摘要
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pc DNA3.1(-)-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.
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
北大核心
2017年第4期373-379,共7页
Chinese Journal of Cancer Biotherapy
基金
国家高技术研究发展计划(863计划)课题资助项目(No.SS2014AA020801)~~
关键词
乳腺癌细胞
Runt相关转录因子2
RNA干扰
低氧环境
细胞凋亡
breast cancer ceils
Runt-related transcription factor 2 （ RUNX2 ）
RNA interference
Hypoxia environment
cell apoptosis
