ZLM-7 通过 miR-212-3p/Sp1/VEGFA 信号轴抑制乳腺癌的发生和血管生成。
ZLM-7 inhibits the occurrence and angiogenesis of breast cancer through miR-212-3p/Sp1/VEGFA signal axis.
机构信息
Molecular Biology Research Centre, Hunan Province Key Laboratory of Basic and Applied Hematology, Hunan Key Laboratory of Animal Models for Human Diseases, School of Life Sciences, Central South University, Changsha, 410008, Hunan, China.
Hunan Key Laboratory of Oral Health Research, Xiangya Stomatological Hospital, Xiangya School of Stomatology, Central South University, Changsha, 410008, Hunan, China.
出版信息
Mol Med. 2020 Nov 13;26(1):109. doi: 10.1186/s10020-020-00239-2.
BACKGROUND
Breast cancer (BC) is a common malignant tumor with poor prognosis. Angiogenesis is related to the growth and progression of solid tumors and associated with prognosis. ZLM-7, SP1, VEGFA and miR-212-3p were associated with BC angiogenesis and proliferation, however the detailed mechanism was not clear. This study aimed to reveal the regulatory mechanism of angiogenesis of BC.
METHODS
BC cell lines were treated with 10 nM ZLM-7 for 8 h. We detected protein expression level by western blot and RNA expression level by qRT-PCR. Overexpression or inhibition of miR-212-3p is performed using miR-212-3p mimics or miR-212-3p inhibitor, Sp1 overexpression using pcDNA3.1 vector. Angiogenesis was analyzed by co-culturing BC cell lines and HUVEC cells. To evaluate regulatory relationship between miR-212-3p and Sp1, dual luciferase assay was performed. Besides, the direct interaction between Sp1 and VEGFA was analyzed by ChIP. Migration and invasion were analyzed by transwell assay and proliferation was detected by clone formation assay. In mice xenograft model developed using BC cells, we also detected angiogenesis marker CD31 through immunohistochemistry.
RESULTS
ZLM-7 up-regulated miR-212-3p and inhibited invasion, migration, proliferation and angiogenesis of BC, while miR-212-3p inhibitor antagonized such effects. Binding sequence was revealed between miR-212-3p and Sp1, and expression of Sp1 was inhibited by miR-212-3p on both protein and mRNA level. Sp1 could interact with VEGFA and promoted its expression. Overexpression of miR-212-3p inhibited migration, invasion, proliferation and angiogenesis of BC cell lines, while Sp1 overexpression showed the opposite effect and could antagonize these effects of miR-212-3p overexpression. ZLM-7 decreased VEGFA expression, which was rescued by co-transfection with miR-212-3p inhibitor. Similar, ZLM-7 could inhibit tumor growth and angiogenesis through the miR-212-3p/Sp1/VEGFA axis in vivo.
CONCLUSIONS
ZLM-7 could directly up-regulate miR-212-3p in BC. MiR-212-3p could inhibit VEGFA expression through Sp1, thereby inhibiting angiogenesis and progression of BC.
背景
乳腺癌（BC）是一种预后不良的常见恶性肿瘤。血管生成与实体瘤的生长和进展有关，并与预后相关。ZLM-7、SP1、VEGFA 和 miR-212-3p 与 BC 血管生成和增殖有关，但详细机制尚不清楚。本研究旨在揭示 BC 血管生成的调节机制。
方法
用 10 nM ZLM-7 处理 BC 细胞系 8 h。通过 Western blot 检测蛋白表达水平，通过 qRT-PCR 检测 RNA 表达水平。使用 miR-212-3p 模拟物或 miR-212-3p 抑制剂过表达 miR-212-3p，使用 pcDNA3.1 载体过表达 Sp1。通过共培养 BC 细胞系和 HUVEC 细胞分析血管生成。为了评估 miR-212-3p 和 Sp1 之间的调节关系，进行了双荧光素酶测定。此外，通过 ChIP 分析了 Sp1 和 VEGFA 之间的直接相互作用。通过 Transwell 测定分析迁移和侵袭，通过克隆形成测定检测增殖。在使用 BC 细胞建立的小鼠异种移植模型中，我们还通过免疫组织化学检测了血管生成标志物 CD31。
结果
ZLM-7 上调 miR-212-3p 并抑制 BC 的侵袭、迁移、增殖和血管生成，而 miR-212-3p 抑制剂拮抗了这种作用。miR-212-3p 和 Sp1 之间存在结合序列，miR-212-3p 在蛋白和 mRNA 水平上抑制 Sp1 的表达。Sp1 可与 VEGFA 相互作用并促进其表达。过表达 miR-212-3p 抑制 BC 细胞系的迁移、侵袭、增殖和血管生成，而过表达 Sp1 则表现出相反的效果，并能拮抗 miR-212-3p 过表达的这种作用。ZLM-7 降低 VEGFA 表达，而 miR-212-3p 抑制剂共转染可挽救该作用。类似地，ZLM-7 可通过体内 miR-212-3p/Sp1/VEGFA 轴抑制肿瘤生长和血管生成。
结论
ZLM-7 可直接上调 BC 中的 miR-212-3p。miR-212-3p 可通过 Sp1 抑制 VEGFA 表达，从而抑制 BC 的血管生成和进展。
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