phenylbutyrate on the proliferation and apoptosis of laryngeal squamous cell carcinoma(LSCC)cell line Hep-2,and to explore the correlation between its mechanism of action and the methylation of Claudins4(CLDN4)gene.Methods Hep-2 cells were treated with 0.5,1.0,2.0,4.0,and 8.0mmol/L sodium phenylbutyrate for 24h.Tetramethylazozolium blue(MTT)colorimetry and flow cytometry were used to detect Hep-2 cell proliferation,cell cycle and apoptosis.Cellular immunofluorescence was used to detecte the caspase-3 expression in Hep-2 cells.Real-time fluorescent quantitative PCR(qRT-PCR)was used to detect DNA methyltransferase(DNMT1,DNMT3a)mRNA expression levels in Hep-2 cells.Western blot method was used to detect the CLDN4 protein expression level in Hep-2 cells.Methylation-specific PCR(MSP)was used to analyze the effect of sodium phenylbutyrate on methylation of CLDN4 gene in Hep-2 cells.Results Phenylbutyrate inhibited the proliferation of Hep-2 cells in a dose-dependent manner,arrested the cell cycle in the G 0/G 1 phase,and induced apoptosis.After 4.0mmol/L sodium phenylbutyrate treated Hep-2 cells,the expression of caspase-3 protein increased significantly.After treatment with Hep-2 cells with sodium phenylbutyrate,the expression level of CLDN4 protein increased with the increase of sodium phenylbutyrate concentration,while the mRNA expression levels of DNMT1 and DNMT3a decreased with the increase of sodium phenylbutyrate concentration.The hypermethylation of CLDN4 gene was reversed by sodium phenylbutyrate in a dose-dependent manner.Conclusion Sodium phenylbutyrate can demethylate CLDN4 gene by inhibiting the expression of methyltransferases DNMT1 and DNMT3a,thereby inhibiting the proliferation and inducing apoptosis of Hep-2 cells.

关 键 词：喉鳞状细胞癌 HEP-2细胞 苯丁酸钠 CLDN4基因 DNA甲基化 

分 类 号：R856.76[医药卫生—航空、航天与航海医学]