Department of Pathology, Cancer Hospital Affiliated to Shanxi Medical University/Shanxi Province Cancer Hospital/ Shanxi Hospital Affiliated to Cancer Hospital Chinese Academy of Medical Sciences, Taiyuan, 030013, Shanxi, China.
Department of Neurosurgery, Cancer Hospital Affiliated to Shanxi Medical University/Shanxi Province Cancer Hospital/ Shanxi Hospital Affiliated to Cancer Hospital Chinese Academy of Medical Sciences, Taiyuan, 030013, Shanxi, China.
Mol Biol Rep. 2024 Mar 5;51(1):385. doi: 10.1007/s11033-024-09247-5.
Glioblastoma, a highly aggressive form of brain cancer, poses significant challenges due to its resistance to therapy and high recurrence rates. This study aimed to investigate the expression and functional implications of CDKN2A, a key tumor suppressor gene, in glioblastoma cells, building upon the existing background of knowledge in this field.
Quantitative reverse transcription PCR (qRT-PCR) analysis was performed to evaluate CDKN2A expression in U87 glioblastoma cells compared to normal human astrocytes (NHA). CDKN2A expression levels were manipulated using small interfering RNA (siRNA) and CDKN2A overexpression vector. Cell viability assays and carmustine sensitivity tests were conducted to assess the impact of CDKN2A modulation on glioblastoma cell viability and drug response. Sphere formation assays and western blot analysis were performed to investigate the role of CDKN2A in glioblastoma stem cell (GSC) self-renewal and pluripotency marker expression. Additionally, methylation-specific PCR (MSP) assays and demethylation treatment were employed to elucidate the mechanism of CDKN2A downregulation in U87 cells.
CDKN2A expression was significantly reduced in glioblastoma cells compared to NHA. CDKN2A overexpression resulted in decreased cell viability and enhanced sensitivity to carmustine treatment. CDKN2A inhibition promoted self-renewal capacity and increased pluripotency marker expression in U87 cells. CDKN2A upregulation led to elevated protein levels of p16INK4a, p14ARF, P53, and P21, which are involved in cell cycle regulation. CDKN2A downregulation in U87 cells was associated with high promoter methylation, which was reversed by treatment with a demethylating agent.
Our findings demonstrate that CDKN2A downregulation in glioblastoma cells is associated with decreased cell viability, enhanced drug resistance, increased self-renewal capacity, and altered expression of pluripotency markers. The observed CDKN2A expression changes are mediated by promoter methylation. These results highlight the potential role of CDKN2A as a therapeutic target and prognostic marker in glioblastoma.
胶质母细胞瘤是一种高度侵袭性的脑癌，由于其对治疗的抵抗力和高复发率，给治疗带来了重大挑战。本研究旨在基于该领域的现有知识背景，研究关键肿瘤抑制基因 CDKN2A 在胶质母细胞瘤细胞中的表达及其功能意义。
采用定量逆转录 PCR（qRT-PCR）分析比较 U87 胶质母细胞瘤细胞与正常人类星形胶质细胞（NHA）中 CDKN2A 的表达。采用小干扰 RNA（siRNA）和 CDKN2A 过表达载体对 CDKN2A 表达进行调控。通过细胞活力测定和卡莫司汀敏感性试验评估 CDKN2A 调节对胶质母细胞瘤细胞活力和药物反应的影响。通过球体形成试验和 Western blot 分析研究 CDKN2A 在胶质母细胞瘤干细胞（GSC）自我更新和多能性标志物表达中的作用。此外，采用甲基化特异性 PCR（MSP）分析和去甲基化处理来阐明 U87 细胞中 CDKN2A 下调的机制。
与 NHA 相比，胶质母细胞瘤细胞中 CDKN2A 的表达显著降低。CDKN2A 过表达导致细胞活力降低，并增强了卡莫司汀治疗的敏感性。CDKN2A 抑制促进了 U87 细胞的自我更新能力，并增加了多能性标志物的表达。CDKN2A 的上调导致细胞周期调节相关蛋白 p16INK4a、p14ARF、P53 和 P21 的蛋白水平升高。U87 细胞中 CDKN2A 的下调与高启动子甲基化有关，用去甲基化剂处理可逆转这种下调。
我们的研究结果表明，胶质母细胞瘤细胞中 CDKN2A 的下调与细胞活力降低、药物耐药性增强、自我更新能力增强以及多能性标志物表达改变有关。观察到的 CDKN2A 表达变化是由启动子甲基化介导的。这些结果突出了 CDKN2A 作为胶质母细胞瘤治疗靶点和预后标志物的潜在作用。
