贵州医科大学学报

2018, v.43;No.216(09) 1012-1018

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靶向OPN基因shRNA慢病毒载体的构建及其对人肺腺癌细胞A549侵袭能力的影响
Construction of shRNA Lentiviral Vector Targeting OPN Gene and Its Effect on Human Lung Adenocarcinoma A549

罗猛;裴登科;孔德淼;刘波;金星;
LUO Meng;PEI Dengke;KONG Demiao;LIU Bo;JIN Xing;Department of Thoracic Surgery,Guizhou People's Hospital Affiliated to Guizhou Medical University;

摘要(Abstract):

目的:通过构建慢病毒介导的骨桥蛋白(OPN)基因沉默shRNA载体,并转染人肺腺癌细胞A549建立OPN基因稳定沉默的肺癌细胞株,为进一步研究OPN与非小细胞肺癌侵袭转移相关分子机制奠定细胞学基础。方法:设计靶向OPN基因的3条siRNA靶点系列,分别合成含有干扰系列的shRNA,与双酶切后的pLVXshRNA2-puro载体连接,构建pLVX-shRNA2-puro-OPN1、pLVX-shRNA2-puro-OPN2和pLVX-shRNA2-puro-OPN3病毒载体,并转化于DH5ɑ感受态细胞,挑阳性克隆质粒测序,通过转染293T细胞,获取慢病毒颗粒,转染人肺腺癌细胞A549,用含嘌呤霉素培养基筛选培养,应用定量PCR及Western Blot检测OPN基因沉默效果,选用沉默效果最佳的pLVX-shRNA2-puro-OPN1建立OPN基因稳定沉默细胞株,并用transwell方法检测各组肺癌细胞株的侵袭能力。结果:经测序鉴定成功构建pLVX-shRNA2-puro-OPN慢病毒载体,定量PCR及Western Blot检测发现转染pLVX-shRNA2-puro-OPN-1、pLVX-shRNA2-puro-OPN2慢病毒载体可明显降低A549细胞株中的OPN mRNA及蛋白水平; OPN基因稳定沉默明显抑制A549细胞株的侵袭能力。结论:成功构建pLVX-shRNA2-puroOPN慢病毒载体,OPN基因稳定沉默明显抑制A549细胞株的侵袭能力。
Objective: To establish a stable silenced lung cancer cell line of OPN gene by constructing a lentiviral vector of osteopontin( OPN) gene silenced shRNA and transfection of human lung adenocarcinoma A549,laying a cytological foundation for further studying the molecular mechanism of OPN and non-small cell lung cancer invasion and metastasis. Methods: Three siRNA target series were designed for OPN gene and the shRNA with interference series was synthesized respectively and connected with plvx-shrna2-puro vector after double enzyme digestion; pLVX shRNA2-puro OPN1,pLVX shRNA2-puro-OPN2 and pLVX-shRNA2-puro-OPN3 viral vector were constructed and converted to DH5 ɑ cells; the positive clonal plasmid was sequenced and the lentiviral particles were obtained by transfection of 293 T cells; human lung adenocarcinoma A549 cancer cells was transfected and the culture of puromycin was selected,a stable silencing cell strain of OPN gene was established by detecting the effects of OPN gene silencing with quantitative PCR and Western Blot and selecting plvx-shrna2-puro-opn1 of the best silencing effect. And transwell method was used to detect the invasiveness of lung cancer cell lines in each group. Results: The plvx-shrna2-puro-opn lentivirus vector was successfully constructed by sequencing. It was detected that the transfection of plvx-shrna2-pur-opn-1 and plvx-shrna2-pur-opn2 lentiviral vector significantly reduced the level of OPN mRNA and protein in A549 cell lines by quantitative PCR and Western Blot assays; the stable silencing of OPN gene significantly inhibited the invasiveness of A549 cell line. Conclusion: The stable silencing of OPN gene significantly inhibits the invasiveness of A549 cell line in condition that plvx-shrna2-puro-opn lentiviral vector is successfully constructed.

关键词(KeyWords): 肺肿瘤;慢病毒感染;肿瘤转移;小发夹RNA;骨桥蛋白质
lung neoplasms;lentiviral infection;neoplasm metastasis;small hairpin RNA;osteopontin

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基金项目(Foundation): 贵州省科技厅项目[黔科合SY字(2013)3002号];贵州省科技厅项目[黔科合J字(2012)2234号]

作者(Author): 罗猛;裴登科;孔德淼;刘波;金星;
LUO Meng;PEI Dengke;KONG Demiao;LIU Bo;JIN Xing;Department of Thoracic Surgery,Guizhou People's Hospital Affiliated to Guizhou Medical University;

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