Verma Brijesh Kumar, Kondaiah Paturu
Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Biological Sciences Building, Bangalore, 560012, India.
J Neurooncol. 2020 Sep;149(2):209-217. doi: 10.1007/s11060-020-03596-4. Epub 2020 Aug 16.
IGFBP2 is one of the highly expressed genes in glioblastoma (GBM). It has both IGF dependent and independent activities. IGF independent actions are mediated by the activation of integrin signalling through its RGD motif present at C-terminal domain. One of the actions of IGFBP2 is to regulate β-catenin by the inactivation of GSK3β, which preferentially accumulates in the cytoplasm. The mechanism of nuclear β-catenin regulation by IGFBP2 and role of cytoplasmic β-catenin is not clear. We aimed to understand the mechanism in GBM cell lines.
The gene expression studies were performed by RT-PCR, western blot analysis; the knockdown of genes was performed by shRNA transfection; RNAIP and luciferase reporter assays were utilized to study the cytoplasmic regulation of genes by β-catenin; neurosphere assays were performed to study the stemness of cells.
IGFBP2 overexpression or treatment in GBM cells regulates β-catenin, TRIM33 (E3 ubiquitin ligase) and Oct4 genes. TRIM33 was induced by IGFBP2. β-catenin was found to accumulate predominantly in the cytoplasm and nuclear β-catenin was depleted by IGFBP2 induced TRIM33. IGFBP2 regulated cytoplasmic β-catenin binds to 3' UTR of Oct4 RNA. IGFBP2 was also able to induce stemness of glioma cells.
IGFBP2 induces TRIM33 which regulates the nuclear β-catenin protein. In addition, IGFBP2 stabilizes the cytoplasmic β-catenin which is involved in the regulation of Oct4 transcript and consequently induction of stemness of glioma cells.
胰岛素样生长因子结合蛋白2（IGFBP2）是胶质母细胞瘤（GBM）中高表达的基因之一。它具有胰岛素样生长因子（IGF）依赖性和非依赖性活性。IGF非依赖性作用是通过其C末端结构域中存在的RGD基序激活整合素信号传导来介导的。IGFBP2的作用之一是通过使糖原合成酶激酶3β（GSK3β）失活来调节β-连环蛋白，β-连环蛋白优先在细胞质中积累。IGFBP2对细胞核β-连环蛋白的调节机制以及细胞质β-连环蛋白的作用尚不清楚。我们旨在了解GBM细胞系中的机制。
通过逆转录聚合酶链反应（RT-PCR）、蛋白质免疫印迹分析进行基因表达研究；通过短发夹RNA（shRNA）转染进行基因敲低；利用RNA免疫沉淀（RNAIP）和荧光素酶报告基因检测来研究β-连环蛋白对基因的细胞质调节；进行神经球检测以研究细胞的干性。
GBM细胞中IGFBP2的过表达或处理可调节β-连环蛋白、TRIM33（E3泛素连接酶）和Oct4基因。TRIM33由IGFBP2诱导。发现β-连环蛋白主要在细胞质中积累，并且IGFBP2诱导的TRIM33使细胞核β-连环蛋白减少。IGFBP2调节的细胞质β-连环蛋白与Oct4 RNA的3'非翻译区（UTR）结合。IGFBP2还能够诱导胶质瘤细胞的干性。
IGFBP2诱导TRIM33，TRIM33调节细胞核β-连环蛋白。此外，IGFBP2使细胞质β-连环蛋白稳定，细胞质β-连环蛋白参与Oct4转录本的调节，从而诱导胶质瘤细胞的干性。
