FAK靶向RNAi重组体的构建及其抗多细胞球体形成效应

Construction of RNAi recombinant targeting focal adhesion kinase and its effect on resisting multicellular spheroids formation in human colon HT29 cells

目的构建RNA干扰(RNAi)重组体抑制黏着斑激酶(FAK)表达,并探讨其对结肠癌多细胞球体(MCSs)形成的影响。方法针对FAK cDNA序列设计并合成1对特异性的含有短发卡的寡核苷酸序列及其对照序列,经退火后插入pGenesil-1中构建重组体。经双酶切鉴定和DNA测序后,用脂质体2000将其转染结肠癌HT29细胞,并用G418稳定筛选,采用Real-time PCR和Western blot检测干扰前后FAK在HT29内的表达变化,并作细胞悬浮培养观测靶向干扰FAK对MCSs形成的影响。结果双酶切和测序鉴定证实插入序列完全正确;靶向干扰FAK后,其mRNA和蛋白表达分别显著下调(79.20±2.97)%和(78.47±4.39)%,且细胞不易聚集形成MCSs。结论成功构建FAK靶向RNAi重组载体,显著地抑制FAK表达,并能有效地抑制MCSs的形成。

Objective To construct a recombinant vector encoding RNA interference （RNAi） targeting focal adhesion kinase （ FAK）, and explore the effect of FAK silencing on the formation of colon carcinoma muhicellular spheroids （MCSs）. Methods One specific pair of oligonucleotides containing short hairpin structure and their negative control sequence based on FAK cDNA sequences were designed and synthesized. After annealed, they were inserted into pGenesil-1 vector to generate the recombinant plasmids. The recombinant plasmids were digested with EcoR Ⅰ and Hind Ⅲ to execute the restriction endonuclease identification, and then DNA sequencing was performed for sequence analysis. The recombinant plasmids were transfected into cultured colon carcinoma HT29 cells using lipofectamineTM 2000. The stably transfected cells were selected in a medium containing geneticin G418. The change of FAK expression in HT29 cells before and after RNAi was detected by real-time polymerase chain reaction （real-time PCR） and Western blot analysis. The cells were cultured as suspended to observe the effect of RNAi targeting FAK on MCSs formation. Results The recombinant plasmids were completely coincided with the designs by the restriction map and the sequence analysis. After RNAi targeting FAK, FAK mRNA and protein expression were decreased significantly to （79.20 ± 2.97 ）% and （78.47 ±4.39）% respectively, and the cells were not so easy to aggregate and form MCSs. Conclusion The recombinant vector of RNAi targeting FAK is constructed successfully, the expression of targeting gene FAK is inhibited markedly, and the formation of MCSs is inhibited efficiently.