HT29细胞黏着斑激酶表达与其对5-氟尿嘧啶敏感性的关系研究

Relationship between the expression of focal adhesion kinase and the sensitivity of HT29 cells to 5-fluorouracil

目的构建人结肠癌细胞株HT29的RNA干扰(RNAi)表达载体以抑制黏着斑激酶(FAK),检测FAK沉默后细胞对5-氟尿嘧啶(5-FU)敏感性的变化。方法利用针对FAK基因的特异性寡核苷酸序列及对照序列构建重组体,酶切并测序鉴定,用脂质体2000转染细胞,G418筛选。采用RT-PCR和免疫组化检测干扰前后细胞内FAK的表达变化,MTT法检测细胞对5-FU敏感性的变化。结果酶切鉴定和测序证实插入序列完全正确。靶向FAK干扰后,免疫组化显示FAK蛋白表达显著降低,RT-PCR显示FAKmRNA的表达下调了76.94%,MTT显示HT29细胞对5-FU敏感性较其他组显著提高,IC50值明显下降(P<0.05)。结论成功构建靶向FAK的RNAi载体并有效抑制了FAK的表达,后者显著提高了HT29细胞对5-FU的敏感性,为寻求新的基因治疗方法提供了可能。

Objective RNA interference （RNAi） expression vector was constructed to inhibit the focal adhesion kinase（FAK）expression in colon carcinoma HT29 cells, and then the sensitivity of the cells to 5-fluorouracil （5-FU） was determined. Methods One specific pair of oligonucleotides with short hairpin and its negative control sequence were designed and synthesized based on FAK cDNA sequences, then they were inserted into pGenesil-l vector to generate the recombinant plasmids. After identification by the restriction endonuclease and DNA sequencing, the recombinant plasmids were transfected into HT29 cells by lipofectamine TM 2000. The stable transfected cells were selected in a medium containing geneticin G418. The change in FAK expression in HT29 cells before and after RNA interference was detected by reverse transcription and polymerase chain reaction （RT-PCR） analysis and SP immunocytochemistry technique. Sensitivity of HT29 cells to 5-FU was determined by MTT assay. Results The recombinant plasmids coincided completely with the designs in the restriction map and the sequence analysis. After FAK being targeted by RNA interference, immunocytochemistry showed the protein expression of FAK was reduced dramatically, and RT-PCR revealed FAK mRNA expression was down-regulated by 76.94% compared to that of untransfected cells. MTT assay also showed that the sensitivity of HT29 cells to 5-FU in transfected pGenesil-1-FAK vector cells was increased, while IC50 declined remarkably （P〈0.05）. Conclusion The recombinant RNA interference vector targeted FAK is successfully constructed. Transfection of this vector may efficiently inhibit the FAK expression, and elevate the sensitivity of HT29 cells to 5-FU. The present study makes it possible to look for a new gene therapy for colorectal carcinomas.