hSETD1A cooperates with beta-catenin to regulate Wnt target genes and control colorectal tumor growth

Glioblastoma multiforme (GBM) is the most prevalent type of adult brain tumor, and one of the deadliest tumors known to mankind. The genetic understanding of GBM is, however, limited, and the molecular mechanisms which facilitate GBM cell survival and growth within the tumor microenvironment are largely unknown. We applied digital karyotyping and single nucleotide polymorphism (SNP) arrays to screen for copy number changes in GBM samples and found that the most frequently amplified region is at chromosome 7p11.2. The high resolution of digital karyotyping and SNP arrays permits the precise delineation of amplicon boundaries and has enabled identification of the minimal region of amplification at 7p11.2, which contains two genes, EGFR and SEC61γ. SEC61γ encodes a subunit of a heterotrimeric protein channel located in the endoplasmic reticulum (ER). In addition to its high frequency of gene amplification in GBMs, SEC61γ is also remarkably overexpressed in 77% of GBMs, but not in lower-grade gliomas. The siRNA-mediated knockdown of SEC61γ expression in tumor cells led to growth suppression and apoptosis. Furthermore, we showed that pharmacological ER stress agents induce SEC61γ expression in GBM cells. Together, these results indicate that aberrant expression of SEC61γ serves significant roles in GBM cell survival, likely via a mechanism that is involved in the cytoprotective ER stress-adaptive response to the tumor microenvironment. hSETD1A was silenced in HCT116 cells using retrovirus harboring shRNA specifically against the hSETD1A gene. 10μg RNA was reverse transcribed to cDNA using the Applied Biosystems High Capacity cDNA Reverse Transcription kit according to the manufacturer’s instructions (Applied Biosystems). cDNA products were treated with 100ng RNaseA and then purified using the Qiagen PCR purification kit according to the manufacturer’s instructions. NimbleGen Human Gene Expression array was purchased from Roche Applied Sciences. cDNA samples were labeled, Cy3 hybridized, and processed at the FSU NimbleGen Microarray Facility at Florida State University.

多形性胶质母细胞瘤（Glioblastoma multiforme, GBM）是成人颅内最常见的肿瘤类型，亦是目前已知致死性最强的肿瘤之一。然而，学界对GBM的遗传学认知仍存在局限，关于肿瘤微环境中促进GBM细胞存活与增殖的分子机制，目前仍不明晰。本研究采用数字核型分析与单核苷酸多态性（single nucleotide polymorphism, SNP）芯片技术，对GBM样本的拷贝数变异进行筛选，发现最频发的扩增区域位于7号染色体短臂1区1带2亚区（7p11.2）。数字核型分析与SNP芯片的高分辨率特性可精准界定扩增子的边界，并成功确定了7p11.2区域内的最小扩增片段，该片段包含EGFR与SEC61γ两个基因。SEC61γ编码定位于内质网（endoplasmic reticulum, ER）的异源三聚体蛋白通道的一个亚基。除在GBM中高频发生基因扩增外，SEC61γ在77%的GBM样本中亦存在显著过表达，而在低级别胶质瘤中则无此表达特征。通过小干扰RNA（small interfering RNA, siRNA）介导的SEC61γ基因敲低，可抑制肿瘤细胞增殖并诱导其凋亡。此外，本研究证实，药理学内质网应激试剂可诱导GBM细胞中SEC61γ的表达。综上，上述结果表明，SEC61γ的异常表达在GBM细胞存活中发挥关键作用，其潜在机制可能与肿瘤微环境下细胞的保护性内质网应激适应性应答通路相关。本研究使用携带有针对hSETD1A基因的特异性短发夹RNA（short hairpin RNA, shRNA）的逆转录病毒，在HCT116细胞中沉默hSETD1A的表达。按照制造商操作说明，使用Applied Biosystems High Capacity cDNA Reverse Transcription试剂盒，将10μg RNA逆转录为cDNA。cDNA产物经100ng RNaseA处理后，依照Qiagen PCR纯化试剂盒的说明书进行纯化。NimbleGen人类基因表达芯片购自Roche Applied Sciences。cDNA样本经标记、Cy3杂交后，于佛罗里达州立大学的FSU NimbleGen微阵列核心实验室完成后续处理。