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目的:利用RNA干扰技术抑制卵巢癌细胞株skov3细胞表皮生长因子受体(EGFR)的表达及细胞的体外增殖活性。方法:根据EGFR mRNA序列设计特异性siRNA插入序列,体外合成该序列后将其定向克隆入线性化质粒pSilencerTM2.1-U6neo,以HifectinII真核转染试剂将重组质粒转染至skov3细胞中,用RT-PCR和免疫细胞化学方法分别鉴定转染前后EGFR基因在mRNA和蛋白水平表达的变化;四甲基偶氮唑蓝法(MTT)测定转染前后细胞增殖活性的改变。结果:DNA测序证实EGFR siRNA表达载体的siRNA插入序列完全正确,该表达载体可特异性抑制EGFR基因的表达;转染后细胞的增殖受到抑制,其中第5天的抑制率达到30%(P<0.05)。结论:靶向EGFR的序列特异性siRNA真核表达载体可有效抑制skov3细胞中EGFR基因的表达和细胞的增殖。
OBJECTIVE: To inhibit the expression of epidermal growth factor receptor (EGFR) and the in vitro proliferative activity of ovarian cancer cell line SKOV3 by RNA interference. Methods: According to the sequence of EGFR mRNA, a specific siRNA insertion sequence was designed and synthesized in vitro. The sequence was cloned into the linearized plasmid pSilencerTM2.1-U6neo. The recombinant plasmid was transfected into skov3 cells with HifectinII eukaryotic transfection reagent. RT-PCR and immunocytochemistry were used to identify the expression of EGFR mRNA at mRNA and protein levels before and after transfection. The changes of cell proliferation were detected by MTT assay. Results: DNA sequencing confirmed that the siRNA insertion sequence of EGFR siRNA expression vector was correct. The expression vector could specifically inhibit the expression of EGFR gene. The proliferation of the transfected cells was inhibited. The inhibition rate reached 5% at day 5 (P < 0.05). CONCLUSION: The sequence-specific siRNA eukaryotic expression vector targeting EGFR can effectively inhibit EGFR gene expression and cell proliferation in SKOV3 cells.