SSAT2通过抑制HIF-1α表达增强肾癌Caki-1细胞化疗敏感性

Spermidine/spermine N^1-acetyltransferase 2 increases sensitivity of renal carcinoma Caki-1 cells to chemotherapy by inhibiting HIF-1α

目的探讨转染精脒/精胺N1乙酰基转移酶2(spermidine/spermine N1-acetyltransferase 2,SSAT2)在低氧条件下对肾癌Caki-1细胞增殖、凋亡和阿霉素敏感性的影响及机制。方法经X-treme GENE HP介导将真核表达质粒pcDNA3.1(D)/V5-His-SSAT2转入肾癌Caki-1细胞,细胞在1%氧浓度下培养,Western blot法检测SSAT2蛋白和低氧诱导因子-1α(hypoxia-inducible factor-1 alpha,HIF-1α)蛋白表达;流式细胞仪检测细胞的凋亡率、死亡率;MTT法检测细胞的增殖,以及不同浓度阿霉素对细胞的抑制率。结果 SSAT2成功转入肾癌Caki-1细胞,与空白细胞组及空载体组比较,转染SSAT2组HIF-1α蛋白表达明显下调(P<0.01),细胞增殖受到明显抑制(P<0.01),细胞凋亡率和死亡率均增加(P<0.05)。阿霉素浓度为2μg/ml时,转染组细胞抑制率可达(67.5±3.7)%,对空白细胞和空载体细胞的抑制率为(54.7±8.4)%、(56.8±7.7)%,差异有统计学意义(P<0.01)。结论转染SSAT2可以通过下调HIF-1α蛋白表达,减少肾癌细胞的增殖,增加肾癌Caki-1细胞的凋亡率、坏死率和对阿霉素的敏感性,针对SSAT2的基因治疗可能成为逆转肾癌耐药的方法之一。

Objective To study the effect of spermidine/spermine N^1-acetyltransferase 2（SSAT2） on the proliferation and apoptosis in renal carcinoma Caki-1 cells and cell sensitivity to adriamycin under hypoxic condition.Methods Plasmid pcDNA3.1（D）/V5-His-SSAT2 was transfected into Caki-1cells with X-treme GENE HP.Caki-1 cells were cultured under hypoxia condition.Expression of SSAT2 and hypoxia-inducible factor-1 alpha（HIF-1α） protein was detected by Western blotting.Apoptotic and necrotic rates of Caki-1 cells were assayed by flow cytometry.Proliferation of Caki-1 cells and inhibitory effect of adriamycin on proliferation of Caki-1 cells were detected by MTT assay.Results The SSAT2 was successfully transfected into Caki-1 cells.The expression level of HIF-1α and the proliferation level of Caki-1 cells were significantly lower while the apoptotic and necrotic rates of Caki-1 cells were significantly higher in SSAT2 transfection group than in blank cell group and empty vector group（P〈0.01）.The inhibitory rate of adriamycin on SSAT2 transfected Caki-1 cells could reach（67.5±3.7）%,while only（54.7±8.4）% and（56.8±7.7）% respectively in blank cell group and empty vector group when adriamycin was administrated at the concentration of 2 μg/ml（P〈0.01）.Conclusion SSAT2 inhibits the proliferation and improves the apoptotic and necrotic rates of Caki-1 cells,enhances the sensitivity of Caki-1 cells to adriamycin by down-regulating the expression of HIF-1α protein.SSAT2 targeting therapy may thus become one of the methods for reversing the drug-resistance of renal carcinoma.