缺氧诱导因子-1α对恶性肿瘤细胞侵袭性生长的影响

目的 探讨缺氧诱导因子-1α(HIF-1α)对恶性肿瘤细胞侵袭性生长的作用机制.方法 人骨肉瘤细胞株经缺氧处理后,通过定点诱变来构建HIF-1α(PP)突变体.CD-1裸鼠双侧皮下注射肿瘤液观察肿瘤发生.细胞种植2周后计算群落数,评价肿瘤细胞的锚定不依赖性生长.细胞增殖使用发光法细胞活力进行检测.细胞侵袭通过计算侵袭性细胞总数.结果 HIF-1α-c-Myc通路和HIF-1α-ARNT通路的功能通过定点诱变的方式在HIF-1α稳定的条件下分别被抑制活性.随着肿瘤抑制活性的降低,HIF-1α(PP)和HIF-1α(PP)+RFC细胞的锚定不依赖性生长均有大幅增加,群落数分别为1480±8和1750±6.HIF-1α(PP)及HIF-1α(PP)+RFC致肿瘤性为100%,而US201及HIF-1α(PP)+VAT则无致肿瘤性.同时,它们的细胞增殖和人工基底膜侵犯也有显著增加,HIF-1α(PP)及HIF-1α(PP)+VAT细胞活性增为11 000±3和10 900±5,而HIF-1α(PP)+RFC及HIF-1α(PP)+RFC+VAT细胞活性为2210±6及1880±8.但是HIF-1α-c-Myc通路的失活阻止了这种增势,侵袭力下降为20%.肿瘤细胞获取侵袭性恶性特质需要H1F-1α-c-Myc通路.结论 HIF-α-c-Myc通路通过基因变异,在肿瘤恶化的缺氧调节中起着关键作用,进而导致恶性肿瘤局部浸润和上皮细胞-间充质转化.

Abstract：

Objective To explore the action mechanism of hypoxia inducible factor-1α (HIF-1α)on human osteosarcoma cells. Methods After hypoxic treatment to human osteosarcoma cell line, additional mutations were made by site-directed mutagenesis to create HIF-1α (PP) mutants. CD-1 nude mice were used for bilateral subcutaneous injections to evaluate the tumor cell tumorigenesis. Cells were seeded for 2 weeks, the total number of colonies per well was calculated and the anchorage-independent growth was analyzed. Cell proliferation was assayed by cell viability assay, and cell invasion was determined by the total number of invading cells and presented as mean ± SEM. Results The functions of the HIF-1α-ARNT pathway and the HIF-1α-c-Myc pathway were inactivated respectively by site-directed mutagenesis in the context of a stabilized HIF-1α. In accordance with diminished tumor-suppressing activities, both HIF-1α(PP) and HIF-1α (PP) + RFC cells exhibited a remarkable gain of anchorage-independent growth, and the total number of colonies per well was 1480 ± 8 and 1750 ± 6 respectively. Tumorigenicity of HIF-1α(PP) and HIF-1 α (PP) + RFC was all 100%, but US201 and HIF-1 α (PP) + VAT had no tumorigenicity. They also showed a marked increase in cell proliferation and Matrigel invasion, the cell viability of HIF1 α (PP) and HIF-1 α (PP) + VAT was 11 000 ± 3 and 10 900 ± 5, but the cell viability of HIF-1 α (PP)+ RFC and HIF-1α (PP) + RFC + VAT was only 2210 ± 6 and 1880 ± 8 respectively. Whereas inactivation of the HIF-1α-c-Myc pathway abrogated such increase, and the tumorigenicity was decreased to 20%.Gain of aggressive malignant traits required the HIF-1α-c-Myc pathway. Conclusion HIF-α-c-Myc pathway plays an essential role in mediating hypoxic effects on malignant progression via genetic alterations,resulting in formation of malignant tumors with aggressive local invasion and epithelial-mesenchymal transition.