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目的观察RNA干扰缺氧诱导因子(HIF)-1α逆转乳腺癌耐药性的作用。方法构建靶向缺氧诱导因子-1α的短发夹状小干扰RNA基因并转染到人乳腺癌耐阿霉素细胞株MCF-7/ADR中。常规MTT法检测细胞存活率,外排实验检测细胞的P-糖蛋白转运功能,逆转录聚合酶链反应(RT-PCR)检测细胞的HIF-1α、多药耐药基因1(mdr-1)mRNA变化,Western印迹法观察干扰前后HIF-1α、P-糖蛋白的变化。结果测序证实成功构建HIF-1α的短发夹状siRNA真核表达载体pSilencer-HIF,转染空质粒ADR/neo不影响肿瘤细胞的耐药性。MTT法检测ADR/shRNA组细胞存活率由76%下降到43%,Rh123荧光强度由22·0%升为86·6%,ADR/shRNA组中HIF-1α、mdr-1mRNA、蛋白水平显著降低(P<0·05),且Spearman相关分析HIF-1α和mdr-1指数在三组中显示呈正相关(r=0·816,P<0·01)。结论成功构建了HIF-1α的短发夹状siRNA真核表达载体pSilencer-HIF,可显著降低乳腺癌MCF-7/ADR细胞中HIF-1α表达从而起到逆转肿瘤耐药的作用。
Objective To observe the role of RNA interference hypoxia inducible factor (HIF) -1α in reversing drug resistance of breast cancer. METHODS: Short hairpin RNA (shRNA) targeting hypoxia inducible factor-1α was constructed and transfected into human breast cancer ADM-MCF-7 / ADR cells. Cell viability was measured by MTT assay, P-glycoprotein transport function was detected by efflux assay, and the expression of HIF-1α, MDR1 (mdr-1) was detected by reverse transcription polymerase chain reaction mRNA changes and Western blotting were used to observe the changes of HIF-1α and P-glycoprotein before and after interference. Results Sequencing confirmed the successful construction of HIF-1α short hairpin siRNA eukaryotic expression vector pSilencer-HIF, transfected empty plasmid ADR / neo does not affect the tumor cell resistance. The cell viability decreased from 76% to 43% and the fluorescence intensity of Rh123 increased from 22.0% to 86.6% in ADR / shRNA group by MTT assay. The mRNA and protein levels of HIF-1α and mdr-1 in ADR / shRNA group were significantly decreased (P <0.05), and the Spearman correlation analysis showed that HIF-1α and mdr-1 index were positively correlated in three groups (r = 0. 816, P <0.01). Conclusion The short hairpin siRNA eukaryotic expression vector pSilencer-HIF of HIF-1α was successfully constructed, which can significantly reduce the expression of HIF-1α in breast cancer MCF-7 / ADR cells and thus play a role in reversing tumor drug resistance.