目的：探讨Runt相关转录因子2(Runt-related transcription factor 2, RUNX2) 在低氧环境下对小鼠乳腺癌4T1细胞凋亡的影响及其作用机制。方法： Real-time PCR和Western blotting分别检测低氧条件对4T1细胞内RUNX1、RUNX2、RUNX3 mRNA和RUNX2蛋白表达的影响；采用小干扰RNA技术与真核重组质粒DNA过表达技术降低或者提高4T1细胞RUNX2的表达，免疫共沉淀法检测4T1细胞中RUNX2与其他蛋白之间的相互结合情况，流式细胞术检测常氧/低氧条件下干扰/过表达RUNX2对4T1细胞凋亡的影响。结果：低氧条件下4T1细胞中RUNX1和RUNX2 mRNA的表达水平上调（P<005），RUNX2的蛋白表达量明显增加；转染RUNX2-siRNA-1415和真核表达载体pcDNA3.1(-)-RUNX2可使4T1细胞内RUNX2水平明显降低或升高；在常氧或低氧条件下，沉默RUNX2 mRNA的表达均导致小鼠乳腺癌4T1细胞凋亡率上升\[常氧：(12.83±0.24)% vs (9.3±0.55)%,P<0.05；低氧：(19.77±0.59)% vs (15.13±0.32)%, P<0.05\]，而过表达RUNX2 均导致4T1细胞凋亡率下降\[常氧：(9.97±0.27)% vs (14.07±0.80)%, P<0.05；低氧：(22.43±1.02)% vs (34.93±071)%, P<0.05\]。在低氧条件下，缺氧诱导因子-1α(hypoxia induced factor-1α,HIF-1α)与RUNX2的表达上升，RUNX2能与HIF-1α形成复合物。结论：在肿瘤低氧微环境中，RUNX2高表达于小鼠乳腺癌4T1细胞中，高表达的RUNX2可能是通过与HIF-1α的相互作用而抑制肿瘤细胞的凋亡。

Objective:To explore the effect of Runt-related transcription factor 2 (RUNX2) on apoptosis of mouse breast cancer 4T1 cells under hypoxia and its mechanism. Methods: Western blotting and Real-time PCR were used to measure the effect of hypoxia on the expression levels of RUNX1, RUNX2, RUNX3 mRNA and RUNX2 protein in 4T1 cells; small interference RNA (siRNA) and eukaryotic recombinant plasmid DNA over-expression technique were used to down-or up-regulate the RUNX2 expression in 4T1 cells respectively; Co-immunoprecipitation was used to detect the binding of RUNX2 and other proteins in 4T1 cells; Flow cytometry was used for the detection of the effect of down-/up-regulation of RUNX2 on apoptosis of 4T1 cells under normoxic/hypoxic condition. Results: The mRNA expression levels of RUNX1 and RUNX2 in 4T1 cells were up-regulated under hypoxic condition (P<0.05), and the protein expression of RUNX2 was significantly elevated (P<0.05). Transfection with RUNX2-siRNA-1415 or pcDNA3.1(-)-RUNX2 could significantly decrease or increase the RUNX2 level in 4T1 cells, respectively; Under normoxic/hypoxic condition, apoptosis rate of 4T1 cells that transfected with siRNA-RUNX2-1415 was significantly increased by comparing with negative control group(normoxia: \[12.83±0.2404\]% vs \[9.3±0.5508\]%, P<0.05; hypoxia: \[19.77±0.59\]% vs \[15.13±032\]%, P<0.05); however, the apoptosis rate of 4T1 cells were decreased by over-expressing RUNX2 (normoxia: \[9.967±02728\]% vs \[14.07±0.7965\]%, P<0.05; hypoxia: \[22.43±1.02\]% vs \[34.93±0.71\]%, P<005). Under hypoxic condition, the expression levels of hypoxic induced factor-1α (HIF-1α) and RUNX2 were significantly elevated, and RUNX2 could bind with HIF-1α to form complex. Conclusion: Under the hypoxic micro-environment, 4T1 cells express the high level of RUNX2, in the meanwhile, high level RUNX2 could inhibit the apoptosis of tumor cells by interacting with HIF-1α.

国家高技术研究发展计划（863计划）课题资助项目（No.SS2014AA020801）