Targeting miR-381-NEFL axis sensitizes glioblastoma cells to temozolomide by regulating stemness factors and multidrug resistance factors

// Zeyou Wang 1,2 , Jing Yang 2 , Gang Xu 2 , Wei Wang 2 , Changhong Liu 2 , Honghui Yang 2 , Zhibin Yu 2 , Qianqian Lei 2 , Lan Xiao 2 , Jing Xiong 2,3 , Liang Zeng 1 , Juanjuan Xiang 1,2 , Jian Ma 1,2 , Guiyuan Li 1,2 and Minghua Wu 1,2 1 Hunan Provincial Tumor Hospital and the Affiliated Tumor Hospital of Xiangya Medical School, Central South University, Changsha, Hunan, China 2 Cancer Research Institute, Central South University, Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Key Laboratory of Carcinogenesis, Ministry of Health, Changsha, Hunan, China 3 Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha, Hunan, China Correspondence: Minghua Wu, email: // Guiyuan Li, email: // Keywords : miRNA, neurofilament light polypeptide, multidrug resistance factor, chemosensitivity, stemness factor Received : August 31, 2014 Accepted : December 12, 2014 Published : December 18, 2014 Abstract MicroRNA-381 (miR-381) is a highly expressed onco-miRNA that is involved in malignant progression and has been suggested to be a good target for glioblastoma multiforme (GBM) therapy. In this study, we employed two-dimensional fluorescence differential gel electrophoresis (2-D DIGE) and MALDI–TOF/TOF-MS/MS to identify 27 differentially expressed proteins, including the significantly upregulated neurofilament light polypeptide (NEFL), in glioblastoma cells in which miR-381 expression was inhibited. We identified NEFL as a novel target molecule of miR-381 and a tumor suppressor gene. In human astrocytoma clinical specimens, NEFL was downregulated with increased levels of miR-381 expression. Either suppressing miR-381 or enforcing NEFL expression dramatically sensitized glioblastoma cells to temozolomide (TMZ), a promising chemotherapeutic agent for treating GBMs. The mechanism by which these cells were sensitized to TMZ was investigated by inhibiting various multidrug resistance factors (ABCG2, ABCC3, and ABCC5) and stemness factors (ALDH1, CD44, CKIT, KLF4, Nanog, Nestin, and SOX2). Our results further demonstrated that miR-381 overexpression reversed the viability of U251 cells exhibiting NEFL-mediated TMZ sensitivity. In addition, NEFL-siRNA also reversed the proliferation rate of U251 cells exhibiting locked nucleic acid (LNA)-anti-miR-381-mediated TMZ sensitivity. Overall, the miR-381-NEFL axis is important for TMZ resistance in GBM and may potentially serve as a novel therapeutic target for glioma.