GBM miR338-p5. Homo sapiens

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors. Despite radical surgery and radiotherapy supported by chemotherapy, the disease still remains incurable with extremely low median survival rate of 12-15 months from the time of initial diagnosis. The main cause of treatment failure is considered to be the presence of cells that are resistant to such treatment. MicroRNAs (miRNAs) as regulators of gene expression are involved in the tumor pathogenesis, including GBM. MiR-338 is a brain specific miRNA which has been described to target pathways involved in proliferation and differentiation. In our study, miR-338-3p and -5p were differentially expressed in GBM tissue in comparison to non-tumor brain tissue. Overexpression of miR-338-3p with miRNA mimic did not show any changes in proliferation rates in GBM cell lines (A172, T98G, U87MG). On the other hand, pre-miR-338-5p notably decreased proliferation and caused cell cycle arrest. Since radiation is currently the main treatment modality in GBM, we combined overexpression of pre-miR-338-5p with radiation, which led to significantly decreased of cell proliferation, and increased cell cycle arrest and apoptosis in comparison to only irradiated cells. To better elucidate the mechanism of action, we performed gene expression profiling analysis that revealed targets of miR-338-5p being Ndfip1, Rheb, ppp2R5a. These genes have been described to be involved in DNA damage response, proliferation and cell cycle regulation. To our knowledge, this is the first study to describe role of miR-338-5p in GBM and its potential to improve sensitivity of GBM to radiation. Overall design: Study was performed on three glioblastoma multiforme cell lines A172, T98G and U87MG. This experiment was performed on Affymetrix GeneChip Human Gene ST 1.0 to elucidate the targets of miRNA-338-5p. Cell lines were seeded 24 hours prior transfection. After transfection with pre-miR338-5p or negative control cell were cultured for 24 hours and harvested. RNA was isolated using MirVana miRNA Isolation Kit (Ambion, USA) and checked for RNA integrity by Bioanalyzer 2100 and purity by ratios 260/280>1.8 and 260/230>1.8 by Nanodrop2000.

多形性胶质母细胞瘤（Glioblastoma multiforme, GBM）是恶性程度最高的脑肿瘤类型。尽管采取根治性手术联合辅助放化疗，该疾病仍无法治愈，初始诊断后的中位生存期仅为12~15个月，预后极差。治疗失败的主要原因被认为是存在对上述治疗产生耐药性的肿瘤细胞。微小RNA（MicroRNAs, miRNAs）作为基因表达的调控因子，参与包括GBM在内的肿瘤发生发展进程。MiR-338是一种脑特异性微小RNA，已有研究证实其可靶向调控细胞增殖与分化相关通路。在本研究中，相较于非肿瘤脑组织，GBM组织中miR-338-3p与miR-338-5p的表达存在显著差异。采用miRNA模拟物过表达miR-338-3p后，GBM细胞系（A172、T98G、U87MG）的增殖速率未出现显著变化。而pre-miR-338-5p则可显著抑制细胞增殖，并诱发细胞周期阻滞。鉴于放疗目前仍是GBM的主要治疗手段，我们将pre-miR-338-5p过表达与放疗联合，结果显示，相较于单纯放疗组，联合组的细胞增殖进一步受到抑制，细胞周期阻滞与细胞凋亡水平均显著升高。为进一步阐明其作用机制，我们开展了基因表达谱分析，结果揭示miR-338-5p的靶基因为Ndfip1、Rheb及ppp2R5a。上述基因已被证实参与DNA损伤应答、细胞增殖及细胞周期调控过程。据我们所知，本研究是首个阐述miR-338-5p在GBM中的作用及其提升GBM放疗敏感性潜力的研究。总体实验设计：本研究采用3株多形性胶质母细胞瘤细胞系A172、T98G及U87MG开展实验。本实验使用Affymetrix GeneChip Human Gene ST 1.0芯片进行基因表达谱分析，以筛选miR-338-5p的靶基因。细胞在转染前24小时进行铺板培养。采用pre-miR338-5p或阴性对照进行转染后，将细胞继续培养24小时后收集。总RNA采用MirVana miRNA Isolation Kit（Ambion公司，美国）提取，通过Bioanalyzer 2100检测RNA完整性，并通过Nanodrop2000检测RNA纯度，要求260/280比值>1.8且260/230比值>1.8。