摘要
目的:构建ShRNA沉默SLC7A11基因,并转染喉癌细胞株,为研究SLC7A11基因在喉鳞状细胞中作用提供基础。方法:针对SLC7A11基因构建3个表达特异性的ShRNA的真核表达载体,通过与包装质粒共转染293T细胞包装慢病毒。将病毒感染喉癌UMCC-5和Hep-2细胞,48h后加入puromycin抗生素进行筛选细胞3代以上,使用荧光显微镜观察筛选后的稳定表达对应shRNA细胞,直至无抗性细胞全部去除。通过RT-PCR及Western blot的方法比较实验组及空白对照的各组细胞SLC7A11基因mRNA和蛋白的表达水平。结果:筛选后的喉癌UMCC-5和Hep-2细胞中全部表达荧光标签,暗示ShRNA-SLC7A11成功感染喉癌细胞系内,然后使用RT-PCR及Western blot检测,两组喉癌细胞中发现Sh-SLC7A11转染后SLC7A11 mRNA水平及蛋白的含量,明显低于Sh-CTRL组(p<0.05),结果显示,与空载质粒组相比,ShRNA慢病毒载体可有效沉默SLC7A11在UMCC-5和Hep-2细胞系中的表达。结论:成功构建并筛选出ShRNA-SLC7A11基因表达的喉癌细胞株,为进一步研究SLC7A11基因在喉癌中的作用奠定了基础。
引文
[1] Siegel RL,Miller KD,Jemal A.Cancer statistics,2016[J].CA Cancer Clin,2016,66(1):7～30.
[2] Graboyes EM,Townsend ME,Kallogjeri D,et al.Evaluation of quality metrics for surgically treated laryngeal squamous cell carcinoma[J].JAMA Otolaryngol Head Neck Surg,2016,142(12):1154～1163.
[3] Lewerenz J,Hewett SJ,Huang Y,et al.The cystine/glutamate antiporter system x(c)(-) in health and disease:from molecular mechanisms to novel therapeutic opportunities[J].Antioxid Redox Signal,2013,18(5):522～555.
[4] Pallast S,Arai K,Wang X,et al.12/15-Lipoxygenase targets neuronal mitochondria under oxidative stress[J].Neurochem,2009,111(3):882～889.
[5] Zhang RR,Pointer KB,Kuo JS.Excitotoxic SLC7A11 Expression Is a Marker of Poor Glioblastoma Survival and a Potential Therapeutic Target[J].Neurosurgery,2015,77(6):N16～17.
[6] Robert SM,Buckingham SC,Campbell SL,et al.SLC7A11 expression is associated with seizures and predicts poor survival in patients with malignant glioma[J].Sci Transl Med,2015,7(289):289～286.
[7] Guo W,Zhao Y,Zhang Z,et al.Disruption of xCT inhibits cell growth via the ROS/autophagy pathway in hepatocellular carcinoma[J].Cancer Lett,2011,312(1):55～61.
[8] Shiozaki A,Iitaka D,Ichikawa D,et al.xCT,component of cysteine/glutamate transporter,as an independent prognostic factor in human esophageal squamous cell carcinoma[J].Journal of gastroenterology,2014,49(5):853～63.
[9] Dixon SJ,Patel DN,Welsch M,et al.Pharmacological inhibition of cystine-glutamate exchange induces endoplasmic reticulum stress and ferroptosis[J].Elife,2014,3:e02523.
[10] 吴伟刚,田圆,刘琢冰,等.SLC7A11基因表达沉默的肝癌细胞株的构建及筛选[J].现代肿瘤医学,2015,23(2):149～152.
