中华皮肤科杂志 ›› 2021, Vol. 54 ›› Issue (4): 294-299.doi: 10.35541/cjd.20200764

• 黑素瘤·论著 • 上一篇    下一篇

皮肤黑素瘤细胞与血管内皮细胞间VEGF-IL-6-STAT3信号交互作用机制

杨景煜1    顾珈榕2，3    郭静1    杨瑞1    王文成1    李云凤1    徐平1    顾金海2   

  1. 1宁夏回族自治区人民医院皮肤科，银川  750021；2宁夏医科大学颅脑疾病重点实验室，银川  750004；3宁波大学医学院预防医学学科系，浙江  315211
  • 收稿日期:2020-07-31 修回日期:2021-01-23 发布日期:2021-03-31
  • 通讯作者: 顾金海 E-mail:gjinhai@126.com
  • 基金资助:
    国家自然科学基金（81160312）；宁夏自然科学基金（2018AAC03268）

Signal crosstalk of VEGF-IL-6-STAT3 between cutaneous melanoma cells and vascular endothe-lial cells

Yang Jingyu1, Gu Jiarong2,3, Guo Jing1, Yang Rui1, Wang Wencheng1, Li Yunfeng1, Xu Ping1, Gu Jinhai2   

  1. 1Department of Dermatology, People′s Hospital of Ningxia Hui Autonomous Region, Yinchuan 750021, China; 2Key Laboratory of Craniocerebral Diseases, Ningxia Medical University, Yinchuan 750004, China; 3Department of Preventive Medicine, School of Medicine, Ningbo University, Ningbo 315211, Zhejiang, China
  • Received:2020-07-31 Revised:2021-01-23 Published:2021-03-31
  • Contact: Gu Jinhai E-mail:gjinhai@126.com
  • Supported by:
    National Natural Science Foundation of China （81160312）; Ningxia Natural Science Foundation （2018AAC03268）

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摘要： 【摘要】 目的 研究皮肤黑素瘤细胞与血管内皮细胞间VEGF-IL-6-STAT3信号交互作用机制。 方法 将血管内皮细胞EC-304分成3组：对照组，常规培养；血管内皮生长因子（VEGF）组，在含VEGF165（50 μg/L）的内皮细胞培养基中培养；A375共培养组，与黑素瘤细胞A375共培养。24 h、48 h、72 h后收集培养液，酶联免疫吸附实验检测白细胞介素6（IL-6）的分泌量。将A375细胞分为4组：对照组，常规培养；A375 + EC-304组，与EC-304共培养；A375 + EC-304 + IL-6组：与EC-304细胞在含STAT3通路激动剂IL-6（50 μg/L）的DMEM中共培养；A375 + EC-304 + JSI-124组：与EC-304在含STAT3通路抑制剂JSI-124（1 μmol/L）的DMEM中共培养。分别在24 h、48 h、72 h后收集细胞，用Western印迹、CCK-8、Transwell侵袭实验分别检测STAT3、p-STAT3蛋白表达、细胞增殖活性及侵袭活性。采用双因素方差分析及t检验统计分析数据。结果 与对照组比较，VEGF组、A375共培养组EC-304培养基中IL-6分泌量均升高（FVEGF = 29.63，P < 0.001；FA375 = 11.09，P = 0.020）。与对照组比较，A375 + EC-304组A375细胞p-STAT3蛋白表达升高（P < 0.001），细胞活性增强（P < 0.001），侵袭细胞数从（86.13 ± 7.24）个增加到（152.66 ± 16.04）个（t = 4.43，P < 0.001）；与A375 + EC-304组比较，A375 + EC-304 + IL-6组细胞p-STAT3蛋白表达升高（P < 0.001），细胞活性增强（P < 0.001），侵袭细胞数量增加至（187.34 ± 14.38）个（t = 2.17，P < 0.001）；与A375 + EC-304组比较，A375 + EC-304 + JSI-124组细胞的p-STAT3蛋白表达降低（P < 0.001），细胞活性减弱（P < 0.001），侵袭细胞数减少至（124.92 ± 8.72）个（t = -1.86，P < 0.001）。结论 皮肤黑素瘤A375细胞与血管内皮细胞之间存在VEGF-IL-6-STAT3信号交互作用机制，这种机制可促进A375细胞的增殖和侵袭。

关键词: 皮肤恶性黑色素瘤, 血管内皮细胞, VEGF, IL-6, STAT3, Crosstalk

Abstract: 【Abstract】 Objective To investigate mechanisms underlying the signal crosstalk of VEGF-IL-6-STAT3 between cutaneous melanoma cells and vascular endothelial cells. Methods EC-304 vascular endothelial cells were divided into 3 groups: control group cultured in conventional endothelial cell-conditioned medium, vascular endothelial growth factor （VEGF） group cultured in endothelial cell-conditioned medium containing 50 μg/L VEGF165, A375 co-culture group co-cultured with a melanoma cell line A375. After 24-, 48- and 72-hour treatment, the culture medium was collected, and enzyme-linked immunosorbent assay was performed to detect the level of interleukin-6 （IL-6）. Cultured A375 cells were divided into 4 groups: control group receiving conventional culture in Dulbecco′s modified Eagle′s medium （DMEM）, A375 + EC-304 group co-cultured with EC-304 cells, A375 + EC-304 + IL-6 group co-cultured with EC-304 cells in DMEM containing 50 μg/L IL-6 （an agonist of the signal transducer and activator of transcription-3 ［STAT3］ pathway ）, A375 + EC-304 + JSI-124 group co-cultured with EC-304 cells in DMEM containing 1 μmol/L JSI-124（a STAT3 pathway inhibitor）. After 24-, 48- and 72-hour treatment, cells were collected, and Western blot analysis, cell counting kit-8 （CCK8） assay and Transwell invasion assay were performed to determine the protein expression of STAT3 and phosphorylated （p）-STAT3, cellular proliferative activity and invasive activity, respectively. Two-way analysis of variance and t test were used for statistical analysis. Results The level of IL-6 significantly increased in the culture medium of EC-304 cells in the VEGF group and A375 co-culture group compared with the control group （FVEGF = 29.63, P < 0.001; FA375 = 11.09, P = 0.020）. Compared with the control group, the A375 + EC-304 group showed significantly enhanced protein expression of p-STAT3 in A375 cells （P < 0.001）, increased cell activity （P < 0.001）, and increased number of invasive cells (152.66 ± 16.04 vs. 86.13 ± 7.24, t = 4.43, P < 0.001）; compared with the A375 + EC-304 group, the A375 + EC-304 + IL-6 group showed significantly increased protein expression of p-STAT3 （P < 0.001）, enhanced cell activity （P < 0.001）, and increased number of invasive cells （187.34 ± 14.38, t = 2.17, P < 0.001）; compared with the A375 + EC-304 group, the A375 + EC-304 + JSI-124 group showed significantly decreased protein expression of p-STAT3 （P < 0.001）, decreased cell activity （P < 0.001）, and decreased number of invasive cells （124.92 ± 8.72, t = -1.86, P < 0.001）. Conclusion There is a signal crosstalk of VEGF-IL-6-STAT3 between cutaneous melanoma cells and vascular endothelial cells, which may play an important role in the proliferation and invasion of A375 cells.

Key words: Cutaneous malignant melanom, Vascular endothelial cell, VEGF, IL-6, STAT3, Crosstalk