HIF-2α在肾透明细胞癌血管生成中的作用

The effect of hypoxia-inducible factor (HIF)-2α on the angiogenesis in clear cell renal cell carcinoma

目的探讨体外水平HIF-2α在肾透明细胞癌血管生成中的作用。方法采用RNAi方法,体外化学合成HIF-2α序列特异的siRNAs,转染肾透明细胞癌786-0细胞抑制HIF-2α表达,Western blotting、ELISA方法检测VEGF表达变化,应用BrdU抗体ELISA比色法研究HIF-2α抑制后肿瘤细胞血管生成能力改变。结果肾透明细胞癌786-0细胞不表达HIF-1a,仅表达HIF-2α,缺氧诱导HIF-2α蛋白表达增加。HIF-2α序列特异的siRNAs特异抑制HIF-2α蛋白的表达,转染效率>80%,抑制率(72.0±8.0)%;HIF-2α抑制后,缺氧诱导的细胞结合型VEGF表达减少,分泌型VEGF在正常氧压情况下抑制率(21.7±3.8)%,缺氧情况下抑制率(37.8±4.7)%,肿瘤细胞在体外水平的血管生成能力降低(42.7±5.0)%。结论HIF-2α在肾透明细胞癌中具有促进肿瘤血管生成的作用,抑制HIF-2α下调细胞结合型与分泌型VEGF表达而降低肾透明细胞癌在体外水平的血管生成能力。

Objective To investigate the effect of HIF-2α on the angiogenesis in vitro in clear cell renal cell carcinoma by the use of RNAi. Methods Synthesized siRNA oligonucleotides that specifically target HIF-2α mRNAs for degradation were transfected into 786-0 clear cell RCC cells. The change of VEGF in protein level under HIF-2α inhibition was investigated using Western Blotting and ELISA, as well as the effect of HIF-2α inhibition on the angiogenesis in vitro using BrdU (colorimetric) cell proliferation ELISA assay. Results 786-0 clear cell RCC cells produce HIF-2α but not HIF-1α, and the expression of HIF-2α is increasing with hypoxic stimulation. Reintroduction of HIF-2α mRNA sequence-specific siRNAs into 786-0 cells with a transfection efficiency of 80% leads to down-regulation of HIF-2α protein levels with the inhibition rate of (72.0±8.0)%. With down-regulation of HIF-2α, the protein expression of hypoxic-induced cell-surface binding VEGF reduced, and the inhibition rate of secreted VEGF by ELISA assay is (21.7±3.8)% under normoxia, and 37.8%±4.7% under hypoxia. In addition, inhibition of HIF-2α decreased the ability of angiogenesis in vitro at a rate of (42.7±5.00)%. Conclusions HIF-2α plays a central role in promoting angiogenesis of clear cell RCC. Inhibition of HIF-2α suppress tumor angiogenesis in vitro through down-regulation of cell-surface binding VEGF and secreted VEGF. These data afford the new approach for early diagnosis and prognosis for clear cell RCC and provide the experimental evidence for HIF isoform-specific inhibitor development and targeted therapy for clear cell RCC.