受体酪氨酸磷酸酶PTPRU以及丝苏氨酸激酶CDKL5在胶质瘤中的功能研究

发布时间：2018-03-13 21:53

本文选题：PTPRU　切入点：胶质瘤　出处：《华东理工大学》2014年博士论文　论文类型：学位论文

【摘要】：PTPRU(受体型蛋白酪氨酸磷酸酶U)属于蛋白磷酸酶超家族中的R2B亚家族。全长型PTPRU由一个MAM结构域、一个Ig样结构域、三个FNⅢ结构域和两个磷酸酶结构域组成。全长型PTPRU在某些类型的癌细胞中发挥抑癌蛋白的作用。它通过同源粘附作用来促进细胞聚集,抑制细胞扩散,还引起β-catenin酪氨酸的去磷酸化并抑制相应下游信号通路而抑制细胞增殖和粘附。然而癌症(包括恶性胶质瘤)中内源性PTPRU的表达情况和功能仍未知。本研究中,我们发现胶质瘤样本中全长型PTPRU的相对表达水平低于丰富的非全长型PTPRU变体。其中一个变体主要表达于细胞核,在多类癌细胞中普遍表达,且表达水平与胶质瘤的恶性级别正相关。使用小发卡RNA敲减人和大鼠胶质母细胞瘤细胞系中的内源性PTPRU导致细胞体外增殖、存活、细胞周期进程、侵袭、迁移、基质非依赖性粘附和血管生成拟态以及颅内肿瘤生长受到抑制。相应的,敲减PTPRU改变了细胞周期调控、凋亡抑制和细胞运动相关蛋白的表达。胶质母细胞瘤中,全长型和非全长型PTPRU变体都能与β-catenin相互作用。敲减PTPRU下调β-catenin的酪氨酸磷酸化,抑制β-catenin与TCF-4的相互作用、β-catenin/TCF/LEF1复合物的转录活性和多个靶基因的表达。在敲减PTPRU的细胞中表达一个持续激活的LEF1/β-catenin融合蛋白能部分回调β-catenin的转录活性和细胞迁移。另外,敲减PTPRU引起E3泛素连接酶c-Cb1的酪氨酸磷酸化上调,促进c-Cb1与FAK的相互作用和FAK的泛素化,或许因此下调FAK和其他局部粘附蛋白的稳定性。综上所述,我们的发现证明内源性PTPRU通过调控β-catenin和局部粘附信号通路参与胶质瘤的生长和运动,为受体磷酸酶从抑癌蛋白转变为促癌蛋白的机制提供了新的证据。CDKL5(周期蛋白依赖性激酶样5)基因的突变与严重的神经发育障碍有关,如伴随有早发性癫痫和婴儿疫挛的雷特综合征。CDKL5蛋白在神经元中的功能和潜在机制已有广泛的研究。神经母细胞瘤细胞中,CDKL5抑制细胞增殖并促进细胞向神经元方向分化,其转录受到MYCN的抑制。本研究中我们发现,CDKL5在神经元属和非神经元属细胞中的表达情况不同。全长型CDKL5特异性的表达于神经元和神经母细胞瘤细胞中,而一种定位于细胞核的CDKL5变体仅在胶质母细胞瘤细胞中表达而不存在于神经元和胶质细胞中。使用特异性的小发卡RNA敲减CDKL5抑制胶质母细胞瘤细胞的体外增殖、存活、迁移和侵袭,也抑制皮下胶质瘤的生长。在用无血清神经干细胞培养基富集的胶质母细胞瘤干细胞样细胞中,CDKL5高表达。通过肿瘤球形成分析和蛋白免疫印迹检测胶质瘤分化标记物发现,敲减CDKL5后,胶质母细胞瘤细胞的干细胞属性受损并出现多向分化。参与调控细胞命运抉择的表观遗传标志也发生了改变。敲减CDKL5下调H3K9三甲基化和Dnmt1,同时上调H3K4二甲基化、H3K9乙酰化和MeCP2。此外,敲减CDKL5上调U87和A172胶质母细胞瘤细胞中DR5的表达,并促进两细胞系响应TRAIL的刺激而发生胱天蛋白酶依赖性的凋亡,而在DR5表达未上调且Bcl-2显著磷酸化的U251细胞中,未出现凋亡诱导作用。综上所述,CDKL5对于维持胶质母细胞瘤的非分化状态和恶性属性而言很重要。
[Abstract]:PTPRU (protein tyrosine phosphatase U) belongs to the R2B subfamily protein phosphatase superfamily. The full-length PTPRU consists of a MAM domain, a Ig like domain, three FN domains and two phosphatase domains. The full-length PTPRU tumor suppressor protein play a role in certain types of in cancer cells. It through homologous adhesion to promote cell aggregation, inhibition of cell proliferation, but also caused by beta -catenin tyrosine phosphorylation and inhibition of corresponding downstream signaling pathways and inhibit the proliferation and adhesion of cells. However, cancer (including malignant glioma) expression and function of endogenous PTPRU is still unknown. In the present study. We found that the glioma samples the relative expression level of PTPRU is lower than the non full-length full-length PTPRU variant rich. One variant was mainly expressed in the nucleus, is generally expressed in many types of cancer cells, and the expression level with glue The tumor malignant degree of positive correlation. The use of small hairpin RNA on endogenous PTPRU glioblastoma cell lines and human rat leads to cell proliferation, survival, cell cycle progression, invasion, migration, matrix dependent adhesion and vasculogenic mimicry and intracranial tumor growth was inhibited. Accordingly, knockdown PTPRU changed the cell cycle regulation, expression of apoptosis inhibition and cell motility related proteins. Glioblastoma, full-length and non full-length PTPRU variant can interact with beta -catenin. On tyrosine phosphorylation of minus PTPRU downregulation of -catenin, -catenin and TCF-4 inhibited the interaction of beta, beta -catenin/TCF/LEF1 transcription activity complex and multiple target genes. The expression of a sustained activation of LEF1/ beta -catenin fusion protein can be part of the transcription activity of -catenin beta callback and cell migration in PTPRU knockdown cells. Outside, knockdown of PTPRU induced tyrosine phosphorylation upregulation of E3 ubiquitin ligase c-Cb1, promote the interaction between FAK and c-Cb1 and FAK ubiquitination, so the stability may be down-regulation of FAK and other focal adhesion proteins. In conclusion, our findings demonstrate that endogenous PTPRU modulates beta -catenin and focal adhesion signaling pathways involved in glioma growth and for the movement, from the transformation of tumor suppressor protein phosphatase receptor mechanism for cancer promoting proteins provides new evidence of.CDKL5 (cyclin dependent kinase 5) mutation and severe neurodevelopmental disorder related genes, such as with extensive studies with early-onset epilepsy and Rett disease with baby syndrome.CDKL5 protein in neurons the function and potential mechanism. Neuroblastoma cells, inhibition of CDKL5 and promote the differentiation of cells into neurons cell proliferation, the inhibition of MYCN transcription by this research. In the study we found that the expression of CDKL5 in neurons and non neuronal cells were different. The full-length type specific expression of CDKL5 in neuron and neuroblastoma cells, and a localized in the nucleus of CDKL5 variant in glioblastoma cell expression but not in neurons and glial cells. The proliferation of small hairpin RNA specific knockdown of CDKL5 inhibited glioblastoma cell survival in vitro, migration and invasion, also inhibited the growth of glioma. Subcutaneous in serum-free neural stem cell culture medium enriched glioblastoma stem like cells, high expression of CDKL5. Found by tumor sphere the formation of Western blotting and detection of glioma differentiation marker, knockdown of CDKL5, glioblastoma stem cells and multipotent differentiation. The damaged property involved in the regulation of cell fate of the epigenetic Mark has also changed. Knockdown of CDKL5 by H3K9 methylation and Dnmt1 methylation, and up regulation of H3K4 two, H3K9 acetylation and MeCP2. expression by CDKL5 in addition, knock up regulation of U87 and A172 glioblastoma cell DR5, apoptosis and promote the response of TRAIL stimulated cell lines two and caspase dependent the expression of DR5 in Bcl-2 was not up-regulated and phosphorylation in U251 cells did not appear apoptosis. In summary, CDKL5 is very important for the maintenance of glioblastoma in an undifferentiated state and malignant properties.

【学位授予单位】：华东理工大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.41

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