Department of Neurosurgery, Shengjing Hospital of China Medical University, Shenyang, 110004, China.
Key Laboratory of Neuro-oncology in Liaoning Province, Shenyang, 110004, China.
J Exp Clin Cancer Res. 2022 May 10;41(1):171. doi: 10.1186/s13046-022-02374-6.
RNA-binding proteins (RBPs) and circular RNAs (circRNAs) play important roles in glioblastoma multiforme (GBM). Aerobic glycolysis is a metabolic characteristic of GBM. However, the roles of RBPs and circRNAs in aerobic glycolysis in GBM remain unclear. The aim of this study is to explore the mechanisms by which RBPs and circRNAs regulate aerobic glycolysis in GBM cells.
RNA sequencing and circRNA microarray analysis were performed to identify RBPs and circRNAs for further study. Mass spectrometry validated the encoded protein and its interacting proteins. Quantitative reverse transcription PCR and western blot assays were used to determine the mRNA and protein expression, respectively. Furthermore, immunofluorescence and fluorescence in situ hybridization assays were used to determine the protein and RNA localization, respectively. Glucose and lactate measurement assays, Seahorse XF glycolysis stress assays and cell viability assays were conducted to investigate the effects on glycolysis and proliferation in GBM cells.
We selected zinc finger CCHC-type and RNA-binding motif 1 (ZCRB1) and circRNA HEAT repeat containing 5B (circHEATR5B) as candidates for this study. These genes were expressed at low levels in GBM tissues and cells. Both ZCRB1 and circHEATR5B overexpression suppressed aerobic glycolysis and proliferation in GBM cells. ZCRB1 overexpression promoted the Alu element-mediated formation of circHEATR5B. In addition, circHEATR5B encoded a novel protein HEATR5B-881aa which interacted directly with Jumonji C-domain-containing 5 (JMJD5) and reduced its stability by phosphorylating S361. JMJD5 knockdown increased pyruvate kinase M2 (PKM2) enzymatic activity and suppressed glycolysis and proliferation in GBM cells. Finally, ZCRB1, circHEATR5B and HEATR5B-881aa overexpression inhibited GBM xenograft growth and prolonged the survival time of nude mice.
This study reveals a novel mechanism of regulating aerobic glycolysis and proliferation in GBM cells through the ZCRB1/circHEATR5B/HEATR5B-881aa/JMJD5/PKM2 pathway, which can provide novel strategies and potential targets for GBM therapy.
RNA 结合蛋白（RBPs）和环状 RNA（circRNAs）在多形性胶质母细胞瘤（GBM）中发挥重要作用。有氧糖酵解是 GBM 的一种代谢特征。然而，RBPs 和 circRNAs 在 GBM 有氧糖酵解中的作用尚不清楚。本研究旨在探讨 RBPs 和 circRNAs 调节 GBM 细胞有氧糖酵解的机制。
进行 RNA 测序和 circRNA 微阵列分析，以鉴定进一步研究的 RBPs 和 circRNAs。质谱验证编码蛋白及其相互作用蛋白。定量逆转录 PCR 和 Western blot 检测分别用于检测 mRNA 和蛋白表达。此外，免疫荧光和荧光原位杂交检测分别用于检测蛋白和 RNA 定位。葡萄糖和乳酸测量检测、 Seahorse XF 糖酵解应激检测和细胞活力检测用于研究对 GBM 细胞糖酵解和增殖的影响。
我们选择锌指 CCHC 型和 RNA 结合基序 1（ZCRB1）和环状 RNA HEAT 重复序列 5B（circHEATR5B）作为本研究的候选物。这些基因在 GBM 组织和细胞中的表达水平较低。ZCRB1 和 circHEATR5B 的过表达均抑制 GBM 细胞的有氧糖酵解和增殖。ZCRB1 过表达促进 Alu 元件介导的 circHEATR5B 的形成。此外，circHEATR5B 编码一种新型蛋白 HEATR5B-881aa，它与 JMJD5 直接相互作用，并通过磷酸化 S361 降低其稳定性。JMJD5 敲低增加丙酮酸激酶 M2（PKM2）酶活性并抑制 GBM 细胞的糖酵解和增殖。最后，ZCRB1、circHEATR5B 和 HEATR5B-881aa 的过表达抑制 GBM 异种移植瘤的生长并延长裸鼠的存活时间。
本研究揭示了一种通过 ZCRB1/circHEATR5B/HEATR5B-881aa/JMJD5/PKM2 通路调节 GBM 细胞有氧糖酵解和增殖的新机制，可为 GBM 治疗提供新的策略和潜在靶点。
