抑制肾上腺髓质素基因的表达对卵巢癌细胞活性及化疗敏感性的影响

Inhibition of adrenomedullin gene via ERK/MMP 9 on chemosensitivity of ovarian cancer cells

目的通过构建靶向于肾上腺髓质素(AM)基因的shRNA并转染卵巢癌细胞,观察抑制AM基因表达对卵巢癌细胞活性及化疗敏感性的影响。方法体外合成针对AM基因的shRNA序列,亚克隆至载体pRNA-U6.1/neo中,构建重组质粒pRNA-U6.1/neo-AM shRNA,转染至卵巢癌细胞中;通过RT-PCR检测转染后细胞内AMmRNA转录水平;MTT法检测细胞增殖活性及对顺铂敏感性变化;Western blot检测细胞外调节蛋白激酶(ERK)活性以及金属基质蛋白酶-9(MMP-9)表达。结果RT-PCR表明,转染后细胞内AMnRNA水平明显下降,与阴性对照组相比,有统计学差异(P<0.05);MTT法结果表明,下调AM的表达后细胞增殖受到抑制,用顺铂处理后,AM shRNA组细胞存活率下降最为显著,与阴性对照组相比,有统计学差异(P<0.05);Western blot结果表明,下调卵巢癌细胞中AM的表达,可以抑制ERK的活性及MMP-9蛋白的表达。结论利用RNAi及基因转染技术下调卵巢癌细胞中AM的表达可以明显抑制卵巢癌细胞的增殖、转移能力并提高其对化疗药物顺铂的敏感性。

Objective To investigate the effect of short hairpin RNA （shRNA） targeting adrenomedullin （AM） gene on chemosensitivity of ovarian cancer cells. Methods Small interfering RNA（siRNA） targeting AM gene was constructed and transfected to ovarian cancer HO8910 cells. The expression of AM was detected by RT-PCR, Cell proliferation was examined by MTT assay and the expression of MMP-9 and ERK was evaluated by Western blot. Results After transfection with siRNA targeting AM, the expression of AM gene in HO8910 cells was inhibited and the expression of MMP-9 and p-ERK was down-regulated. Meanwhile, the chemosensitivity of HO8910 cells was increased. Conclusion Silencing of AM gene in ovarian cancer HO8910 cells increases the chemosensitivity of the cells, which is associated with the down-regulation of MMP-9 and p-ERK proteins.