低氧处理不同时间对人肺腺癌A549细胞增殖的影响及相应缺氧模型探讨
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摘要
目的:观察低氧处理不同时间对人肺腺癌A549细胞增殖的影响,探讨合理的人肺腺癌细胞株A549体外模拟缺氧时间。方法:将人肺腺癌细胞A549细胞株在低氧环境下分别培养12 h、24 h、48 h、72 h,设置常氧对照组,通过CCK8法测定A549细胞存活率,RT-PCR和免疫印迹分别检测细胞缺氧诱导因子-1α(hypoxia-inducible factor-1α, HIF-1α)和血管内皮生长因子(vascularendothelial growth factor, VEGF)mRNA及蛋白的表达。结果:低氧24 h组A549细胞存活率最高,低氧48 h、72 h组A549细胞存活率呈时间依赖性明显下降(P<0.001)。自低氧12 h起,A549细胞HIF-1αmRNA和VEGFmRNA的表达开始随低氧时间延长而显著增加(P均<0.001);HIF-1α和VEGF蛋白表达自24 h开始随低氧时间延长而显著增加(P均<0.001)。结论:低氧诱导的A549细胞存活率呈时间依赖性降低,而HIF-1α、VEGF表达呈时间依赖性增高,人肺癌细胞株A549缺氧模型最适时间为24 h。
ABSTRCT Objective: To observe the effect of different time of hypoxia treatment on the proliferation of human lung adenocarcinoma A549 cells. To explore the optimal time for the hypoxic model in vitro for human lung adenocarcinoma cell line A549.Methods: Human lung adenocarcinoma A549 cells were cultured under hypoxic environment for 12 h, 24 h, 48 h and 72 h respectively and set up a normoxic group as contrast. The proliferation of A549 cells was detected by CCK8 assay. The transcriptional mR NA and protein levels of hypoxia-inducible factor-1α(HIF-1α) and vascular endothelial growth factor(VEGF) were examined by RT-PCR and western blot. Results: The survival rate of the 24 h group was the highest, and the cells' proliferation of 48 h and 72 h group significantly decreased in a time-dependent manner(P<0.001). The mR NA expression of HIF-1α and VEGF began to increase significantly from 12 h while the protein expression rose significantly from 24 h. After the above time points the mR NA and protein expression both increased with the prolongation of hypoxic time(P <0.001). Conclusions: The viability of hypoxia-induced A549 cells decreased in a time-dependent manner while the expression of HIF-1α and VEGF increased with the extension of hypoxic time. The optimal time for A549 hypoxic model in our study was 24 h.
ABSTRCT Objective: To observe the effect of different time of hypoxia treatment on the proliferation of human lung adenocarcinoma A549 cells. To explore the optimal time for the hypoxic model in vitro for human lung adenocarcinoma cell line A549.Methods: Human lung adenocarcinoma A549 cells were cultured under hypoxic environment for 12 h, 24 h, 48 h and 72 h respectively and set up a normoxic group as contrast. The proliferation of A549 cells was detected by CCK8 assay. The transcriptional mR NA and protein levels of hypoxia-inducible factor-1α(HIF-1α) and vascular endothelial growth factor(VEGF) were examined by RT-PCR and western blot. Results: The survival rate of the 24 h group was the highest, and the cells' proliferation of 48 h and 72 h group significantly decreased in a time-dependent manner(P<0.001). The mR NA expression of HIF-1α and VEGF began to increase significantly from 12 h while the protein expression rose significantly from 24 h. After the above time points the mR NA and protein expression both increased with the prolongation of hypoxic time(P <0.001). Conclusions: The viability of hypoxia-induced A549 cells decreased in a time-dependent manner while the expression of HIF-1α and VEGF increased with the extension of hypoxic time. The optimal time for A549 hypoxic model in our study was 24 h.
引文
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[22]Tian HY,Li ZX,Li HY,et al.Effects of 14 single herbs on the induction of caspase-3 in tumor cells:a brief review[J].Chin J Integr Med,2013,19(8):636-640
[23]Cui R,Kim T,Fassan M,et al.Micro RNA-224 is implicated in lung cancer pathogenesis through targeting caspase-3 and caspase-7[J].Oncotarget,2015,6(26):21802-21815
[24]沈红,朱祎娜,秦湧,等.低氧降低肺腺癌A549细胞紫杉醇敏感性及其机制的探讨[J].肿瘤,2017,37(02):135-142Shen Hong,Zhu Yi-na,Qin Yong,et al.Inhibitory effect of hypoxia on chemosensitivity of human lung cancer A549 cells to paclitaxel and its possible mechanism[J].Tumor,2017,37(02):135-142
[25]杨士芳,李晓明,安弘,等.HIF-1α介导了间歇低氧对人肺腺癌A549细胞体外生物学行为的影响[J].中国病理生理杂志,2016,(11):1958-1965Yang Shi-fang,Li Xiao-ming,An Hong,et al.HIF-1αmediates effect of intermittent hypoxia on biological behavior of A549 cells in vitro[J].Chinese Journal of Pathophysiology,2016,(11):1958-1965
[26]Gozal D,FarréR,Nieto FJ.Obstructive sleep apnea and cancer:Epidemiologic links and theoretical biological constructs[J].Sleep Med Rev,2016,27:43-55
[2]袁源,钟竑,梅举,等.人非小细胞肺癌中缺氧诱导因子1α的表达及临床意义[J].现代生物医学进展,2010,10(15):2814-2817Yuan Yuan,Zhong Hong,Mei Ju,et al.Expression and Clinical Significance of HIF-1αin Non-small Cell Lung Cancer[J].Progress in Modern Biomedicine,2010,10(15):2814-2817
[3]Jackson AL,Zhou B,Kim WY.HIF,hypoxia and the role of angiogenesis in non-small cell lung cancer[J].Expert Opin Ther Targets,2010,14(10):1047-1057
[4]Jain RK.Antiangiogenesis strategies revisited:from starving tumors to alleviating hypoxia[J].Cancer Cell,2014,26(5):605-622
[5]张莉,李清泉.低氧对人肺腺癌A549细胞生长的影响[J].实用医学杂志,2007,(04):451-453Zhang Li,Li Qing-quan.Effect of hypoxia on cell growth of human lung adenocarcinoma cell line A549[J].The Journal of Practical Medcine,2007,(04):451-453
[6]Ahluwalia A,Tarnawski AS.Critical role of hypoxia sensor--HIF-1αin VEGF gene activation.Implications for angiogenesis and tissue injury healing[J].Curr Med Chem,2012,19(1):90-97
[7]Alevizakos M,Kaltsas S,Syrigos KN.The VEGF pathway in lung cancer[J].Cancer Chemother Pharmacol,2013,72:1169-1181
[8]Li X,Liu X,Xu Y,et al.KLF5 promotes hypoxia-induced survival and inhibits apoptosis in non-small cell lung cancer cells via HIF-1α[J].Int J Oncol,2014,45(4):1507-1514
[9]Li W,Chen YQ,Shen YB,et al.HIF-1αknockdown by mi RNAdecreases survivin expression and inhibits A549 cell growth in vitro and in vivo[J].Int J Mol Med,2013,32:271-280
[10]Shen H,Feng GZ,Cui J,et al.Clinical implications of serum hypoxia inducible factor-1αand vascular endothelial growth factor in lung cancer[J].Tumori,2015,101(4):404-411
[11]沈红,崔进,秦湧,等.晚期非小细胞肺癌患者化疗前后血清HIF-1α和VEGF的变化及临床意义[J].现代肿瘤医学,2015,23(6):791-794Shen Hong,Cui Jin,Qin Yong,et al.Clinical study of the changes of serum HIF-1αand VEGF before and after chemotherapy for patients with non-small cell lung cancer[J].Journal of Modern Oncology,2015,23(6):791-794
[12]Jing XG,Chen TF,Huang C,et al.Mi R-15a expression analysis in non-small cell lung cancer A549 cells under local hypoxia microenvironment[J].Eur Rev Med Pharmacol Sci,2017,21(9):2069-2074
[13]Rogoziński P,Bruliński K,Malinowski E,et al.Is the vascular-endothelial growth factor responsible for the malignancy of lung cancer?[J].Pol Merkur Lekarski,2012,33(196):213-216
[14]Ajdukovic'J.HIF-1:a big chapter in the cancer tale[J].Exp Oncol,2016,38(1):9-12
[15]Yousefi B,Rahmati M,Ahmadi Y.The roles of p53R2 in cancer progression based on the new function of mutant p53 and cytoplasmic p21[J].Life Sci,2014,99(1-2):14-17
[16]Cathcart MC,Gately K,Cummins R,et al.Thromboxane synthase expression and correlation with VEGF and angiogenesis in non-small cell lung cancer[J].Biochim Biophys Acta,2014,1842(5):747-755
[17]郭树鹏,杨柳,张莉琳,等.HIF-1α信号通路在肿瘤细胞调控中的研究进展[J].现代生物医学进展,2015,15(16),3145-3148Guo Shu-peng,Yang Liu,Zhang Li-lin,et al.Research Progress in Regulation of HIF-1αSignaling Pathways in Cancers[J].Progress in Modern Biomedicine,2015,15(16):3145-3148
[18]Frezzetti D,Gallo M,Maiello MR,et al.VEGF as a potential target in lung cancer[J].Expert Opin Ther Targets,2017,21(10):959-966
[19]Naikoo NA,Dil-Afroze,Rasool R,et al.Upregulation of vascular endothelial growth factor(VEGF),its role in progression and prognosis of non-small cell lung carcinoma[J].Cancer Genet,2017,216-217:67-73
[20]曲耘,吴荣,张晓晔.NF-κB/p65 si RNA对小鼠Lewis肺癌移植瘤细胞凋亡和Bax表达的影响[J].中国医科大学学报,2017,46(02):145-148Qu Yun,Wu Rong,Zhang Xiao-ye.Effects of NF-κB/p65 si RNA on Apoptosis and Bax Expression in Lung Tumour Cell Xenografts of Nude Mouse[J].Journal of China Medical University,2017,46(02):145-148
[21]Ahmed K,Tabuchi Y,Kondo T.Hyperthermia:an effective strategy to induce apoptosis in cancer cells[J].Apoptosis,2015,20(11):1411-1419
[22]Tian HY,Li ZX,Li HY,et al.Effects of 14 single herbs on the induction of caspase-3 in tumor cells:a brief review[J].Chin J Integr Med,2013,19(8):636-640
[23]Cui R,Kim T,Fassan M,et al.Micro RNA-224 is implicated in lung cancer pathogenesis through targeting caspase-3 and caspase-7[J].Oncotarget,2015,6(26):21802-21815
[24]沈红,朱祎娜,秦湧,等.低氧降低肺腺癌A549细胞紫杉醇敏感性及其机制的探讨[J].肿瘤,2017,37(02):135-142Shen Hong,Zhu Yi-na,Qin Yong,et al.Inhibitory effect of hypoxia on chemosensitivity of human lung cancer A549 cells to paclitaxel and its possible mechanism[J].Tumor,2017,37(02):135-142
[25]杨士芳,李晓明,安弘,等.HIF-1α介导了间歇低氧对人肺腺癌A549细胞体外生物学行为的影响[J].中国病理生理杂志,2016,(11):1958-1965Yang Shi-fang,Li Xiao-ming,An Hong,et al.HIF-1αmediates effect of intermittent hypoxia on biological behavior of A549 cells in vitro[J].Chinese Journal of Pathophysiology,2016,(11):1958-1965
[26]Gozal D,FarréR,Nieto FJ.Obstructive sleep apnea and cancer:Epidemiologic links and theoretical biological constructs[J].Sleep Med Rev,2016,27:43-55
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