摘要
目的:构建整合素β1基因特异的RNA干扰质粒,探讨抑制整合素β1蛋白表达对非小细胞肺癌(non-small cell lungcarcinoma,NSCLC)细胞生物学行为的影响。方法:根据GenBank数据库提供的整合素β1基因核苷酸序列,选择设计1条能转录短发夹RNA(small hairpin RNA,shRNA)的DNA序列,命名为17-2;同时设计1条非特异性序列作为阴性对照,命名为N。将设计序列与pUC19质粒载体连接,转化感受态大肠埃希菌,挑选阳性克隆,抽取重组质粒。2种重组质粒分别转染NSCLC耐药细胞株PC-9/AB2细胞,用G418筛选后挑选单克隆并扩增获得稳定转染株。荧光显微镜、实时荧光定量PCR(real-timefluorescent quantitative PCR,RFQ-PCR)和Western印迹法检测整合素β1 mRNA及蛋白表达水平。细胞划痕实验和黏附实验比较整合素β1对细胞迁移和黏附能力的影响。结果:G418筛选出稳定转染2种质粒的PC-9/AB2细胞,荧光显微镜下可见满视野带绿色荧光的细胞,转染17-2组细胞中整合素β1的mRNA及蛋白表达水平明显低于转染N组细胞及母细胞PC-9/AB2。划痕实验和黏附实验表明,整合素β1抑制可以使细胞迁移和黏附能力明显降低。结论:成功构建针对整合素β1基因的特异性shRNA真核表达载体,转染NSCLC细胞后可抑制整合素β1蛋白表达。整合素β1表达抑制后细胞迁移和黏附能力明显降低。
Objective：To construct a specific small hairpin RNA（shRNA） expressing vectors against human receptor for advanced glycation end product（RAGE） gene and study its inhibitory effect on the proliferation of androgen-independent prostate cancer cells DU145.Methods：Four RAGE specific oligonucleotides were designed and synthesized.These oligonucleotides were annealed to forill double strand DNA fragments and this fragment was cloned into psi-U6 plasmid.The recombinants were transfected into RAGE-overexpressing sub DU145-2C1 cells.Cellular morphology and transfection efficiency were observed under fluorescence microscope.The inhibitory effect of RAGE shRNA construct on RAGE mRNA and protein expression was examined with semi-quantitative reverse transcriptase-polymerase chain reaction（RT-PCR） and Western blotting,respectively.The cellular proliferation was detected with cell counting kit-8（CCK-8）.Scratch test was used to observe the migration of DU145 cells.Results：RAGE shRNA expression plasmids were successfully constructed and transfected into sub DU145-2C1 cells.It can effectively inhibit the expression of RAGE mRNA（P0.05）.The inhibitory effects of shRNA RAGE-1（R1） was the most stronger.The RAGE mRNA expression was inhibited by 84% and RAGE protein expression was inhibited by 27%.Compared with negative control,the proliferation potential was significantly decreased in shRNA RAGE-transfected cells.The cell migration capability had no significant changes.Conclusion：RAGE shRNA effectively inhibited the expression of RAGE mRNA and protein and suppressed the proliferation of DU145 cells in vitro.
出处
《肿瘤》
CAS
CSCD
北大核心
2010年第3期188-193,共6页
Tumor
基金
国家自然科学基金资助项目(编号:30873023)
关键词
癌
非小细胞肺
RNA干扰
整合素类
基因表达
细胞运动
Prostatic neoplasms Glycosylation end products advanced RNA small interfering Cell DU145
