我们先前报道了TRPM7通过STAT3调节胶质瘤细胞的干细胞特性。此外，我们证明了FOSL1是TRPM7的反应基因，FOSL1基因作为一个致癌基因促进胶质瘤的增殖和侵袭。在本研究中，我们通过流式细胞术确定了FOSL1对胶质瘤干细胞标记物CD133和ALDH1的影响，并通过极限稀释法（ELDA）确定了其对干细胞活性的维持。为了进一步揭示TRPM7激活FOSL1基因转录以促进胶质瘤干细胞特性的机制，我们构建了一个FOSL1启动子及其GAS突变体，并进行了荧光素酶报告基因实验和ChIP-qPCR实验，使用胶质瘤细胞系和胶质瘤患者源性异种移植瘤。我们在胶质瘤患者脑组织微阵列（TMA）中通过免疫组织化学染色（IHC）检测GSC标记物ALDH1和TRPM7以及FOSL1。我们发现FOSL1敲除会降低GSC标记物CD133和ALDH1的表达，并且FOSL1对于维持胶质瘤细胞中的干细胞活性是必需的。实验还表明，-328至-336和-378至-386 GAS元件的突变明显降低了FOSL1启动子活性。恒定活化的STAT3增加了而优势负性的STAT3降低了FOSL1启动子活性。此外，TRPM7的过表达增强了而TRPM7的沉默减少了FOSL1启动子活性。ChIP-qPCR实验揭示了在经恒定活化的STAT3刺激的胶质瘤细胞的细胞核裂解物中，STAT3能够分别结合到两个GAS元件。我们证明FOSL1的Lys-116残基位于其DNA结合域内，去乙酰化会增加FOSL1的转录活性。我们发现TRPM7、ALDH1和FOSL1蛋白的表达与恶性胶质瘤分级相关，TRPM7蛋白的表达与ALDH1和FOSL1的表达在胶质瘤患者中相关。这些综合结果表明，TRPM7诱导了FOSL1的转录激活，这一过程通过STAT3的作用介导，该机制在胶质瘤干细胞特性中具有重要意义。这些结果表明，类似于GSC标记物ALDH1和TRPM7，FOSL1是胶质瘤患者的一个诊断标记和潜在药物靶点。（c）2023.作者。

We previously reported that TRPM7 regulates glioma cells' stemness through STAT3. In addition, we demonstrated that FOSL1 is a response gene for TRPM7, and the FOSL1 gene serves as an oncogene to promote glioma proliferation and invasion.In the present study, we determined the effects of FOSL1 on glioma stem cell (GSC) markers CD133 and ALDH1 by flow cytometry, and the maintenance of stem cell activity by extreme limiting dilution assays (ELDA). To further gain insight into the mechanism by which TRPM7 activates transcription of the FOSL1 gene to contribute to glioma stemness, we constructed a FOSL1 promoter and its GAS mutants followed by luciferase reporter assays and ChIP-qPCR in a glioma cell line and glioma patient-derived xenoline. We further examined GSC markers ALDH1 and TRPM7 as well as FOSL1 by immunohistochemistry staining (IHC) in brain tissue microarray (TMA) of glioma patients.We revealed that FOSL1 knockdown reduces the expression of GSC markers CD133 and ALDH1, and FOSL1 is required to maintain stem cell activity in glioma cells. The experiments also showed that mutations of - 328 to - 336 and - 378 to - 386 GAS elements markedly reduced FOSL1 promoter activity. Constitutively active STAT3 increased while dominant-negative STAT3 decreased FOSL1 promoter activity. Furthermore, overexpression of TRPM7 enhanced while silencing of TRPM7 reduced FOSL1 promoter activity. ChIP-qPCR assays revealed that STAT3, present in nuclear lysates of glioma cells stimulated by constitutively activated STAT3, can bind to two GAS elements, respectively. We demonstrated that deacetylation of FOSL1 at the Lys-116 residue located within its DNA binding domain led to an increase in FOSL1 transcriptional activity. We found that the expression of TRPM7, ALDH1, and FOSL1 protein is associated with grades of malignant glioma, and TRPM7 protein expression correlates to the expression of ALDH1 and FOSL1 in glioma patients.These combined results demonstrated that TRPM7 induced FOSL1 transcriptional activation, which is mediated by the action of STAT3, a mechanism shown to be important in glioma stemness. These results indicated that FOSL1, similar to GSC markers ALDH1 and TRPM7, is a diagnostic marker and potential drug target for glioma patients.© 2023. The Author(s).