Table_1_Cycling Quiescence in Temozolomide Resistant Glioblastoma Cells Is Partly Explained by microRNA-93 and -193-Mediated Decrease of Cyclin D.XLS

Glioblastoma multiforme (GBM) is a fatal malignancy of the central nervous system, commonly associated with chemoresistance. The alkylating agent Temozolomide (TMZ) is the front-line chemotherapeutic agent and has undergone intense studies on resistance. These studies reported on mismatch repair gene upregulation, ABC-targeted drug efflux, and cell cycle alterations. The mechanism by which TMZ induces cell cycle arrest has not been well-established. TMZ-resistant GBM cells have been linked to microRNA (miRNA) and exosomes. A cell cycle miRNA array identified distinct miRNAs only in exosomes from TMZ-resistant GBM cell lines and primary spheres. We narrowed the miRs to miR-93 and -193 and showed in computational analyses that they could target Cyclin D1. Since Cyclin D1 is a major regulator of cell cycle progression, we performed cause-effect studies and showed a blunting effects of miR-93 and -193 in Cyclin D1 expression. These two miRs also decreased cell cycling quiescence and induced resistance to TMZ. Taken together, our data provide a mechanism by which GBM cells can exhibit TMZ-induced resistance through miRNA targeting of Cyclin D1. The data provide a number of therapeutic approaches to reverse chemoresistance at the miRNA, exosomal and cell cycle points.

多形性胶质母细胞瘤（Glioblastoma multiforme, GBM）是一类致死性中枢神经系统恶性肿瘤，常与化疗耐药密切相关。烷化剂替莫唑胺（Temozolomide, TMZ）是其一线化疗药物，针对其耐药性的相关研究已得到广泛开展。既往研究已报道错配修复基因上调、靶向ATP结合盒（ATP-binding cassette, ABC）转运蛋白的药物外排以及细胞周期改变等耐药机制，但TMZ诱导细胞周期阻滞的具体机制尚未完全阐明。现有研究已将TMZ耐药的GBM细胞与微小RNA（microRNA, miRNA）及外泌体（exosomes）建立了关联。本研究通过细胞周期miRNA芯片分析，仅在TMZ耐药GBM细胞系及原代肿瘤球的外泌体中鉴定到特征性微小RNA。我们将候选微小RNA筛选至miR-93与miR-193，并通过计算分析证实二者可靶向调控细胞周期蛋白D1（Cyclin D1）。鉴于细胞周期蛋白D1是细胞周期进程的核心调控因子，我们开展了因果验证实验，结果显示miR-93与miR-193可抑制细胞周期蛋白D1的表达。此外，这两种微小RNA还可降低细胞周期静息状态，诱导细胞对TMZ产生耐药性。综上，本研究数据阐明了GBM细胞可通过微小RNA靶向调控细胞周期蛋白D1，进而产生TMZ诱导的化疗耐药的具体机制。本研究同时提供了多种可从微小RNA、外泌体及细胞周期层面逆转肿瘤化疗耐药的治疗策略。