Contribution of microRNA-1275 to Claudin11 suppression via polycomb-mediated silencing mechanism in human glioma stem-like cells

Glioblastomas show heterogeneous histological features, in which tumor cells show distinct phenotypic states that confer different functional attributes. This functional and morphological heterogeneity characterizes aggressive glioblastoma; however, molecular mechanisms underlying the heterogeneity are poorly understood. Glioma stem-like cells (GSCs) are considered able to aberrantly differentiate into diverse cell types and may contribute the establishment of tumor heterogeneity. Using the GSC model, we investigated the differentially expressed microRNAs(miRNAs) and associated epigenetic mechanism that regulate the differentiation of GSCs. MiRNA-microarray showed that 13 and 34 miRNAs were commonly upregulated and downregulated in two independent GSC lines during differentiation, respectively. Among those miRNAs, quantitative-PCR analysis showed that miR-1275 was consistently downregulated during the GSC differentiation along with the upregulation of its target, CLDN11, a marker of oligodendroglial-lineage differentiation. Compellingly, inhibition of miR-1275 with specific antisense oligonucleotide (anti-miR-1275) in GSC increased the expression of CLDN11, together with significant growth suppression. Epigenetic analysis revealed that gain of histone H3 lysine 27 trimethylation (H3K27me3) and loss of H3K9Ac in the pri-miR-1275 promoter were closely associated with the miR-1275 expression. Treatment of 3-Dezaneplanocin-A, an inhibitor of H3K27 methyltransferase, impaired the GSC differentiation in parallel with sustained miR-1275 expression. Our results illuminated the epigenetic regulatory pathways of miR-1275 closely associated with oligodendroglial differentiation, which may contribute to the tissue heterogeneity formation of glioblastomas. Inhibition of miR-1275 induces the GSC differentiation and suppresses tumor cell proliferation, miR-1275 may be a potential therapeutic target for glioblastomas. Human miRNA Microarray V3 kits (G4470C; Agilent Technologies, Santa Clara, California) were used according to the manufacturer’s protocols. This microarray system contains probes for all 866 human and 89 human viral miRNAs reported from the Sanger database v12.0 (http://microrna.sanger.ac.uk/sequences/). Each miRNA species is printed 20 times with replicate probes on the array. Total RNA was isolated with TRIzol reagent (Invitrogen).　One hundred ng of total RNA was labeled with pCp-Cy3 (Agilent Technologies) and 15 units of T4 RNA ligase (GE Healthcare, Little Chalfont, Buckinghamshire, UK) at 16°C for 2 hours. Labeled samples were purified with MicroBio-Spin 6 columns (Bio-Rad, Richmond, CA) and hybridized to microarray at 55 °C with rotation at 20 rpm for 20 hours. Microarrays were scanned by an Agilent Scanner (Agilent Scan Control 7.0 software) and analyzed using Agilent Feature Extraction 10.7 software for miRNA microarray.

胶质母细胞瘤（Glioblastomas）具有异质性组织学特征，其肿瘤细胞呈现各异的表型状态，进而赋予不同的功能属性。这种功能与形态异质性是侵袭性胶质母细胞瘤的典型特征，但该异质性背后的分子机制仍不甚明晰。胶质瘤干细胞样细胞（Glioma stem-like cells, GSCs）被认为可异常分化为多种细胞类型，或参与肿瘤异质性的形成。本研究采用GSC模型，探究了调控GSC分化的差异表达微小RNA（microRNAs, miRNAs）及其相关表观遗传机制。miRNA微阵列分析显示，在两个独立的GSC系分化过程中，分别有13种和34种miRNA呈现普遍上调和下调。其中，实时定量PCR分析表明，miR-1275在GSC分化过程中持续下调，同时其靶基因CLDN11——少突胶质细胞谱系分化标志物——的表达呈上调趋势。值得注意的是，在GSC中使用特异性反义寡核苷酸（anti-miR-1275）抑制miR-1275后，CLDN11的表达水平升高，同时肿瘤细胞增殖受到显著抑制。表观遗传分析发现，pri-miR-1275启动子区域的组蛋白H3赖氨酸27三甲基化（H3K27me3）水平升高，以及H3赖氨酸9乙酰化（H3K9Ac）水平降低，与miR-1275的表达密切相关。用H3K27甲基转移酶抑制剂3-去氮腺嘌呤核苷（3-Dezaneplanocin-A）处理GSC后，其分化过程受到损害，同时miR-1275的表达持续维持于较高水平。本研究结果阐明了与少突胶质细胞分化密切相关的miR-1275表观遗传调控通路，该通路或参与胶质母细胞瘤的组织异质性形成。抑制miR-1275可诱导GSC分化并抑制肿瘤细胞增殖，提示miR-1275有望成为胶质母细胞瘤的潜在治疗靶点。
本研究按照厂商操作流程，使用人类miRNA微阵列V3试剂盒（G4470C；安捷伦科技公司，加利福尼亚州圣克拉拉）完成实验。该微阵列系统包含Sanger数据库v12.0版本中收录的全部866种人类miRNA以及89种人类病毒miRNA的探针，阵列上每种miRNA均设置20次重复探针打印。总RNA采用TRIzol试剂（Invitrogen）提取。取100 ng总RNA，使用pCp-Cy3（安捷伦科技）与15单位T4 RNA连接酶（通用电气医疗集团，英国白金汉郡利特尔查尔方特）于16℃下标记2小时。标记后的样品采用MicroBio-Spin 6层析柱（伯乐公司，加利福尼亚州里士满）纯化，随后于55℃、20 rpm旋转条件下与微阵列杂交20小时。微阵列扫描采用安捷伦扫描仪（配套Agilent Scan Control 7.0软件），数据分析使用安捷伦Feature Extraction 10.7软件完成miRNA微阵列分析。