Chen Hanchun, Lu Qiong, Chen Chao, Di Yunhai, Li Ya'Nan, Min Weijie, Yu Zhengquan, Dai Dongwei
Department of Neurosurgery, Suzhou Kowloon Hospital, Shanghai Jiaotong University School of Medicine, Suzhou, Jiangsu 215021, P.R. China.
Department of Laboratory Medicine, Changhai Hospital, Second Military Medical University, Shanghai 200433, P.R. China.
Exp Ther Med. 2019 Aug;18(2):1285-1290. doi: 10.3892/etm.2019.7680. Epub 2019 Jun 18.
Mutations of the β-catenin gene are common in various cancer types. MicroRNA (miR)-24 suppresses gene expression during the cell cycle. However, the effects of miR-24 on the cell viability and autophagy of glioma cells, and how these biological processes are regulated by β-catenin are largely unclear. The current study aimed to investigate the role of β-catenin in regulating the effects of miR-24 on the cell viability and autophagy of glioma cells. The expression levels of microtubule-associated proteins 1A/1B light chain 3B (LC3B) and Beclin1 were detected by immunohistochemistry and western blotting. Glioma C6 cells were transfected with miR-24 mimics, miR-24 inhibitors and negative control miRNAs. C6 cells transfected with miR-24 mimics or negative control miRNAs were treated with the β-catenin inhibitor, XAV-939. An MTT assay was utilized to evaluate the viability of C6 cells. The expression of miR-24 and mRNA expression of autophagy related 4a cysteine peptidase (ATG4A) were detected by quantitative polymerase chain reaction analysis. The protein expression of LC3B and Beclin1 decreased significantly in glioma tissue and glioma C6 cells compared with normal brain tissue. Compared with the negative control group, C6 cells transfected with miR-24 mimics exhibited significantly higher cell viability at 24 and 48 h, and those transfected with miR-24 inhibitors exhibited significantly lower cell viability at 48 h. XAV-939 decreased the stimulatory effects of miR-24 mimics on the viability of C6 cells. The expression of miR-24 significantly decreased and ATG4A mRNA significantly increased in C6 cells transfected with XAV-939 compared with those transfected with the negative control miRNA. XAV-939 attenuated the miR-24-induced decrease of the protein expression of LC3B and Beclin1, and decreased the stimulatory effects of miR-24 mimics on cell viability. In addition, XAV-939 attenuated the miR-24-induced decrease of autophagy marker expression by attenuating miR-24 expression and increasing ATG4A mRNA expression in glioma C6 cells. To the best of our knowledge, the present study is the first to demonstrate whether β-catenin regulates the intracellular effects of miR-24 on the viability and autophagy of glioma cells. The results also provide some mechanistic basis to the pharmaceutical targeting of WNT signaling in high grade glial tumors.
β-连环蛋白基因的突变在多种癌症类型中很常见。微小RNA（miR）-24在细胞周期中抑制基因表达。然而，miR-24对胶质瘤细胞的细胞活力和自噬的影响，以及这些生物学过程如何受β-连环蛋白调控，目前尚不清楚。本研究旨在探讨β-连环蛋白在调节miR-24对胶质瘤细胞的细胞活力和自噬影响中的作用。通过免疫组织化学和蛋白质印迹法检测微管相关蛋白1A/1B轻链3B（LC3B）和Beclin1的表达水平。用miR-24模拟物、miR-24抑制剂和阴性对照微小RNA转染胶质瘤C6细胞。用β-连环蛋白抑制剂XAV-939处理转染了miR-24模拟物或阴性对照微小RNA的C6细胞。采用MTT法评估C6细胞的活力。通过定量聚合酶链反应分析检测miR-24的表达和自噬相关4a半胱氨酸蛋白酶（ATG4A）的mRNA表达。与正常脑组织相比，胶质瘤组织和胶质瘤C6细胞中LC3B和Beclin1的蛋白表达显著降低。与阴性对照组相比，转染miR-24模拟物的C6细胞在24小时和48小时时细胞活力显著更高，而转染miR-24抑制剂的C6细胞在48小时时细胞活力显著更低。XAV-939降低了miR-24模拟物对C6细胞活力的刺激作用。与转染阴性对照微小RNA的C6细胞相比，转染XAV-939的C6细胞中miR-24的表达显著降低，ATG4A mRNA显著增加。XAV-939减弱了miR-24诱导的LC3B和Beclin1蛋白表达的降低，并降低了miR-24模拟物对细胞活力的刺激作用。此外，XAV-939通过减弱胶质瘤C6细胞中miR-24的表达并增加ATG4A mRNA的表达，减弱了miR-24诱导的自噬标志物表达的降低。据我们所知，本研究首次证明了β-连环蛋白是否调节miR-24对胶质瘤细胞活力和自噬的细胞内效应。这些结果也为高级别胶质瘤中WNT信号通路的药物靶向提供了一些机制基础。
