Glioma stem cells (GSCs) are glioma cells with stemness and are responsible for a variety of malignant behaviors of glioma. Evidence has shown that signals from tumor microenvironment (TME) enhance stemness of glioma cells. However, identification of the signaling molecules and underlying mechanisms has not been completely elucidated. Human samples and glioma cell lines were cultured in vitro to determine the effects of adenovirus (ADV) infection by sphere formation, RT-qPCR, western blotting, FACS and immunofluorescence. For in vivo analysis, mouse intracranial tumor model was applied. Bioinformatics analysis, gene knockdown by siRNA, RT-qPCR and western blotting were applied for further mechanistic studies. Infection of patient-derived glioma cells with ADV increases the formation of tumor spheres. ADV infection upregulated stem cell markers and in turn promoted the capacities of self-renewal and multi-lineage differentiation of the infected tumor spheres. These ADV infected tumor spheres had stronger potential to form xenograft tumors in immune-compromised mice. GSCs formation could be promoted by ADV infection via TLR9, because TLR9 was upregulated after ADV infection, and knockdown of TLR9 reduced ADV-induced GSCs. Consistently, MYD88, as well as total STAT3 and phosphorylated (p-)STAT3, were also upregulated in ADV-induced GSCs. Knockdown of MYD88 or pharmaceutical inhibition of STAT3 attenuated stemness of ADV-induced GSCs. Moreover, we found that ADV infection upregulated lncRNA NEAT1. Knockdown of NEAT1 impaired stemness of ADV-induced GSCs. Lastly, HMGB1, a damage associated molecular pattern (DAMP) that triggers TLR signaling, also upregulated stemness markers in glioma cells. ADV, which has been developed as vectors for gene therapy and oncolytic virus, promotes the formation of GSCs via TLR9/NEAT1/STAT3 signaling.

中文翻译：

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腺病毒感染通过TLR9/NEAT1/STAT3通路促进胶质母细胞瘤细胞形成胶质瘤干细胞。

胶质瘤干细胞（GSCs）是具有干性的胶质瘤细胞，负责胶质瘤的多种恶性行为。有证据表明，来自肿瘤微环境 (TME) 的信号增强了神经胶质瘤细胞的干性。然而，信号分子的鉴定和潜在机制尚未完全阐明。体外培养人类样本和神经胶质瘤细胞系，通过球体形成、RT-qPCR、蛋白质印迹、FACS 和免疫荧光来确定腺病毒 (ADV) 感染的影响。对于体内分析，应用了小鼠颅内肿瘤模型。生物信息学分析、siRNA 基因敲低、RT-qPCR 和蛋白质印迹被应用于进一步的机制研究。用 ADV 感染患者来源的神经胶质瘤细胞会增加肿瘤球的形成。ADV 感染上调了干细胞标志物，进而促进了受感染肿瘤球体的自我更新和多向分化能力。这些 ADV 感染的肿瘤球体具有更强的潜力在免疫受损的小鼠中形成异种移植肿瘤。ADV 通过 TLR9 感染可以促进 GSC 的形成，因为 ADV 感染后 TLR9 上调，而 TLR9 的敲低减少了 ADV 诱导的 GSC。一致地，MYD88 以及总 STAT3 和磷酸化 (p-)STAT3 在 ADV 诱导的 GSC 中也上调。MYD88 的敲低或 STAT3 的药物抑制减弱了 ADV 诱导的 GSC 的干性。此外，我们发现 ADV 感染上调了 lncRNA NEAT1。敲除 NEAT1 会损害 ADV 诱导的 GSC 的干性。最后，HMGB1，一种触发 TLR 信号的损伤相关分子模式 (DAMP)，还上调了胶质瘤细胞中的干细胞标记。ADV 已被开发为基因治疗和溶瘤病毒的载体，通过 TLR9/NEAT1/STAT3 信号促进 GSC 的形成。