沙利度胺抑制肿瘤细胞分泌VEGF/bFGF机制的探讨*

doi:10.12047/j.cjap.6262.2022.032

中国应用生理学杂志 ›› 2022, Vol. 38 ›› Issue (2): 169-174.doi: 10.12047/j.cjap.6262.2022.032

沙利度胺抑制肿瘤细胞分泌VEGF/bFGF机制的探讨^*

黄海宁^1,2, 汪汇⁴, 龙超良², 张浩^2,4△, 汪海^2,3,4△

1.济宁医学院附属医院, 山东济宁 272029;
2.北京赛德维康医药研究院, 北京 100039;
3.江苏师范大学健康科学学院, 徐州 221116;
4.中国人民解放军总医院, 北京 100853

收稿日期:2021-11-29 修回日期:2022-03-26 出版日期:2022-03-28 发布日期:2022-08-29
通讯作者: ^△Tel: 010-66931557, 010-66932651; E-mail: zhanghal197@hotmail.com, wh9588@sina.com
基金资助:
^*国家973计划项目子课题(2011CB518206);济宁医学院教师科研扶持基金(JYFC2019FKJ161 )

Mechanism of the inhibitory effects of thalidomide on expressions of VEGF and bFGF

HUANG Hai-ning^1,2, WANG Hui⁴, LONG Chao-liang², ZHANG Hao^2,4△, WANG Hai^2,3,4△

1. Affiliated Hospital of Jining Medical University, Jining 272029;
2. Thadweik Academy of Medicine, Beijing 100039;
3. College of Heacth Science, Jiangsu Normal University, Xuzhou 221116;
4. General Hospital of PLA, Beijing 100853, China

Received:2021-11-29 Revised:2022-03-26 Online:2022-03-28 Published:2022-08-29

摘要/Abstract

摘要： 目的: 探讨Cereblon(CRBN)对沙利度胺抑制人肺癌A549细胞及人肝癌HepG2细胞分泌VEGF/bFGF的影响。方法: 采用慢病毒介导的短发夹RNA(shRNA)干扰技术建立稳定敲低CRBN的A549细胞系(A549_CRBN)及HepG2细胞系(HepG2_CRBN)并通过实时定量PCR(Real-time PCR)和蛋白质印记(Western blot)实验验证。将A549细胞分为阴性对照组(A549_luciferase)、CRBN低表达组(A549_CRBN);HepG2细胞分为阴性对照组(HepG2_luciferase)、CRBN低表达组(HepG2_CRBN),以上细胞按照 3×10⁵cells/well接种到6孔板中,放入37℃,5%CO2的培养箱中培养24 h,分别加入1 ml含100 μmol/L沙利度胺(thalidomide组)和1 ml 1‰ DMSO(control组)的培养液,继续培养24 h再行后续实验,每组设计3个复孔。MTS法检测沙利度胺对细胞增殖的影响;Real-time PCR检测VEGF、bFGF、c-jun mRNA表达,ELISA法检测VEGF、bFGF蛋白表达。结果: 与对照组比较,沙利度胺在浓度为1、10、50、100 μmol/L 时对A549 及HepG2细胞的增殖能力无显著影响(P＞0.05)。与A549_CRBN或HepG2_CRBN组比较,A549_luciferase及HepG2_luciferase组分泌的VEGF及bFGF均显著降低(P＜0.05)。与A549_luciferase或HepG2_luciferase细胞的对照组比较,沙利度胺可抑制A549_luciferase和HepG2_luciferase细胞的VEGF和bFGF的表达(P＜0.05),而对A549_CRBN和HepG2_CRBN细胞中VEGF和bFGF的表达无显著抑制作用;与HepG2_luciferase细胞的对照组比较,沙利度胺可抑制HepG2_luciferase细胞的c-Jun表达(P＜0.01),而对HepG2_CRBN细胞的c-Jun表达无显著抑制作用。结论: 沙利度胺对A549和HepG2细胞VEGF和bFGF表达的抑制作用可能是通过CRBN介导的,而c-Jun可能是抑制作用的关键转录因子之一。

关键词: 沙利度胺, 血管新生, 细胞培养, 血管内皮生长因子(VEGF), 碱性成纤维细胞生长因子 (bFGF), cereblon(CRBN)

Abstract: Objective: To investigate the inhibitory effects of thalidomide on the expressions of VEGF and bFGF in human lung adenocarcinoma A549 cells and human hepatocellular carcinomas HepG2 cells mediated by cereblon (CRBN). Methods: shRNA technology was used to construct the A549 cell line (A549_CRBN) and HepG2 cell line (HepG2_CRBN) with stable knockdown of CRBN, which was verified by real-time PCR and Western blot. A549 cells were divided into negative control group (A549_luciferase) and CRBN down-regulation group (A549_CRBN); HepG2 cells were divided into negative control group (HepG2_luciferase) and CRBN down-regulation group (HepG2_CRBN). The above cells were seeded into 6-well plates at 3×10⁵ cells/well, and cultured in a 37℃, 5% CO₂ incubator for 24 h. Then, 1 ml medium containing 100 μmol/L thalidomide (thalidomide group) and 1 ml medium containing 1‰ DMSO (control group) were added respectively, and the culture was continued for 24 hours before subsequent experiments. Each group was designed with three replicate wells. The effect of thalidomide on the activity of A549 cell line was detected by MTS assay. Real-time PCR was performed to detect mRNA expression levels of VEGF, bFGF and c-jun. ELISA assay was performed to detect protein expressions of VEGF and bFGF. Results: Compared with the control group, thalidomide at the concentrations of 1, 10, 50 and 100 μmol/L had no significant effects on the proliferation of A549 and HepG2 cells (P＞0.05). VEGF and bFGF levels in the A549_CRBN or HepG2_luciferase groups were significantly lower than those in the A549_CRBN or HepG2_CRBN groups (P＜0.05). Compared with the control group of the A549_luciferase or HepG2_luciferase, thalidomide inhibited the expressions of VEGF and bFGF in A549_luciferaseand HepG2_luciferase cells (P＜ 0.05), but did not inhibit the expressions of VEGF and bFGF in A549_CRBN and HepG2_CRBN cells. Compared with the control group of the HepG2_luciferase, thalidomide inhibited c-Jun expression in HepG2_luciferase cells (P＜0.01), but did not significantly inhibit c-Jun expression in HepG2_CRBN cells. Conclusion: The inhibitory effects of thalidomide on VEGF and bFGF expressions may be mediated by CRBN in A549 and HepG2 cells, and c-Jun may be one of the key transcription factors responsible for this inhibition.

Key words: thalidomide, angiogenesis, cell culture, VEGF, bFGF, cereblon(CRBN)

中图分类号:

R966

黄海宁, 汪汇, 龙超良, 张浩, 汪海. 沙利度胺抑制肿瘤细胞分泌VEGF/bFGF机制的探讨^*[J]. 中国应用生理学杂志, 2022, 38(2): 169-174.

HUANG Hai-ning, WANG Hui, LONG Chao-liang, ZHANG Hao, WANG Hai. Mechanism of the inhibitory effects of thalidomide on expressions of VEGF and bFGF[J]. CJAP, 2022, 38(2): 169-174.

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沙利度胺抑制肿瘤细胞分泌VEGF/bFGF机制的探讨^*

Mechanism of the inhibitory effects of thalidomide on expressions of VEGF and bFGF

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