靶向沉默IGF1R基因miR30-shRNA慢病毒的构建及其抗肝癌细胞生长

Construction of lentiviral vector of microRNA targeting IGF1R gene and its inhibition effect on liver cancer cell growth

目的研究靶向人胰岛素样生长因子1类受体(IGFIR)基因miR30-shRNA慢病毒对肝癌细胞生长的影响。方法针对已经筛选确定的IGF1R基因干扰有效序列,合成靶序列的OligoDNA,退火形成双链DNA,与经XhoI和EcoRI酶切后的pPRIME载体连接产生pPRIME-IGF1R-miR30-shRNA。用后者,psPAX2及pMD2G3质粒共转染包装细胞293T细胞,包装产生慢病毒。慢病毒PRIME-IGF1R-miR30-shRNA大量扩增,氯化铯梯度离心纯化,测病毒滴度。RT-PCR及Western blot检测IGF1R表达,CCK-8法检测细胞生长。结果成功构建PRIME-IGF1R-miR30-shRNA,滴度为4.58×109PFU/mL;该慢病毒能抑制肝癌细胞IGF1R表达及生长。结论IGF1R基因miR30-shRNA慢病毒靶向转染可有效地封闭肝癌细胞的IGF1R表达,降低肝癌细胞的增殖能力,为以IGFIR基因为靶点的肝癌生物治疗的进一步研究奠定了实验基础。

Objective To study the effects of lentiviral vector of microRNA targeting IGF 1 R gene on growth of liver cancer cells. Methods The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and cloned into the pPRIME vector, named pPRIME-IGFIR-miR30-shRNA. The viruses were propagated on 293T cells. Viruses were purified by CsCI gradient according to standard techniques, and functional PFU titers were determined by plaque assay on 293 cells. The effect of pPRIME-IGFIR-miR30-shRNA on IGFIR expression of Hep3B. SMMC7721 cells was detected by RT-PCR and Western blot. The antitumor potential of PRIME-IGFIR-miR30-shRNA to Hep3B, SMMC7721 cells was evaluated by CCK-8 assay and Tunel. Results PRIME-IGFIR-miR30-shRNA was constructed successfully. Functional PFU titers of pPRIME-IGFIR-miR30-shRNA were 4. 58 ×10^9 pFU/ml. PRIME-IGFIR-miR30-shRNA inhibited IGFIR expression and the proliferation of Hep3 B. SMMC7721 cells. Conclusions PRIME-IGFIR-miR30-shRNA expressing IGFIR-siRNA can inhibit IGFIR expression and may be used for further investigation of gene therapy of liver cancer.