摘要
目的：三叶因子3（trefoil factor 3,TFF3）除具有黏膜保护作用外还与恶性肿瘤的形成、生长、转移有关。本研究通过慢病毒表达质粒介导shRNA,瞬时转染自身表达TFF3的甲状腺乳头状癌K1细胞,筛选出了靶向人TFF3的最有效的siRNA序列。方法：分别在人TFF3基因mRNA的132、170、258和537bp处作为潜在靶位点,合成4条siRNA转录模板的发夹结构（shRNA1-4）以及1条阴性对照（shRNAC）,体外退火后插入pLVX-shRNA-puro构建重组质粒,酶切鉴定,并测序。瞬时转染K1细胞、Real-time PCR及western-blot等检测TFF3mRNA和蛋白在转染细胞的表达。结果：shRNA 1-2两条发夹结构序列有基因突变,shRNA 3-4对K1细胞TFF3表达有不同程度的抑制效应（P〈0.01）。其中shRNA3（TFF3 258-276）表现出了最高的沉默效率（转染效率76.83%时,mRNA水平的沉默效率达60.67%;蛋白质水平的沉默效率达56.44%,均P〈0.01）。结论：成功构建了pLVX-shRNA-puro-TFF3慢病毒质粒,并筛选出了最有效的序列,为进一步研究TFF3的功能奠定了基础。
Objective：Trefoil factor 3 plays a pivot role in oncogenic transformation, growth and metastatic extension of solid tumours besides mucosal protection. We screened the best siRNA sequence targeting human TFF3 by the transient-transfection of the lentiviral mediated shRNA into thyroid carcinoma K1 cells which secrete TFF3 themselves. Method： Four siRNA transcription template hairpin structure target potential sites in human TFF3 mRNA sequence（132,170,258 and 537 bp, seperately） were selected and synthesized,as well as one negative shR- NA（shRNAC）. After annealing in vitro,insert pLVX-shRNA- puro construct recombinant plasmid, then enzyme digestion and sequencing analysis. The lentiviral-shRNAs were transient-transfected into K1 ceils. TFF3 mRNA and protein levels were test by real-time PCR and western blot respectively in K1 cells at 48h post transient-trans- fected. Result：Genetic mutations in two sequences of shRNA1 - 2, so the follow-up study terminated. The TFF3 expression were obviously inhibited in K1 cells at 48 hours post transient-transfected of shRNA3 and shRNA4. TFF3（258--276）showed the highest silencing effieiency（TFF3 mRNA reduced 60.67 % and TFF3 protein reduced 56.44% ,P〈0.01） when the transfection efficiency was 76.83%. Conclusion.. pLVX-shRNA-puro-TFF3 expres- sion plamid were successfully constructed and the highest efficiency sequences were screened. All these laid a foun- dation for further study about the function of TFF3 gene.
出处
《临床耳鼻咽喉头颈外科杂志》
CAS
北大核心
2016年第2期130-134,共5页
Journal of Clinical Otorhinolaryngology Head And Neck Surgery
基金
2013年度省级重大医学科研课题(No:zd2013052)
关键词
三叶因子3
慢病毒载体
SHRNA
瞬时转染
trefoil factor
lentiviral vector
short hair RNA
transient transfection
