针对前列腺癌HIF—1α的2种pSUPER RNAi焦统的构建及鉴定  

Construction and identification of two pSUPER RNAi systems of inhibiting HIF - 1 α activity of Prostate Cancer

作　　者：刘荣福[1] 耿爱民[1] 李博安[1] 

机构地区：[1]福建医科大学附属厦门第一医院泌尿外科

出　　处：《国际泌尿系统杂志》2010年第2期148-153,共6页International Journal of Urology and Nephrology

基　　金：本课题受厦门市科技局科研基金资助（课题编号：3502Z20084002）

摘　　要：目的构建2种抑制HIF—1α的siRNA表达载体。方法根据GenBank中HIF—1α序列信息并结合2种pSUPER载体的共同的酶切位点结构，化学合成2对编码短发夹RNA序列的靶向HIF-1α基因的寡核苷酸链，各60个碱基、退火、克隆到经BglⅠ、HindⅢ双酶切的2种pSUPER载体上，获得的2种重组RNAi质粒转化感受态大肠杆菌，挑选阳性克隆，抽取重组质粒，EcoRⅠ、HindⅢ双酶切电泳、测序鉴定，培养前列腺癌PC-3细胞通过Western blot检测2种重组RNAi载体干扰效果。结果将重组构建的2种pSUPER质粒经双酶切电泳分析及插入基因片段进行序列分析，目的基因片段成功插入到设计位点，并且序列完全一致。2种重组质粒可显著降低前列腺癌PC-3细胞蛋白的表达。结论2种重组RNAi载体的成功构建，为进一步研究HIF—1α在前列腺癌发病机理及增殖、转移中的功能奠定了基础，同时可以用于其在其他肿瘤的功能研究。Objectives To construct expressing vector of two siRNA in order to inhibit HIF - 1αtactivity. Methods According to the HIF -1α sequence and the same constructure of two pSUPER vectors, Sixty base - pair oligos for hairp in RNA expression which targeted HIF - 1α gene were chemically synthesized and annealed. The two recombinant RNAi vectors were then transformed into competent Ecoli , the positive clones were selected and recombinant. The two pSUPER RNAi plasmids were extracted. The two pSUPER RNAi plasmids were digested with EcoR Ⅰ and Hind Ⅲ, and loaded in agarose gel electrophoresis. The recombinant vectors were transfected into the Prostate Cancer PC - 3 ceils , and detechted the expression levels of HIF - 1 α by Western blot. Results Recombinant the two pSUPER RNAi plasmids were identified by digestion with EcoR Ⅰ and Hind Ⅲ, and confirmed by sequencing analysis, we knew the annealed oligos were inserted into the two pSUPER vectors, and the insertion sequence were exactly correct. The levels of the whole Prostate Cancer PC - 3 cells proteins extremely down - regulated by the two recombinant vectors. Conclusions The two pSUPER RNAi systems have been constructed successfully. These will facilitate the studying of HIF - 1 α activity inhibition and function in the pathogenesis, infiltration, proliferation and transformation of the prostate cancer, meantime, they can help us to study the functions of HIF - 1α in other carcinomas.

关 键 词：前列腺肿瘤 低氧 

分 类 号：R737.25[医药卫生—肿瘤]