Calreticulin regulates vascular endothelial growth factor-A mRNA stability in gastric cancer cells
Calreticulin regulates vascular endothelial growth factor-A mRNA stability in gastric cancer cells
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DOI：
10.1371/journal.pone.0225107
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发表时间：
2019-11-14
期刊：
影响因子：
3.7
通讯作者：
Lee, Hsinyu
中科院分区：
文献类型：
--
作者：
Lee, Po-Chu;Chiang, Jui-Chung;Lee, Hsinyu
钙网蛋白 (CRT) 和血管内皮生长因子-A (VEGF-A) 对于血管生成至关重要，并介导胃癌的多种恶性行为。在这项研究中，我们报告胃癌患者中 CRT 与 VEGF-A 呈正相关。此外，CRT和VEGF-A的高表达与患者的病理分期、进展和不良预后显着相关。因此，我们试图阐明 CRT 影响胃癌中 VEGF-A 的机制。首先，我们证明了新的发现，即 CRT 的敲低降低了两种胃癌细胞系 AGS 和 MKN45 中 VEGF-A mRNA 的稳定性。 AU-Rich 元件 (ARE) 被认为在维持 VEGF-A mRNA 稳定性中发挥着至关重要的作用。荧光素酶报告基因检测显示，CRT 的敲低显着降低了具有 VEGF-A ARE 序列的海肾荧光素酶的活性。此外，RNA 结合/电泳迁移率变动测定的竞争结果表明，CRT 通过与 ARE 结合，与 VEGF-A mRNA 形成 RNA-蛋白质复合物。此外，当用来自CRT敲低细胞的条件培养基处理时，人脐静脉内皮细胞（HUVEC）的增殖率显着降低；这是由外源 VEGF-A 重组蛋白拯救的。我们的结果表明，CRT 参与 VEGF-A ARE 结合蛋白复合物以稳定 VEGF-A mRNA，从而促进血管生成和胃癌的进展。
Calreticulin (CRT) and vascular endothelial growth factor-A (VEGF-A) are crucial for angiogenesis, and mediate multiple malignant behaviors in gastric cancer. In this study, we report that CRT is positively correlated with VEGF-A in gastric cancer patients. Moreover, high expressions of both CRT and VEGF-A are markedly associated with the pathological stage, progression, and poor prognosis in the patients. Therefore, we sought to elucidate the mechanism by which CRT affects VEGF-A in gastric cancer. Firstly, we demonstrate the novel finding that knockdown of CRT reduced VEGF-A mRNA stability in two gastric cancer cell lines, AGS and MKN45. The AU-Rich element (ARE) is believed to play a crucial role in the maintenance of VEGF-A mRNA stability. Luciferase reporter assay shows that knockdown of CRT significantly decreased the activity of renilla luciferase with VEGF-A ARE sequence. Additionally, competition results from RNA-binding/electrophoretic mobility shift assay indicate that CRT forms an RNA-protein complex with the VEGF-A mRNA by binding to the ARE. In addition, the proliferation rate of human umbilical vein endothelial cells (HUVEC) was significantly reduced when treated with conditioned medium from CRT knockdown cells; this was rescued by exogenous VEGF-A recombinant protein. Our results demonstrate that CRT is involved in VEGF-A ARE binding protein complexes to stabilize VEGF-A mRNA, thereby promoting the angiogenesis, and progression of gastric cancer.