Expression Microarray Analysis Reveals Alternative Splicing of LAMA3 and DST Genes in Head and Neck Squamous Cell Carcinoma

PurposePrior studies have demonstrated tumor-specific alternative splicing events in various solid tumor types. The role of alternative splicing in the development and progression of head and neck squamous cell carcinoma (HNSCC) is unclear. Our study queried exon-level expression to implicate splice variants in HNSCC tumors.Experimental DesignWe performed a comparative genome-wide analysis of 44 HNSCC tumors and 25 uvulopalatopharyngoplasty (UPPP) tissue samples at an exon expression level. In our comparison we ranked genes based upon a novel score—the Maximum-Minimum Exon Score (MMES) – designed to predict the likelihood of an alternative splicing event occurring. We validated predicted alternative splicing events using quantitative RT-PCR on an independent cohort.ResultsAfter MMES scoring of 17,422 genes, the top 900 genes with the highest scores underwent additional manual inspection of expression patterns in a graphical analysis. The genes LAMA3, DST, VEGFC, SDHA, RASIP1, and TP63 were selected for further validation studies because of a high frequency of alternative splicing suggested in our graphical analysis, and literature review showing their biological relevance and known splicing patterns. We confirmed TP63 as having dominant expression of the short DeltaNp63 isoform in HNSCC tumor samples, consistent with prior reports. Two of the six genes (LAMA3 and DST) validated by quantitative RT-PCR for tumor-specific alternative splicing events (Student's t test, PConclusionAlternative splicing events of oncologically relevant proteins occur in HNSCC. The number of genes expressing tumor-specific splice variants needs further elucidation, as does the functional significance of selective isoform expression.研究背景：既往研究已在多种实体瘤类型中证实了肿瘤特异性可变剪接（alternative splicing）事件。但可变剪接在头颈部鳞状细胞癌（head and neck squamous cell carcinoma, HNSCC）的发生与进展中所发挥的作用尚不明确。本研究通过分析外显子水平的表达谱，探究头颈部鳞状细胞癌（HNSCC）肿瘤中的剪接变体。 实验设计：我们对外显子表达水平的44例HNSCC肿瘤组织与25例悬雍垂腭咽成形术（uvulopalatopharyngoplasty, UPPP）组织样本开展了全基因组比较分析。本次对比分析基于一种新型评分——最大-最小外显子得分（Maximum-Minimum Exon Score, MMES）——对基因进行排序，该评分旨在预测可变剪接事件发生的概率。我们通过定量RT-PCR（quantitative RT-PCR）在独立队列中对预测得到的可变剪接事件进行了验证。 研究结果：对17422个基因进行MMES评分后，我们对得分最高的前900个基因开展了图形化分析，以人工审阅其表达模式。基于图形化分析显示的高频率可变剪接现象，结合文献报道的生物学相关性与已知剪接模式，我们选取了LAMA3、DST、VEGFC、SDHA、RASIP1及TP63这6个基因进行进一步验证研究。我们证实TP63在HNSCC肿瘤样本中优势表达短亚型DeltaNp63，这与既往研究结果一致。通过定量RT-PCR验证，6个基因中的2个（LAMA3与DST）存在肿瘤特异性可变剪接事件（Student t检验，P[原文信息不全]）。 结论：肿瘤学相关蛋白的可变剪接事件在HNSCC中普遍存在。表达肿瘤特异性剪接变体的基因数量，以及选择性异构体表达的功能意义，均有待进一步阐明。