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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 (TFF3) is associated with the formation, growth and metastasis of malignant tumors in addition to mucosal protection. In this study, lentivirus-mediated plasmid mediated shRNA, transiently transfected TFF3 self-expressed thyroid papillary carcinoma K1 cells, screened out the most effective siRNA targeting human TFF3. Methods: Hairpin structure (shRNA1 ~ 4) and a negative control (shRNAC) of four siRNA transcription templates were synthesized at 132, 170, 258 and 537bp of human TFF3 gene mRNA respectively and annealed in vitro Insert pLVX-shRNA-puro construct recombinant plasmid, identified by restriction enzyme digestion, and sequenced. Transient transfection of K1 cells, Real-time PCR and western-blot were used to detect the expression of TFF3 mRNA and protein in transfected cells. Results: shRNA 1 ~ 2 had the gene mutation in the two hairpin structure sequences. ShRNA 3 ~ 4 inhibited the TFF3 expression in K1 cells to varying degrees (P <0.01). Among them shRNA3 (TFF3 258 ~ 276) showed the highest silencing efficiency (silencing efficiency was 60.67% at mRNA level of 76.83% and silencing efficiency of protein at level of 56.44%, both P <0.01). CONCLUSION: The pLVX-shRNA-puro-TFF3 lentiviral plasmid was successfully constructed and the most effective sequence was screened out, which laid the foundation for further study on the function of TFF3.