国际肿瘤学杂志

国际肿瘤学杂志 ›› 2019, Vol. 46 ›› Issue (12): 711-717.doi: 10.3760/cma.j.issn.1673-422X.2019.12.002

• 论著 • 上一篇    下一篇

HIF-1介导的线粒体能量代谢参与丙泊酚调控肾透明细胞癌细胞增殖及凋亡

李正民1  张玉明2  张振1  张茹1  朱婧2  王君3   

  1. 1空军军医大学唐都医院麻醉科，西安  710038； 2陕西省人民医院麻醉科，西安  710068； 3陕西省肿瘤医院麻醉科，西安  710061
  • 收稿日期:2019-09-29 修回日期:2019-11-12 出版日期:2019-12-08 发布日期:2019-12-09
  • 通讯作者: 王君 E-mail:wangjun0519@126.com
  • 基金资助:

    陕西省自然科学基础研究计划(2018JM7047035、2018JM7121)；

    陕西省人民医院科研孵化基金（2018YXQ-07）

Mitochondrial energy metabolism mediated via HIF-1 involves the proliferation and apoptosis of renal clear cell carcinoma cells regulated by propofol

Li Zhengmin1  Zhang Yuming2  Zhang Zhen1 Zhang Ru1  Zhu Jing2  Wang Jun3   

  1. 1Department of Anesthesiology, Tangdu Hospital, Air Force Medical University, Xi′an 710038, China;  2Department of Anesthesiology, Shaanxi Provincial People′s Hospital, Xi′ an 710068, China;  3Department of Anesthesiology, Shaanxi Tumor Hospital, Xi′an 710061, China
  • Received:2019-09-29 Revised:2019-11-12 Online:2019-12-08 Published:2019-12-09
  • Contact: Wang Jun E-mail:wangjun0519@126.com
  • Supported by:

    Natural Science Basic Research Program of Shaanxi (2018JM7047035, 2018JM7121); 

    Incubation Fund of Shaanxi Provincial People′s Hospital (2018YXQ-07)

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摘要： 目的  探讨低氧诱导因子-1（HIF-1）介导的线粒体能量代谢在丙泊酚调控肾透明细胞癌细胞增殖及凋亡过程中的作用。方法  选用不表达VHL基因的人肾透明细胞癌细胞株RCC4为研究对象。将pcDNA3VHL质粒和pcDNA3空质粒转染入RCC4细胞中，分别获得稳定表达外源性VHL蛋白的RCC4-VHL(+)细胞和不表达VHL蛋白的RCC4-VHL(-)细胞。随后将两种细胞暴露于0、25、50和100 μmol/L的丙泊酚中，Western blotting法检测两种细胞中HIF-1蛋白表达，流式细胞仪检测细胞增殖活性和细胞凋亡率，细胞能量代谢分析仪检测线粒体能量代谢情况。结果  与RCC4-VHL(-)细胞相比，RCC4-VHL(+)细胞中HIF-1α蛋白的相对表达量显著降低（分别为0.05±0.02、1.23±0.10，t=16.016，P＜0.001）。丙泊酚浓度为50 μmol/L和100 μmol/L时，RCC4-VHL(+)细胞的增殖活性显著低于RCC4-VHL(-)细胞（50 μmol/L：0.10±0.02 vs. 0.13±0.04，t=3.502，P=0.032；100 μmol/L：0.05±0.02 vs. 0.10±0.01，t=6.771，P=0.017），而凋亡率显著高于RCC4-VHL(-)细胞［50 μmol/L：（35.50±1.84）% vs. (22.15±1.06）%，t=7.082，P=0.004；100 μmol/L：(54.35±2.97）% vs. (35.10±3.25）%，t=10.241，P＜0.001］。与0 μmol/L丙泊酚相比，100 μmol/L的丙泊酚可增加RCC4-VHL(+)细胞中HIF-1α蛋白表达（0.93±0.05 vs. 0.04±0.02，t=18.500，P＜0.001）。与RCC4-VHL(-)细胞相比，RCC4-VHL(+)细胞中耗氧率（OCR）［（130.42±11.81）pmol/min vs. (48.27±7.66）pmol/min，t=11.672，P＜0.001］、基础有氧呼吸［（98.55±8.09）pmol/min vs. (41.63±6.21) pmol/min，t=11.162，P＜0.001］、有氧呼吸最大值［（226.79±13.51）pmol/min vs. (70.18±6.82）pmol/min，t=20.697，P＜0.001］、非线粒体呼吸［（28.36±4.29）pmol/min vs. (8.92±1.70）pmol/min，t=8.426，P=0.001］和质子漏耗氧速率［（23.85±5.08）pmol/min vs. (7.80±1.24）pmol/min，t=6.139，P=0.006］均显著增加，而胞外酸化率（ECAR）显著降低［（26.76±4.35）mpH/min vs. (39.48±5.17）mpH/min，t=3.765，P=0.010］。与0 μmol/L丙泊酚相比，100 μmol/L的丙泊酚可显著减少RCC4VHL(+)细胞中OCR［（72.44±8.15）pmol/min vs. (131.56±9.04）pmol/min，t=9.751，P＜0.001］、基础有氧呼吸［（54.31±5.35）pmol/min vs. (96.49±6.86）pmol/min，t=9.697，P＜0.001］、有氧呼吸最大值［（116.71±12.39）pmol/min vs. (219.53±11.80）pmol/min，t=12.019，P＜0.001］、非线粒体呼吸［（13.25±4.01）pmol/min vs. (29.04±5.11）pmol/min，t=4.862，P=0.002］和质子漏耗氧速率［（10.24±3.79）pmol/min vs. (22.92±4.12）pmol/min，t=4.530，P=0.003］，并显著升高ECAR［（37.69±3.75）mpH/min vs. (25.87±4.03）mpH/min，t=4.294，P=0.004］。结论  VHL缺失可上调HIF-1蛋白表达，HIF-1可通过介导线粒体能量代谢途径参与丙泊酚调控RCC4细胞的增殖和凋亡。

关键词: 缺氧诱导因子1, 能量代谢, 二异丙酚, 细胞增殖, 肾透明细胞癌

Abstract: Objective  To investigate the role of mitochondrial energy metabolism mediated via hypoxia-inducible factor-1 (HIF-1) in the proliferation and apoptosis of renal clear cell carcinoma cells regulated by propofol. Methods  We chose human renal clear cell carcinoma cell line RCC4 as the research object, which did not express VHL gene. The pcDNA3VHL plasmid and the pcDNA3 empty plasmid were respectively transfected into RCC4 cells to obtain RCC4-VHL(+) cells stably expressing the exogenous VHL protein and RCC4-VHL(-) cells without expressing the VHL protein. These two kinds of cells were then exposed to propofol at dosage of 0, 25, 50 and 100 μmol/L. HIF-1 protein expression was detected by Western blotting in the two kinds of cells, cell proliferation activity and apoptosis rate were detected by flow cytometry, and mitochondrial energy metabolism was detected by energy metabolism analyzer. Results  Compared with RCC4-VHL(-) cells, the relative expression of HIF-1α protein in RCC4-VHL(+) cells was significantly decreased (0.05±0.02 vs. 1.23±0.10, t=16.016, P＜0.001). When propofol concentrations were 50 μmol/L and 100 μmol/L, the proliferation activity of RCC4-VHL(+) cells was significantly lower than that of RCC4-VHL(-) cells (50 μmol/L: 0.10±0.02 vs. 0.13±0.04, t=3.502, P=0.032; 100 μmol/L: 0.05±0.02 vs. 0.10±0.01, t=6.771, P=0.017), and the apoptotic rate was significantly higher than that of RCC4-VHL (-) cells ［50 μmol/L: (35.50±1.84)% vs. (22.15±1.06)%, t=7.082, P=0.004; 100 μmol/L: (54.35±2.97)% vs. (35.10±3.25)%, t=10.241, P＜0.001). Compared with 0 μmol/L propofol, 100 μmol/L propofol increased HIF-1α protein expression in RCC4-VHL (+) cells (0.93±0.05 vs. 0.04±0.02, t=18.500, P＜0.001). Compared with RCC4-VHL(-) cells, the oxygen consumption rate (OCR) ［(130.42±11.81) pmol/min vs. (48.27±7.66) pmol/min, t=11.672, P＜0.001］, basal aerobic respiration ［(98.55±8.09) pmol/min vs. (41.63±6.21) pmol/min, t=11.162, P＜0.001］, aerobic maximum ［(226.79±13.51) pmol/min vs. (70.18±6.82) pmol/min, t=20.697, P＜0.001］, non-mitochondrial respiration ［(28.36±4.29) pmol/min vs. (8.92±1.70) pmol/min, t=8.426, P=0.001］ and oxygen consumption rate of proton leak ［(23.85±5.08) pmol/min vs. (7.80±1.24) pmol/min, t=6.139, P=0.006］ were significantly increased in RCC4-VHL(+) cells, while the extracellular acidification rate (ECAR) was significantly decreased ［(26.76±4.35) mpH/min vs. (39.48±5.17) mpH/min, t=3.765, P=0.010］. Compared with 0 μmol/L propofol added in RCC4-VHL(+) cells, 100 μmol/L propofol decreased OCR ［(72.44±8.15) pmol/min vs. (131.56±9.04) pmol/min, t=9.751, P＜0.001］, basal aerobic respiration ［(54.31±5.35) pmol/min vs. (96.49±6.86) pmol/min, t=9.697, P＜0.001］, aerobic maximum ［(116.71±12.39) pmol/min vs. (219.53±11.80) pmol/min, t=12.019, P＜0.001］, non-mitochondrial respiration ［(13.25±4.01) pmol/min vs. (29.04±5.11) pmol/min, t=4.862, P=0.002］ and oxygen consumption rate of proton leak ［(10.24±3.79) pmol/min vs. (22.92±4.12) pmol/min, t=4.530, P=0.003］, and increased ECAR significantly ［(37.69±3.75) mpH/min vs. (25.87±4.03) mpH/min, t=4.294, P=0.004］. Conclusion  Loss of VHL up-regulates expression of HIF-1 protein, and mitochondrial energy metabolism mediated via HIF-1 involves the proliferation and apoptosis of RCC4 cells regulated by propofol.

Key words: Hypoxia-inducible factor 1, Energy metabolism, Propofol, Cell proliferation, Renal clear cell carcinoma