PHD3对缺氧状态A549细胞株中HIF-1α表达及其生物学特性的影响

Effect of PHD3 on HIF-1α expression and biological characteristics in hypoxic non-small cell lung cancer A549 cells

目的探讨PHD3对缺氧状态非小细胞肺癌HIF-1α表达的调控作用及对其生物学特性的影响。方法采用氯化钴分别处理人非小细胞肺癌(NSCLC) A549细胞株12 h和24 h,建立A549细胞缺氧模型。用Western blot和免疫细胞化学法检测缺氧A549细胞中的PHD3、HIF-1α蛋白表达的水平;分别采用质粒转染和siRNA干扰法建立PHD3过表达和低表达的A549细胞缺氧模型,用Western blot法检测PHD3过表达和低表达变化对A549细胞中的HIF-1α蛋白表达水平的影响,CCK-8法检测细胞增殖能力,Transwell法检测细胞转移、侵袭能力。结果与氯化钴处理12 h比较,缺氧状态24 h非小细胞肺癌A549细胞中HIF-1α的表达水平升高(0. 185±0. 076 vs 0. 476±0. 114,P <0. 05),而PHD3的表达却逐渐减少(0. 235±0. 114vs 0. 127±0. 065,P <0. 05)。PHD3的过表达显著降低了HIF-1α的表达(0. 036±0. 018 vs 0. 017±0. 012,P <0. 01),PHD3低表达可增加HIF-1α的表达(0. 048±0. 021 vs 0. 453±0. 027,P <0. 05)。在缺氧条件下,PHD3表达升高会导致A549细胞的增殖、侵袭显著降低,而PHD3降低会导致A549肺癌细胞的增殖、迁移和侵袭显著增加。结论 PHD3能调控缺氧状态非小细胞肺癌A549细胞的增殖、迁移和侵袭能力,可能通过调控HIF-1α的表达来实现。

Objective To explore the effects of prolyl hydroxylase3(PHD3)expression on hypoxia-inducible factor(HIF)-1αexpression and biological characteristics in hypoxic non-small cell lung cancer A549 cells.Methods A model of hypoxia was established in the human non-small cell lung cancer(NSCLC)A549 cell line by treatment with 200 μmol/L CoCl2 for 12h and 24h,and verified by Western blot and immunocytochemical staining.The expression levels of HIF-1αwere determined by Western blot after overexpression or depletion of PHD3 in A549 cells.In addition,cell viability and invasion were measured with CCK-8and Transwell assays,respectively.Results The expression of HIF-1alpha in hypoxic NSCLC A549cells was higher after treatment for24h than that for12h(0.476±0.114 vs 0.185±0.076,P<0.05),while the expression of PHD3was lower after treatment for24h than that for12h(0.127±0.065vs0.235±0.114,P<0.05).The expression of HIF-1αwas decreased after overexpression of PHD3(0.036±0.018 vs 0.017±0.012,P<0.01),while the expression of HIF-1αwas increased after the low expression of PHD3(0.048±0.021 vs 0.453±0.027,P<0.05).Under hypoxic conditions,the increased expression of PHD3significantly decreased the proliferation and invasion of A549 cells,while the decreased expression of PHD3 significantly increased the proliferation,migration and invasion of A549 lung cancer cells.Conclusion PHD3can regulate the proliferation,migration and invasion of non-small cell lung cancer A549cells in hypoxic state,possibly by regulating the expression of HIF-1α.