Regulation of CD44E by DARPP-32-dependent activation of SRp20 splicing factor in gastric tumorigenesis.
Regulation of CD44E by DARPP-32-dependent activation of SRp20 splicing factor in gastric tumorigenesis.
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CD44E 是胃癌中经常过度表达的 CD44 变体。胃癌中调节 CD44 剪接和表达的机制仍不清楚。在此，我们研究了 DARPP-32（多巴胺和 cAMP 调节的磷蛋白，Mr 32000）通过调节 CD44 剪接促进肿瘤生长的作用。进行了定量荧光素酶报告基因、定量实时 RT-PCR (qRT-PCR)、蛋白质印迹、免疫共沉淀、泛素化和肿瘤异种移植实验。 Western blot 和 qRT-PCR 结果表明，内源性 DARPP-32 的敲低显着降低了 CD44 V8-V10 (CD44E) 的表达。使用定量剪接荧光素酶报告系统，我们检测到 DARPP-32 过度表达后报告活性显着增加 (p < 0.001)。相反，敲低内源性 DARPP-32 会显着减弱剪接活性 (p < 0.001)。进一步的实验表明，DARPP-32调节SRp20剪接因子的表达，并与其共存于同一蛋白质复合物中。用洋地黄毒苷抑制选择性剪接，然后进行免疫沉淀和免疫印迹，表明 DARPP-32 在调节 SRp20 蛋白稳定性中发挥重要作用。内源性 DARPP-32 的敲除证实了 DARPP-32 调节 SRp20 依赖性 CD44E 剪接。使用肿瘤异种移植小鼠模型，敲除内源性 DARPP-32 显着降低 SRp20 和 CD44E 蛋白水平，同时减少肿瘤生长。这些细胞中 SRp20 表达的重建挽救了肿瘤的生长。此外，我们还证明了人胃原发性肿瘤中 DARPP-32、SRp20 和 CD44E 表达水平频繁共过表达且呈正相关。我们的新发现首次确立了 DARPP-32 在调节胃癌细胞剪接因子中的作用。 DARPP-32-SRp20 轴在调节 CD44E 剪接变体促进胃肿瘤发生方面发挥着关键作用。
CD44E is a frequently overexpressed variant of CD44 in gastric cancer. Mechanisms that regulate CD44 splicing and expression in gastric cancer remain unknown. Herein, we investigated the role of DARPP-32 (dopamine and cAMP-regulated phosphoprotein, Mr 32000) in promoting tumor growth through regulation of CD44 splicing. Quantitative luciferase reporter, quantitative real-time RT-PCR (qRT-PCR), Western blot, co-immunoprecipitation, ubiquitination, and tumor xenograft experiments were performed. Western blot and qRT-PCR results indicated that knockdown of endogenous DARPP-32 markedly reduces expression of CD44 V8-V10 (CD44E). Using a quantitative splicing luciferase reporter system, we detected a significant increase in the reporter activity following DARPP-32 overexpression (p < 0.001). Conversely, knocking down endogenous DARPP-32 significantly attenuated the splicing activity (p < 0.001). Further experiments showed that DARPP-32 regulates the expression of SRp20 splicing factor and co-exists with it in the same protein complex. Inhibition of alternative splicing with digitoxin followed by immunoprecipitation and immunoblotting indicated that DARPP-32 plays an important role in regulating SRp20 protein stability. The knockdown of endogenous DARPP-32 confirmed that DARPP-32 regulates the SRp20-dependent CD44E splicing. Using tumor xenograft mouse model, knocking down endogenous DARPP-32 markedly reduced SRp20 and CD44E protein levels with a decreased tumor growth. The reconstitution of SRp20 expression in these cells rescued tumor growth. In addition, we also demonstrated frequent co-overexpression and positive correlation of DARPP-32, SRp20 and CD44E expression levels in human gastric primary tumors. Our novel findings establish for the first time the role of DARPP-32 in regulating splicing factors in gastric cancer cells. The DARPP-32–SRp20 axis plays a key role in regulating the CD44E splice variant that promotes gastric tumorigenesis.
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影响因子：
3.8
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Mukherjee, Kaushik;Peng, Dunfa;Brifkani, Zaid;Belkhiri, Abbes;Pera, Manuel;Koyama, Tatsuki;Koehler, Elizabeth A. S.;Revetta, Frank L.;Washington, Mary K.;El-Rifai, Wael
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