CD44基因RNA干扰慢病毒载体的构建与鉴定  

Construction and identification of a lentiviral vector for RNA interference of the CD44 gene

作　　者：沈晨凌[1] 向明亮[1] 聂琛 胡海霞 马衍[1] 吴皓[1] 

机构地区：[1]上海交通大学医学院附属新华医院耳鼻咽喉头颈外科,上海交通大学医学院耳科学研究所,上海200092

出　　处：《中国耳鼻咽喉颅底外科杂志》2014年第5期379-383,388,共6页Chinese Journal of Otorhinolaryngology-skull Base Surgery

基　　金：国家自然科学基金资助项目(81271088);上海市自然科学基金资助项目(11ZR1423600)

摘　　要：目的构建CD44基因RNA干扰(RNA interference,RNAi)慢病毒表达载体并在下咽癌FaDu细胞株上鉴定其沉默效率,为研究目的基因沉默后下咽癌肿瘤细胞致瘤性的改变提供稳定可靠的载体。方法针对目的基因CD44mRNA的序列,按照RNA干扰序列的设计原则,设计、合成4个CD44基因特异性小分子干扰iRNA(small interference RNA,siRNA)靶点,将其合成短发卡hRNA(short hairpin RNA,shRNA)并退火成双链DNA,与慢病毒载体重组形成shRNA表达载体,利用PCR和测序鉴定获取正确克隆。将筛选出的最有效重组载体与慢病毒包装质粒共转染293T细胞并测定病毒滴度。随后将其感染下咽癌FaDu细胞株细胞,采用Real-time PCR检测靶基因的沉默效率。结果对LV-CD44-shRNA载体进行双酶切鉴定,证实短发夹RNA正确插入慢病毒载体,DNA测序证实插入序列正确,CD44基因RNA干扰重组慢病毒载体经293T细胞成功包装后,其病毒滴度为8E+8TU/ml。RNA干扰下咽癌FaDu细胞CD44基因后,CD44 mRNA水平显著下降,干扰效率大于70%。结论成功构建了CD44基因的shRNA慢病毒表达载体,能够在细胞水平有效沉默靶基因,为CD44基因沉默后下咽癌肿瘤细胞生物学改变的研究打下了较坚实的基础。Objective To construct a lentiviral vector for RNA interference（ RNAi） of the CD44 gene and to identify the silencing efficiency in the FaDu hypopharyngeal cancer cell line. To provide a stable and reliable vector for studying the tumorigenicity of hypopharyngeal cancer cells after silencing CD44. Methods In accordance with the study,4 specific sequences of siRNA targeting CD44 gene were confirmed,the complementary DNA containing both sense and antisense oligonucleotides of the targeting sequences were designed,synthesized,annealed and inserted into the lentiviral vector. The correct clonings were confirmed by PCR and sequencing. The most effective recombinant lentivirus vector was screened and the recombinant plasmid and a lentivirus packaging mix were co-transfected into 293T cells to obtain packaged lentivirus particles. Viral titer was then determined. The silencing efficiency of target gene in FaDu cells was detected by Real-time PCR. Results Double restriction digestion analysis and DNA sequencing showed that the shRNA sequence was successfully inserted into the lentivirus vector. The recombinant lentiviral vector harboring shRNA targeting the CD44 gene was successfully transfected into 293 cells. The recombinant lentivirus harvested from 293T cells had a titer of 8E ＋ 8TU / ml. After silencing CD44 in FaDu cells,the expression level of CD44 mRNA decreased significantly, and the RNAi efficiency was greater than 70 %.Conclusion A lentiviral shRNA expression vector targeting the CD44 gene is successfully constructed and may effectively silence the target gene at cellular level,which lays a solid foundation for studying the tumorigenicity of hypopharyngeal cancer cells after silencing CD44.

关 键 词：下咽癌 CD44 RNA干扰 慢病毒 

分 类 号：Q784[生物学—分子生物学] R739.63[医药卫生—肿瘤]