MTHFD1L对舌鳞癌细胞增殖、凋亡和体内成瘤的影响及机制研究

Effects of MTHFD1L on proliferation,apoptosis and tumorigenesis of tongue squamous cell carcinoma cells and its mechanism

目的:探讨亚甲基四氢叶酸脱氢酶1(MTHFD1L)敲减对Cal27细胞增殖、克隆形成、凋亡、成瘤的影响及机制。方法:从TCGA和GTEx数据库下载相关数据,比较头颈鳞癌(HNSC)和正常组织中MTHFD1L表达差异和生存分析,并应用GEPIA数据库验证差异表达及预测生存曲线。利用慢病毒小干扰RNA技术敲减MTHFD1L表达,通过实时荧光定量聚合酶链反应(RT-qPCR)和蛋白质印迹分析(Western blot)检测MTHFD1L敲减效率。Celigo细胞计数法、MTT法、平板克隆实验、Annexin V-APC单染法分别研究敲减MTHFD1L对Cal27细胞增殖、克隆形成和凋亡的影响。经腋窝下注射Cal27细胞建立裸鼠舌癌模型,比较瘤体重量和体积,并用活体成像仪比较荧光强度。RT-qPCR和Western blot检测p38α、IL1α表达水平。结果:MTHFD1L在HNSC中的表达量高于正常组织,且MTHFD1L高表达与预后不良有关。MTHFD1L敲减率达95.1%,Celigo细胞计数法、MTT法、平板克隆结果显示MTHFD1L敲减后Cal27细胞增殖速率和克隆形成能力受到明显抑制。Annexin V-APC单染法结果提示MTHFD1L敲除明显促进Cal27细胞凋亡。接种经MTHFD1L敲减Cal27细胞的裸鼠瘤体体积、重量、荧光强度及成瘤率明显降低。Cal27细胞经MTHFD1L敲减后,p38α蛋白和mRNA表达量明显降低,而IL1α明显升高。结论:MTHFD1L基因敲减明显抑制舌鳞癌细胞增殖,诱导凋亡,并抑制小鼠体内成瘤。

Objective:To investigate the effect of methylenetetrahydrofolate dehydrogenase 1(MTHFD1 L)knockdown on the proliferation,clone formation,apoptosis and tumorigenesis of Cal27 and its mechanism.Methods:Data related to head and neck squamous cell carcinoma(HNSC)was downloaded from TCGA and GTEx databases to compare the difference of MTHFD1 L expression and survival analysis between HNSC and normal tissues.GEPIA database was used to verify differential expression and predict survival curves.The expression of MTHFD1 L was knocked down by lentivirus small interfering RNA technology,and the knockdown efficiency of MTHFD1 L was detected by RT-qPCR and Western blot analysis.The effects of MTHFD1 L knockdown on Cal27 cell proliferation,clonal formation and apoptosis were investigated by Celigo cell count assay,MTT assay,plate cloning assay and Annexin V-APC staining,respectively.The nude mouse tongue cancer model was established by injecting Cal27 cells into the armpit.The weight,volume and fluorescence intensity were compared.The expression levels of p38αand IL1αwere detected by RT-qPCR and Western blot.Results:The expression of MTHFD1 L in HNSC was higher than that in normal tissues,and the high expression of MTHFD1 L was associated with poor prognosis.MTHFD1 L knockdown rate was 95.1%.The results of Celigo cell counting,MTT and plate cloning showed that MTHFD1 L knockdown inhibited the proliferation rate and clonal formation ability of Cal27.Annexin V-APC staining showed that MTHFD1 L knockdown significantly promoted apoptosis of Cal27.The tumor volume,weight,fluorescence intensity and tumor formation rate of nude mice inoculated with MTHFD1 L knockdown Cal27 cells decreased significantly.After MTHFD1 L knockdown,the expression of p38αand mRNA in Cal27 cells decreased significantly,while the expression of IL1αincreased significantly.Conclusion:MTHFD1 L knockdown inhibited tongue squamous cell carcinoma cell proliferation,induced apoptosis,and inhibited tumor formation in mice.