胶质瘤中BMPs/smad1信号路径的表达及功能研究
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摘要
骨形态发生蛋白(Bone morphogenetic proteins，BMPs)在机体的生长发育以及组织细胞的定向分化过程中具有重要的调节作用，而sma和mad的同源蛋白(sma and mad homologues，smads)是介导BMPs转录调节信号的胞内重要的信号分子。目前研究表明BMPs／smad1信号路径参与了许多肿瘤的发生发展，但是有关其在胶质瘤中的表达及功能作用还知之甚少。
我们研究组利用自行设计合成的、含有218个与神经系统发育相关的寡核苷酸微阵列芯片，检测到Smad1在胶质瘤组织中的表达比正常脑组织明显降低，据此，我们研究组采用60例不同级别胶质瘤组织标本(20例Ⅱ级胶质瘤、20例Ⅲ级胶质瘤、20例胶质母细胞瘤)、20例正常脑组织标本及U251胶质母细胞瘤细胞系，利用Western蛋白印记杂交(Western blot，WB)，反转录聚合酶链反应(Reverse transcriptase-polymerasechainreaction，RT-PCR)，免疫组化等技术研究BMPs／Smad1相关信号分子在不同级别胶质瘤中的表达，结果显示磷酸化-Smad1(phosphorylated-smad1，P-smad1)的蛋白表达随着胶质瘤恶性级别的增高而降低，而其上游信号分子骨形态发生蛋白受体-IB亚单位(Bone morphogenetic proteins receptor-IB subunit，BMPR-IB)mRNA在大部分恶性胶质瘤中低表达，在以上研究基础上我们进一步通过对U251细胞BMPR-IB和Smad1等基因的瞬时转染，观察到过表达BMPR-IB、smad1可活化U251细胞中的smad1及BMPs信号通路并诱导瘤细胞的分化和凋亡，同时，我们应用外源BMP-2刺激U251细胞，观察到BMP-2可以时间依赖方式引起瘤细胞的增殖抑制和分化，并伴有BMPR-IB和P-smad1表达的上调。
上述研究结果提示胶质瘤的恶性进展过程中伴有smad1的磷酸化活化障碍，而其上游信号分子BMPR-IB的低表达是引起smad1活化障碍的因素之一。过表达BMPR-IB、smad1或外源BMP-2刺激均可引起U251细胞中smad1及BMPs信号路径的活化，而BMPs／smad1信号路径的活化可引起胶母细胞瘤细胞系U251的增殖抑制、分化和凋亡。该项研究为胶质瘤的诊断和新的治疗靶点的选择提供了线索和实验依据。
BMPs play an essential role in cell fate determination, and smads proteins play akey role in transforming BMPs signal transduction. Although extensively investigated inmany tumors, smad1-associated pathway has only received limited study in glioma.
In this study, we have examined one glioblastoma cell line-U251 and tissuespeciments of 60 gliomas: 20 astrocytomas(AST), 20 anaplastic astrocytomas(AAST),and 20 gliomblastoma multiform gradeⅣ(GBM).. We found that phospho-smad1decreased in U251 cell line and most malignant glioma tissues, and smad1 decreased inU251 and GBM tissues by Western blotting analysis compared with normal braintissues. Using RT-PCR and Western blot we detected the lower expression of BMPR-IBon most glioma tissues as well as U251 cells. Transient transfection of BMPR-IB,smad1, or both activated smad1 and caused transient apoptosis and differentiation ofU251 cells, accompanied by a significant increase in the activity of BRE-luciferasereporter and the expression levels of phospho-smad1 and GFAP compared withtransfection of empty vector. Treatment with an inhibitor smad protein-smad6significantly inhibited these effects. We then studied the effects of BMP-2 on U251 cells,and found that BMP-2 treatment of U251 cells delayed cell growth and inducedglioblastoma cell differentiation. Of note, we found a significant up-regulation ofBMPR-IB and P-smad1 in response to BMP-2.
Our results suggested that the lower expressions of BMPR-IB and smad1 blockedthe activation of BMPs/smad1 signaling pathway in gliomas, over-expression ofBMPR-IB and smad1 could induce apoptosis and differentiation of U251 cells throughtheir activation of smad1. Our results also suggest that BMP-2 could regulate the growthand differentiation of gliomblastoma by autocrine or paracrine mode. BMPs/smad1signal pathway might provide a new therapeutic strategy for gliomas.
我们研究组利用自行设计合成的、含有218个与神经系统发育相关的寡核苷酸微阵列芯片，检测到Smad1在胶质瘤组织中的表达比正常脑组织明显降低，据此，我们研究组采用60例不同级别胶质瘤组织标本(20例Ⅱ级胶质瘤、20例Ⅲ级胶质瘤、20例胶质母细胞瘤)、20例正常脑组织标本及U251胶质母细胞瘤细胞系，利用Western蛋白印记杂交(Western blot，WB)，反转录聚合酶链反应(Reverse transcriptase-polymerasechainreaction，RT-PCR)，免疫组化等技术研究BMPs／Smad1相关信号分子在不同级别胶质瘤中的表达，结果显示磷酸化-Smad1(phosphorylated-smad1，P-smad1)的蛋白表达随着胶质瘤恶性级别的增高而降低，而其上游信号分子骨形态发生蛋白受体-IB亚单位(Bone morphogenetic proteins receptor-IB subunit，BMPR-IB)mRNA在大部分恶性胶质瘤中低表达，在以上研究基础上我们进一步通过对U251细胞BMPR-IB和Smad1等基因的瞬时转染，观察到过表达BMPR-IB、smad1可活化U251细胞中的smad1及BMPs信号通路并诱导瘤细胞的分化和凋亡，同时，我们应用外源BMP-2刺激U251细胞，观察到BMP-2可以时间依赖方式引起瘤细胞的增殖抑制和分化，并伴有BMPR-IB和P-smad1表达的上调。
上述研究结果提示胶质瘤的恶性进展过程中伴有smad1的磷酸化活化障碍，而其上游信号分子BMPR-IB的低表达是引起smad1活化障碍的因素之一。过表达BMPR-IB、smad1或外源BMP-2刺激均可引起U251细胞中smad1及BMPs信号路径的活化，而BMPs／smad1信号路径的活化可引起胶母细胞瘤细胞系U251的增殖抑制、分化和凋亡。该项研究为胶质瘤的诊断和新的治疗靶点的选择提供了线索和实验依据。
BMPs play an essential role in cell fate determination, and smads proteins play akey role in transforming BMPs signal transduction. Although extensively investigated inmany tumors, smad1-associated pathway has only received limited study in glioma.
In this study, we have examined one glioblastoma cell line-U251 and tissuespeciments of 60 gliomas: 20 astrocytomas(AST), 20 anaplastic astrocytomas(AAST),and 20 gliomblastoma multiform gradeⅣ(GBM).. We found that phospho-smad1decreased in U251 cell line and most malignant glioma tissues, and smad1 decreased inU251 and GBM tissues by Western blotting analysis compared with normal braintissues. Using RT-PCR and Western blot we detected the lower expression of BMPR-IBon most glioma tissues as well as U251 cells. Transient transfection of BMPR-IB,smad1, or both activated smad1 and caused transient apoptosis and differentiation ofU251 cells, accompanied by a significant increase in the activity of BRE-luciferasereporter and the expression levels of phospho-smad1 and GFAP compared withtransfection of empty vector. Treatment with an inhibitor smad protein-smad6significantly inhibited these effects. We then studied the effects of BMP-2 on U251 cells,and found that BMP-2 treatment of U251 cells delayed cell growth and inducedglioblastoma cell differentiation. Of note, we found a significant up-regulation ofBMPR-IB and P-smad1 in response to BMP-2.
Our results suggested that the lower expressions of BMPR-IB and smad1 blockedthe activation of BMPs/smad1 signaling pathway in gliomas, over-expression ofBMPR-IB and smad1 could induce apoptosis and differentiation of U251 cells throughtheir activation of smad1. Our results also suggest that BMP-2 could regulate the growthand differentiation of gliomblastoma by autocrine or paracrine mode. BMPs/smad1signal pathway might provide a new therapeutic strategy for gliomas.
引文
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