通过帕博西尼调节 lncRNA SNHG15/CDK6/miR-627 通路，克服胶质母细胞瘤中替莫唑胺耐药并减少与胶质瘤相关的小胶质细胞 M2 极化。

BACKGROUND: Accumulating evidence demonstrates the oncogenic roles of lncRNA (long non-coding RNA) molecules in a wide variety of cancer types including glioma. Equally important, However, tumorigenic functions of lncRNA in glioma remain largely unclear. A recent study suggested lncRNA SNHG15 played a role for regulating angiogenesis in glioma but its role in the tumor microenvironment (TME) was not investigated. METHODS: First, we showed that SNHG15 was upregulated in GBM cells and associated with a poor prognosis for the patients of GBM using public databases. Next, we collected temozolomide sensitive (TMZ-S) and resistant (TMZ-R) clinical samples and demonstrated that co-culturing TMZ-R cells with HMC3 (microglial) cells promoted M2-polarization of HMC3 and the secretion of pro-GBM cytokines TGF-β and IL-6. RESULTS: Comparative qPCR analysis of TMZ-S and TMZ-R cells showed that a significantly higher level of SNHG15, coincidental with a higher level of Sox2, β-catenin, EGFR, and CDK6 in TMZ-R cells. Subsequently, using bioinformatics tool, a potential mechanistic route for SNHG15 to promote GBM tumorigenesis was by inhibiting tumor suppressor, miR-627-5p which leads to activation of CDK6. Gene-silencing technique was employed to demonstrate that suppression of SNHG15 indeed led to the suppression of GBM tumorigenesis, accompanied by an increase miR-627-5p and decreased its two oncogenic targets, CDK6 and SOX-2. In addition, SNHG15-silenced TMZ-R cells became significantly sensitive towards TMZ treatment and less capable of promoting M2-phenotype in the HMC3 microglial cells. We then evaluated the potential anti-GBM activity of CDK6 inhibitor, palbociclib, using TMZ-R PDX mouse models. Palbociclib treatment significantly reduced tumorigenesis in TMZ-R/HMC3 bearing mice and SNHG15 and CDK6 expression was significantly reduced while miR-627-5p level was increased. Additionally, palbociclib treatment appeared to overcome TMZ resistance as well as reduced M2 markers in HMC3 cells. CONCLUSION: Together, we provided evidence supporting the usage of CDK6 inhibitor for TMZ-resistant GBM cases. Further investigation is warranted for the consideration of clinical trials.

背景：越来越多的证据表明，长链非编码 RNA（lncRNA）分子在包括神经胶质瘤在内的多种癌症类型中具有致癌作用。同样重要的是，lncRNA 在神经胶质瘤中的致瘤功能仍很大程度上不清楚。最近的一项研究表明，lncRNA SNHG15 可以调节神经胶质瘤中的血管生成，但它在肿瘤微环境（TME）中的作用尚未被研究。

方法：首先，我们使用公共数据库显示 SNHG15 在 GBM 细胞中上调，并与 GBM 患者的不良预后相关。接下来，我们收集了替莫唑胺敏感（TMZ-S）和耐药（TMZ-R）临床样本，并证明 TMZ-R 细胞与 HMC3（小胶质细胞）共培养可促进 HMC3 的 M2 极化和促 GBM 细胞因子 TGF-β和 IL-6 的分泌。

结果：TMZ-S 和 TMZ-R 细胞的比较 qPCR 分析显示，SNHG15 水平显著升高，同时 TMZ-R 细胞中 Sox2、β-catenin、EGFR 和 CDK6 水平也升高。随后，通过生物信息学工具，发现 SNHG15 促进神经胶质瘤发生的潜在机制途径是抑制肿瘤抑制因子 miR-627-5p，从而激活 CDK6。基因沉默技术用于证明抑制 SNHG15 确实会抑制神经胶质瘤的发生，同时增加 miR-627-5p 并降低其两个致癌靶标 CDK6 和 SOX-2。此外，沉默 SNHG15 的 TMZ-R 细胞对 TMZ 治疗变得更加敏感，并且在 HMC3 小胶质细胞中促进 M2 表型的能力降低。然后，我们使用 TMZ-R PDX 小鼠模型评估 CDK6 抑制剂 palbociclib 的潜在抗 GBM 活性。Palbociclib 治疗显著降低了携带 TMZ-R/HMC3 的小鼠的肿瘤发生，同时 SNHG15 和 CDK6 的表达明显降低，而 miR-627-5p 的水平升高。此外，Palbociclib 治疗似乎克服了 TMZ 耐药性，并降低了 HMC3 细胞中的 M2 标志物。

结论：综上所述，我们提供了支持使用 CDK6 抑制剂治疗 TMZ 耐药性 GBM 病例的证据。需要进一步研究以考虑临床试验。