青藤碱衍生物调变多发性骨髓瘤细胞趋化因子受体的实验研究

居身傲男, 王莹莹, 唐勇, 姚一芸, 吴英理, 朱琦

  1. 1.上海交通大学医学院附属第九人民医院口腔颌面-头颈肿瘤科，上海 200011
    2.上海交通大学医学院病理生理学系，细胞分化与凋亡教育部重点实验室，上海 200025
    3.上海交通大学医学院附属第九人民医院黄浦分院血液肿瘤内科，上海 200011
  • 收稿日期:2022-12-22 修回日期:2023-05-04 出版日期:2023-06-30 发布日期:2023-07-26
  • 通信作者: 朱琦
  • 作者简介:居身傲男（ORCID: 0000-0002-3675-5099），上海交通大学医学院内科学研究生。
  • 基金资助:
    国家自然科学基金(81870156)

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摘要/Abstract

摘要：

背景与目的：多发性骨髓瘤（multiple myeloma，MM）细胞表面CXC族趋化因子受体（CXC motif chemokine receptor，CXCR）与骨髓微环境内趋化因子相互作用参与MM细胞生存、增殖和髓外侵袭。青藤碱衍生物YL064通过靶向MM细胞内信号转导通路分子发挥其生物学效应。本研究探究青藤碱衍生物YL064对MM细胞表面CXCR3的调变作用及其生物学效应。方法：以MM细胞系H929和MM1.S细胞为模型，利用慢病毒载体构建过表达CXCR3的H929-OE和MM1.S-OE细胞，采用克隆形成和体外迁移实验检测并比较H929、H929-OE、MM1.S和MM1.S-OE细胞克隆形成和迁移率；采用流式细胞术检测YL064处理后H929、H929-OE、MM1.S和MM1.S-OE细胞凋亡率；进一步以不同浓度YL064处理H929和MM1.S细胞，采用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction，RTFQ-PCR）和蛋白质印迹法（Western blot）检测细胞内CXCR3基因转录和蛋白水平及其下游信号转导通路关键分子细胞外调节蛋白激酶（extracellular regulated protein kinase，ERK）和p-蛋白激酶B（p-protein kinase B，p-AKT）蛋白水平的改变。结果：CXCR3过表达H929-OE和MM1.S-OE细胞克隆形成率分别为81.33%±5.79%和73.00%±4.90%，显著高于H929的58.33%±3.30%和MM1.S细胞的41.00%±3.14%；H929-OE和MM1.S-OE细胞迁移率分别为7.90%±0.81%和23.00%±1.63%，显著高于H929的4.63%±0.37%和MM1.S细胞的14.63%±1.04%；经YL064处理后，H929-OE和MM1.S-OE细胞凋亡率分别为29.80%±0.30%和14.2%±0.26%，显著低于H929的33.40%±0.25%和MM1.S细胞的21.60%±0.21%；上述结果比较差异有统计学意义（P均<0.05）。进一步研究发现YL064能够降低H929和MM1.S细胞克隆形成率和迁移率，同时抑制细胞内CXCR3基因转录并下调CXCR3、ERK和AKT蛋白表达。结论：CXCR3可以促进MM细胞增殖和迁移，青藤碱衍生物YL064能够下调MM细胞内CXCR3表达并抑制其下游信号转导通路进而干扰其增殖和迁移并诱导凋亡。

关键词: 多发性骨髓瘤, 青藤碱衍生物, CXCR3

Abstract:

Background and purpose: The interaction of CXC motif chemokine receptor (CXCR) on the cell surface of multiple myeloma (MM) with chemokines in the bone marrow microenvironment is involved in proliferation, survival and extramedullary invasion of MM cells. Sinomenine derivative YL064 exerts its biological effects by targeting intracellular signaling regulators in MM cells. This study aimed to explore possible modulating and biological effects of sinomenine derivative YL064 on CXCR3 in MM cells. Methods: The MM cell lines H929 and MM1.S were used as in vitro models. H929-OE and MM1.S-OE cells with stable overexpression of CXCR3 were constructed with lentivirus vector. The effects of CXCR3 on clonal formation and migration of MM cells were detected by clone spot formation and transwell migration assay. Meanwhile, flow cytometry was used to analyze apoptotic rates of H929, H929-OE, MM1.S and MM1.S-OE cells treated with different concentrations of YL064. Furthermore, real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) and Western blot assay were applied to detect expression levels of CXCR3 and its downstream signal regulators extracellular regulated protein kinase (ERK) and p-protein kinase B (p-AKT) in H929 and MM1.S cells before and after the treatment with YL064. Results: The clonal formation rates of H929-OE and MM1.S-OE cells with CXCR3 overexpression reached to 81.33%±5.79% and 73.00%±4.90%, which were significantly higher compared with H929 (58.33%±3.30%) and MM1.S cells (41.00%±3.14%). The migration proportion of H929-OE and MM1.S-OE cells were 7.90%±0.81% and 23.00%±1.63%, which were significantly higher compared with H929 (4.63%±0.37%) and MM1.S cells (14.63%±1.04%). After treatment with YL064, the apoptotic rates of H929-OE and MM1.S-OE cells were 29.80%±0.30% and 14.20%±0.26%, which were lower than those of H929 (33.40%±0.25%) and MM1.S cells (21.60%±0.21%). It was also shown that YL064 could reduce clonal formation and migration, inhibit CXCR3 gene transcription and downregulate expressions of CXCR3, ERK and AKT in H929 and MM1.S cells. Conclusions: CXCR3 might promote proliferation and invasion of MM cells. Sinomenine derivative YL064 could downregulate expressions of CXCR3 and its downstream signal regulators in MM cells, reduce abilities of clonal formation and migration, and induce apoptosis.

Key words: Multiple myeloma, Sinomenine derivative, CXCR3

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