Circadian Period 2 (Per2) downregulate inhibitor of differentiation 3 (Id3) expression via PTEN/AKT/Smad5 axis to inhibits glioma cell proliferation

In this study, we employed multiple laboratory techniques to acknowledge the biological activities and processes of Per2 and Id3 in glioma. We analyzed TCGA and CGGA databases for seeking association among Per2, Id3, and clinical features in glioma. Immunohistochemistry and Western blot were used to detect protein expression levels. CCK-8 assay, colony formation assay, Transwell assay, the wound healing assay, flow cytometric, and Xenograft nude mice were used to acknowledge the impact of Per2 and Id3 on biological behavior of glioma. The results showed that the Per2 mRNA expression was negatively correlated with the WHO grade, while the Id3 mRNA expression was positively correlated with the WHO grade in patients with glioma in TCGA and CGGA databases. Per2 and Id3 maintained separate prognostic abilities and had a negative connection in human glioma. In the clinical sample study, Per2 and Id3 were validated at the protein level with the same results compared to the mRNA expression level in TCGA and CGGA. By using a wide range of functional examples, overexpression of Per2 restrains malignant biological behaviors in glioma cells by many ways, while Id3 promotes malignant biological behaviors in glioma cells. Furthermore, overexpression of Per2 can inhibit Id3 expression via regulating PTEN/AKT/Smad5 signaling pathway and thereby abolish malignant biological behaviors that are caused by Id3 overexpression. These results suggested that Per2 inhibits glioma cell proliferation through regulating PTEN/AKT/Smad5/Id3 signaling pathway, which may be a viable therapeutic target for glioma.本研究采用多种实验室技术，探究胶质瘤中Per2与Id3的生物学活性及过程。我们通过分析癌症基因组图谱（The Cancer Genome Atlas, TCGA）与中国胶质瘤基因组图谱（Chinese Glioma Genome Atlas, CGGA）数据库，探究胶质瘤中Per2、Id3与临床特征之间的关联；采用免疫组化（immunohistochemistry）与蛋白质免疫印迹（Western blot）检测蛋白表达水平；运用CCK-8检测法（CCK-8 assay）、集落形成实验（colony formation assay）、Transwell实验（Transwell assay）、划痕愈合实验（wound healing assay）、流式细胞术（flow cytometry）以及裸鼠异种移植模型（Xenograft nude mice），阐明Per2与Id3对胶质瘤生物学行为的影响。研究结果显示，在TCGA与CGGA数据库的胶质瘤患者中，Per2 mRNA表达水平与WHO分级呈负相关，而Id3 mRNA表达水平则与WHO分级呈正相关；Per2与Id3各自具备独立的预后价值，且二者在人脑胶质瘤中呈负相关关系。在临床样本研究中，我们在蛋白水平验证了上述结论，其结果与TCGA、CGGA数据库中的mRNA表达结果一致。通过多类功能实验证实，过表达Per2可通过多种途径抑制胶质瘤细胞的恶性生物学行为，而Id3则可促进胶质瘤细胞的恶性生物学行为。此外，过表达Per2可通过调控PTEN/AKT/Smad5信号通路（PTEN/AKT/Smad5 signaling pathway）抑制Id3的表达，从而抵消Id3过表达所诱导的恶性生物学行为。上述结果表明，Per2可通过调控PTEN/AKT/Smad5/Id3信号通路抑制胶质瘤细胞增殖，有望成为胶质瘤潜在的治疗靶点。