| 刘 捷,施露露,刘春燕,帅 优,束永前.慢病毒载体沉默ADAMTS6人非小细胞肺癌稳转株的构建[J].,2019,19(8):1401-1405 |
| 慢病毒载体沉默ADAMTS6人非小细胞肺癌稳转株的构建 |
| Construction of ADAMTS6 Stable Knockdown Non-small Cell Lung Cancer Cell Line Using Lentiviral Vector |
| 投稿时间：2018-09-26 修订日期：2018-10-22 |
| DOI：10.13241/j.cnki.pmb.2019.08.001 |
| 中文关键词: ADAMTS6 慢病毒载体 肿瘤数据库 小发卡RNA 非小细胞肺癌 |
| 英文关键词: ADAMTS6 Lentiviral vector Tumor database Short hairpin RNA NSCLC |
| 基金项目:国家重点研究发展计划项目(ZDZX2017ZL-01)；南京医科大学高水平创新团队项目(JX102GSP201727) |
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| 中文摘要: |
| 摘要 目的：通过数据库预测ADAMTS6在非小细胞肺癌(Non-small-cell lung cancer，NSCLC)组织中的表达及其与NSCLC患者临床预后的关系，构建ADAMTS6的shRNA干扰载体并建立ADMATS6的NSCLC稳定敲减细胞株。方法：通过Oncomine数据库分析ADAMTS6在NSCLC组织和肺正常组织的表达差异，通过Kaplan-Meier Plotter数据库分析ADAMTS6的表达水平与临床NSCLC患者预后关系，设计合成ADAMTS6的shRNA干扰序列，shRNA模板退火并与双酶切pGLV3-GFP线性化载体连接，转化挑取阳性菌落后送测序。干扰质粒进行病毒包装并感染人NSCLC细胞株NCI-H358，使用嘌呤霉素进行稳定敲减细胞株筛选。荧光观察慢病毒感染细胞密度，通过qRT-PCR和Western blot检测ADAMTS6的mRNA和蛋白水平的敲减效果。结果：Oncomine数据库分析结果显示NSCLC组织中ADAMTS6 mRNA表达较正常肺组织显著升高(P<0.001)；Kaplan-Meier Plotter数据库分析结果显示高表达ADAMTS6的NSCLC患者预后较低表达ADAMTS6的NSCLC患者差(P<0.05)；pGLV3-GFP载体双酶切线性化后与shRNA退火模板连接成功，测序结果正确。荧光观察显示慢病毒感染细胞密度在95 %左右，通过qRT-PCR和Western blot检测ADAMTS6慢病毒干扰质粒已成功敲减ADAMTS6的mRNA和蛋白水平。结论：ADAMTS6的高表达可能与NSCLC患者的不良临床预后密切相关。本研究构建了ADAMTS6的慢病毒感染质粒，并成功建立NCI-H358稳定敲减细胞株，为进一步研究ADAMTS6在NSCLC中的作用及机制奠定了基础。 |
| 英文摘要: |
| ABSTRACT Objective: To predict the expression of ADAMTS6 in non-small cell lung cancer (NSCLC) tissue and the prognosis of NSCLC patients via database, constract the ADAMTS6 shRNA vector and establish ADMATS6 stable knockdown cell line. Methods: The different expression of ADAMTS6 in NSCLC tissue and normal lung tissue was analyzed through the Oncomine database. The relationship between the expression level of ADAMTS6 and the prognosis of patients with NSCLC was analyzed by Kaplan-Meier Plotter database. Subsequently, the shRNA interference sequences of ADAMTS6 were designed and synthesized, and then was cloned into the pGLV3-GFP vector linearized by digestion with the restriction enzymes. The positive transformed bacteria were picked and sent for DNA sequencing. The lentiviral vector was subjected to virus packaging and then infected the NSCLC cell line NCI-H358. The stable knockdown cell line was selected through puromycin. The density of lentivirus-infected cells were observed by fluorescence microscope, and the knockdown effect of mRNA and protein levels of ADAMTS6 were detected by qRT-PCR and Western blot analysis, respectively. Results: The mRNA levels of ADAMTS6 were significantly higher in NSCLC tissue than in normal lung tissue according to the Oncomine database (P<0.001). Kaplan-Meier Plotter database analysis showed that NSCLC patients with high expression of ADAMTS6 had a poorer prognosis than patients with low expression (P<0.05). The shRNA annealing template was successfully cloned into the restriction digestion and linearized pGLV3-GFP vector. The DNA sequencing result was correct. Fluorescence observation showed that the density of lentivirus-infected cells was about 95%. The mRNA and protein levels of ADAMTS6 were successfully knocked down detected by qRT-PCR and Western blot analysis. Conclusion: The high expression of ADAMTS6 might be related to the poor clinical prognosis of NSCLC patients. We constructed the ADAMTS6 lentiviral vector and successfully established the stable knockdown cell line to lay the foundation for the future study of ADAMTS6 in NSCLC. |
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