胚胎干性基因PAX3通过调控Wnt信号通路在神经胶质瘤细胞中发挥癌基因作用的分子机制

Wnt/β-catenin signal pathway plays an important role in the pathogenesis and development of gliomas. As an embryonic stem cell gene, PAX3 plays an important role in the oncogenesis. In the our preliminary study, it has been demonstrated that PAX3 is highly expressed in glioma, and that it promotes proliferation and invasion of glioma cells, and suppresses tumor apoptosis, thereby playing an oncogene-like role. However, the defined molecular mechanism of PAX3 regulating proliferation, invasion and apoptosis of glioma cells remain largely unknown. Furthermore, our previous research has demonstrated that β-catenin expression was dramatically down-regulated after PAX3 expression was suppressed by siRNA; The interactions between PAX3 and β-catenin were detected with co-immunoprecipitation. Therefore we propose a hypothesis: PAX3 plays an oncogene role by regulating the Wnt/β-catenin signaling pathways in glioma cells. In this study, we introduce the related molecular biological techniques in the level of cells, tissues and nude mice, such as gene silencing and plasmid overexpression technique, and aim to investigate the specific molecular mechanism of PAX3 regulating the malignant biological behaviors of glioma via Wnt/β-catenin signal pathway. This study may provide theoretical support and innovative clinical thoughts for the study on pathogenesis and target therapy of glioma.

Wnt信号通路在神经胶质瘤的发生发展过程中发挥重要作用。PAX3作为胚胎干性基因在肿瘤发生发展过程中扮演重要角色。我们的前期研究已证实：PAX3在胶质瘤细胞和组织中高表达，其通过促进胶质瘤细胞增殖和侵袭，抑制凋亡而发挥癌基因作用，然而其发挥癌基因作用的具体分子机制不明确。预实验结果初步证实在胶质瘤U87细胞中PAX3与β-catenin蛋白表达负相关并且存在相互作用。据此我们提出假说：PAX3可能是通过调控Wnt/β-catenin信号通路在胶质瘤细胞中发挥癌基因作用。本课题拟通过基因沉默和过表达技术在细胞、组织和裸鼠水平采用一系列分子生物学实验技术研究PAX3通过介导Wnt信号通路调控神经胶质瘤细胞促增殖和抗凋亡等恶性生物学行为的具体分子机制。通过本项目的研究，阐明PAX3致癌的具体分子机制，为神经胶质瘤的发病机制研究提供实验基础，并为胶质瘤基因靶向治疗提供新的靶标。

前期的研究结果表明PAX3基因在神经胶质瘤细胞中高表达，高表达的PAX3预示着胶质瘤更差的预后，且PAX3可通过促进细胞增殖和侵袭，抑制凋亡在胶质瘤细胞中发挥促癌作用。本研究中，我们通过免疫组化及western blot检测47例胶质瘤标本中PAX3蛋白的表达，培养胶质瘤U87细胞，CO-IP检测 PAX3及β-catenin的相互作用，构建PAX3过表达U87稳转细胞株，检测PAX3改变表达后β-catenin及WNT信号通路下游分子的表达变化，以及胶质瘤细胞增殖，周期及凋亡等生物学行为的变化。.结果：PAX3高表达于高级别胶质瘤且预示着患者更差的预后，PAX3高表达与IDH突变及MGMT甲基化无明确相关性，同时PAX3和β-catenin存在正相关性；在胶质瘤U87细胞中，CO-IP实验证实PAX3和β-catenin存在相互作用。另外，我们发现PAX3过表达可通过改变Wnt信号通路重要分子β-catenin及Myc、VEGF、cyclinD1、MMP7、wnt1的表达从而促进细胞增殖和细胞周期，抑制细胞凋亡。与此同时，我们也证实了PAX3与β-catenin在促进胶质瘤细胞U87增殖和周期，抑制凋亡方面也存在反馈调节机制。我们的实验进一步证实了PAX3在胶质瘤中促进肿瘤生长及进展，其可能是通过与β-catenin分子相互作用从而调节WNT信号通路发挥相关的作用的。.另外，我们发现了一种长链非编码RNA FER1L4在高表达于高级别胶质瘤且其高表达预示着胶质瘤较差的预后，同时LncRNA FER1L4可通过竞争性内源性RNA的机制，吸附miR-372，从而上调E2F1，进而促进胶质瘤细胞周期和细胞增殖行为。受该项目资助，发表SCI论文4篇，接收2篇，发表中文核心期刊2篇，获专利1项。获浙江省医坛新秀人才资助1名，培养硕士研究生1名。