Zhao Zijin, Liu Miaomiao, Long Wenyong, Yuan Jian, Li Haoyu, Zhang Chi, Tang Guodong, Jiang Weixi, Yuan Xianrui, Wu Minghua, Liu Qing
Department of Neurosurgery, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha, 410008, Hunan, China.
Neurosurgical Medical Central, Central South University, Changsha, China.
Cancer Cell Int. 2021 Aug 28;21(1):456. doi: 10.1186/s12935-021-02153-x.
The regulatory roles of long non-coding RNA (lncRNA) CRNDE in temozolomide (TMZ) chemoresistance to glioblastoma multiforme (GBM) are still poorly understood. Therefore, the function, characteristics, and possible mechanism of CRNDE in TMZ-induced chemoresistance to GBM were explored.
Firstly, the expression level of CRNDE in 58 cases of glioma tissue specimens and 30 cases of normal brain tissues were tested by qRT-PCR. Meanwhile, the correlation between CRNDE expression level, the clinicopathological characteristics, and survival time of patients with glioma were analyzed. Then, the CRNDE expression in various glioma cell lines was detected, and CRNDE knockdown cell models were constructed. Subsequently, to explore the effect of CRNDE on chemosensitivity to TMZ, cell viability was detected by the CCK-8 assay and IC values, and cell proliferation was detected by cell clone assay and EdU assay, as well as cell survival was detected by apoptosis with flow cytometry under TMZ treatment. Further, the expression of drug-resistance protein ABCG2, autophagy related proteins, and PI3K/Akt/mTOR pathway were measured by western blot or qRT-PCR in TMZ-treated glioma cells. Finally, the mouse tumor xenograft model was established and the tumor volume and weight were measured, and ABCG2 expression was conducted by immunohistochemistry assay.
The integrated results demonstrated lncRNA CRNDE was a poor prognosis factor for GBM patient, which was upregulated in patients who were resistant to TMZ, and closely associated with chemotherapeutic response status to TMZ treatment. Further, functional assays revealed that knockdown of CRNDE could notably reduce glioma cell viability and proliferation, and elevate cell apoptosis to enhance the chemosensitivity to TMZ in vitro and in vivo. Mechanistically, the depression of CRNDE could diminish the expression of LC3 II/I, Beclin1 and Atg5 and increase the p62 expression level to inhibit autophagy due to the activation of PI3K/Akt/mTOR pathway as well as highly correlated with ABCG2 expression.
Overall, the study provided that lncRNA CRNDE is a reliable clinical predictor of outcome and prognosis and a potential biomarker for predicting TMZ treatment response in GBM by modulating the autophagy through PI3K/Akt/mTOR pathway and ABCG2 expression which may be a novel therapeutic target for regulating TMZ sensitivity to GBM.
长链非编码RNA（lncRNA）CRNDE在多形性胶质母细胞瘤（GBM）对替莫唑胺（TMZ）的化疗耐药中的调控作用仍不清楚。因此，本研究探讨了CRNDE在TMZ诱导的GBM化疗耐药中的功能、特征及可能机制。
首先，采用qRT-PCR检测58例胶质瘤组织标本和30例正常脑组织中CRNDE的表达水平。同时，分析CRNDE表达水平与胶质瘤患者临床病理特征及生存时间的相关性。然后，检测各种胶质瘤细胞系中CRNDE的表达，并构建CRNDE敲低细胞模型。随后，为探讨CRNDE对TMZ化疗敏感性的影响，采用CCK-8法和IC值检测细胞活力，通过细胞克隆实验和EdU实验检测细胞增殖，并在TMZ处理下通过流式细胞术检测细胞凋亡来评估细胞存活情况。此外，采用蛋白质免疫印迹法或qRT-PCR检测TMZ处理的胶质瘤细胞中耐药蛋白ABCG2、自噬相关蛋白以及PI3K/Akt/mTOR信号通路的表达。最后，建立小鼠肿瘤异种移植模型，测量肿瘤体积和重量，并通过免疫组织化学检测ABCG2的表达。
综合结果表明，lncRNA CRNDE是GBM患者预后不良的因素，在对TMZ耐药的患者中上调，且与TMZ治疗的化疗反应状态密切相关。此外，功能实验表明，敲低CRNDE可显著降低胶质瘤细胞活力和增殖，并提高细胞凋亡率，从而增强体外和体内对TMZ的化疗敏感性。机制上，CRNDE的抑制可降低LC3 II/I、Beclin1和Atg5的表达，增加p62表达水平，通过激活PI3K/Akt/mTOR信号通路抑制自噬，且与ABCG2表达高度相关。
总体而言，本研究表明lncRNA CRNDE是GBM患者预后和结局的可靠临床预测指标，通过PI3K/Akt/mTOR信号通路和ABCG2表达调节自噬，可能是预测GBM对TMZ治疗反应的潜在生物标志物，这可能是调节GBM对TMZ敏感性的新治疗靶点。
