The effect of HES6 silencing on the gene expression profiles of the glioblastoma A172 and LN405 cell lines

Malignant glioma is the most common type of primary brain tumor diagnosed annually in 16,000 individuals in the United States. We performed a systematic large-scale transcriptomics data mining study of 9,783 Affymetrix samples from the GeneSapiens database in order to identify those genes that are most glioma-specific as compared to other cancers and normal tissues. We searched for genes that are highly expressed in 322 glioblastoma multiforme tissue samples and 66 anaplastic astrocytomas as compared to 425 samples from histologically normal central nervous system. Transcription cofactor HES6 (Hairy and Enhancer of Split 6) emerged as one of the most glioma-specific genes. In immunostaining of a tissue microarray series, HES6 was expressed in 335 (98.8%) out of the 339 clinical glioma samples. Recurrent grade 2 astrocytomas and grade 2-3 oligodendrogliomas showed higher levels of HES6 immunoreactivity than the corresponding primary tumors. Functional studies implied a critical role for HES6 in supporting survival of glioma cells, as evidenced by 60% reduced cancer cell viability and induction of Caspase 3/7 activity after HES6 silencing by RNA interference in A172 and LN405 cells. The biological role and consequences of HES6 silencing and overexpression was explored with genome-wide analyses, which indicated a key role for HES6 in e.g. p53, c-myc, and NF-?B transcriptional networks. We conclude that HES6 has a critical role in sustaining glioma cell growth, survival, migration and possibly angiogenesis. HES6 is a potential therapeutic target and biomarker for glioma. A172 and LN405 cells (240 000 per well on 6-well plates) were transfected for 12-24 h with siHES6 pool, or three individual siRNAs (HES6_1, HES6_2, HES6_3 from Qiagen) or AllStars negative control siRNA (Qiagen) at 30 nM using SiLentFect (Bio-Rad Laboratories, Hercules, CA). One to two biological replicate transfections were performed. LN405 cells were also used for creating stable cell lines by transfecting either pEYFP-C1-mock or pEYFP-HES6 with Fugene6 (Roche) and selecting the positive cells with 600ug/ml of G418 (Sigma). The cells were kept in culture in the presence of 400ug/ml of G418. Total RNA was isolated with RNeasy (Qiagen) or MiRVana? Total RNA Isolation kit (Ambion). RNA quality was evaluated by using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA).

恶性胶质瘤（Malignant glioma）是美国年确诊病例达16000例的最常见原发性脑肿瘤。本研究针对GeneSapiens数据库中的9783份Affymetrix样本开展系统性大规模转录组数据挖掘分析，旨在筛选出相较于其他癌症及正常组织、胶质瘤特异性最高的基因。我们对比了322份多形性胶质母细胞瘤组织样本、66份间变性星形细胞瘤样本，以及425份组织学正常的中枢神经系统样本，以筛选出在前者中高表达的基因。转录辅因子HES6（Hairy and Enhancer of Split 6）成为最具胶质瘤特异性的基因之一。在组织微阵列系列的免疫染色实验中，339份临床胶质瘤样本里有335份（98.8%）呈现HES6阳性表达。复发的2级星形细胞瘤及2~3级少突胶质细胞瘤的HES6免疫反应性水平高于对应原发肿瘤。功能研究证实HES6在胶质瘤细胞存活中发挥关键作用：在A172与LN405细胞中通过RNA干扰沉默HES6后，癌细胞存活率下降60%，且可诱导Caspase 3/7活性升高。本研究通过全基因组分析探究了HES6沉默与过表达的生物学作用及效应，结果显示HES6在p53、c-myc及NF-κB等转录网络中扮演关键角色。本研究结论表明，HES6在维持胶质瘤细胞增殖、存活、迁移及潜在的血管生成过程中发挥关键作用，可作为胶质瘤潜在的治疗靶点与生物标志物。实验方法如下：将A172与LN405细胞（6孔板每孔接种240000个细胞）以30nM浓度的siHES6混合序列、3条单独的siRNA（购自Qiagen的HES6_1、HES6_2、HES6_3）或AllStars阴性对照siRNA（Qiagen），使用SiLentFect转染试剂（Bio-Rad Laboratories，加利福尼亚州赫拉克勒斯市）转染12~24小时，每组设置1~2次生物学重复转染。此外，以Fugene6转染试剂（罗氏）将pEYFP-C1空载质粒或pEYFP-HES6重组质粒转染LN405细胞，通过600μg/ml G418（Sigma）筛选阳性转染细胞，并以400μg/ml G418维持细胞培养。总RNA提取采用RNeasy总RNA提取试剂盒（Qiagen）或MiRVana™总RNA分离试剂盒（Ambion）。RNA质量通过Agilent 2100生物分析仪（Agilent Technologies，加利福尼亚州帕洛阿尔托市）进行评估。