安徽医科大学学报

目的 探究双皮质素(DCX)在胶质瘤中的作用机制，筛选DCX过表达相关差异基因，为胶质瘤靶向治疗提供新靶点。方法 以大鼠胶质瘤细胞C6为靶细胞，构建LV-DCX-EGFP、LV-Cas9-Puro和LV-Ctrl-EGFP慢病毒，经Cas9双载体系统转染、筛选建立DCX过表达及对照C6细胞系。利用转录组测序获取差异基因表达谱，通过iDEP、DESeq2筛选差异基因(|Log₂(FC)|>2,P<0.05);运用STRING、Cytoscape构建蛋白互作网络并筛选关键基因；借助DAVID进行基因本体(GO)功能富集分析；利用BUSCA及UniProt分析关键基因亚细胞定位；绘制ROC曲线验证关键基因诊断价值；通过Enrichr预测转录因子及miRNA调控网络；采用RT-qPCR验证关键基因表达。结果 筛选得到45个上调、47个下调差异基因，明确POU2F3等6个上调及UBB等5个下调关键基因。GO功能富集显示，上调基因参与脂质生物合成的正调节等过程，下调基因涉及碱性磷酸酶活性调节等功能。关键基因主要分布在细胞核和胞质中。除RPS17外AUC值均>0.7,具有良好的诊断价值。RT-qPCR验证部分上调基因POU2F3、LPAR6、SREBF1 mRNA表达显著升高(P<0.001),部分下调基因UBB、ACTB、UBE2I mRNA表达也显著升高(P<0.001),这种差异可能与转录及转录后调控相关。结论 成功筛选胶质瘤中DCX过表达相关差异基因及其调控网络，为揭示DCX在胶质瘤发展中的作用机制提供理论依据，为潜在治疗策略开发奠定基础。

Objective To investigate the role mechanism of doublecortin(DCX) in glioma and screen DCX overexpression-related differentially expressed genes, so as to provide new targets for glioma targeted therapy. Methods Using rat glioma C6 cells as the target cells, LV-DCX-EGFP, LV-Cas9-Puro, and LV-Ctrl-EGFP lentiviruses were constructed. DCX overexpressing and control C6 cell lines were established through Cas9 dual-vector system transfection and screening. Transcriptome sequencing was used to obtain the differentially expressed gene profiles. iDEP and DESeq2 were applied to screen differentially expressed genes with the threshold of |Log_2FC|>2 and P<0.05. STRING and Cytoscape were utilized to construct protein-protein interaction networks and screen key genes. DAVID was employed for gene ontology(GO) enrichment analysis. BUSCA and UniProt were used to predict the subcellular localization of key genes. Receiver operating characteristic(ROC) curves were plotted to verify the diagnostic value of key genes. Enrichr was adopted to predict the regulatory networks of transcription factors and miRNAs. RT-qPCR was performed to validate the expression of key genes. Results A total of 45 upregulated and 47 downregulated differentially expressed genes were screened, and 6 upregulated key genes including POU2F3 and 5 downregulated key genes including UBB were identified. GO enrichment analysis showed that upregulated genes were enriched in biological processes such as the positive regulation of lipid biosynthesis, whereas downregulated genes were linked to molecular functions including the regulation of alkaline phosphatase activity. Key genes were mainly distributed in the nucleus and cytoplasm. Except for RPS17, all key genes had an AUC value>0.7, indicating good diagnostic value. RT-qPCR validation showed that the mRNA expression levels of some upregulated genes(POU2F3, LPAR6, SREBF1) significantly increased(P<0.001), while the mRNA expression levels of some downregulated genes(UBB, ACTB, UBE2I) also significantly increased(P<0.001), which might be related to transcriptional and post-transcriptional regulation. Conclusion Differentially expressed genes and their regulatory networks related to DCX overexpression in glioma are successfully screened, providing a theoretical basis for revealing the role mechanism of DCX in glioma development and laying a foundation for the development of potential therapeutic strategies.