整合染色體免疫沉澱定序與基因表現分析揭露PAICS在神經母細胞瘤中為MYCN重要的下游調控基因

Abstract

神經母細胞瘤 (Neuroblastoma : 簡稱NBL或NB) 是一種兒童癌症，其源發於胚胎神經脊當中的交感神經系統，臨床期數越高則越難被治癒。在臨床特徵中MYCN致癌基因異常放大是評估神經母細胞瘤不良預後的重要指標，且MYCN在腫瘤學中也被強調其會透過大量轉錄調控使細胞不正常增生。為了探究MYCN所調控的基因在神經母細胞瘤的進程中扮演的重要性，我們首先在大量表現MYCN的SK-N-BE (2) –C細胞株中，利用染色質免疫沉澱定序 (ChIP-Seq) 分析MYCN結合的啟動子區域，再從基因表達綜合數據庫 (Gene Expression Omnibus, GEO)中整合神經母細胞瘤患者的基因數據，並找到那些會和MYCN大量表現有高度相關的基因。結合ChIP-Seq和GEO分析結果，我們選擇了PAICS為候選基因來做更進一步的實驗。我們使用了tet-21n會受四環黴素調控MYCN (tet-off)表現的細胞株，並利用即時聚合酶鏈式反應 (Real-time PCR)偵測，發現PAICS（磷酸氨基咪唑羧化酶、磷酸氨基咪唑琥珀酰甲酰胺合成酶）與嘌呤生合成相關的代謝酶，其在MYCN被抑制時會顯著下降，以及在不同MYCN表現量的組織和細胞也觀察到MYCN與PAICS有正向表現相關，這表示MYCN可能會直接活化PAICS此下游基因，進一步使用啟動子冷光分析也發現PAICS具有受MYCN調節的啟動子存在。降低PAICS基因的表現量亦顯示抑制細胞生長的效果。我們的結果表明，在惡化的神經母細胞瘤中PAICS可能為腫瘤發生和癌症生物學研究裡一個具有潛力的治療靶標。

Neuroblastoma is a common neural crest-derived childhood cancer of sympathetic nervous system, which would be difficult to cure if progressed to higher stages. Among several clinical features, v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN) amplification is a prognostic marker for advanced neuroblastoma, which through massive transcriptional regulation, underlies dysregulated cell proliferation as part of the tumor biology. To investigate MYCN-regulated genes crucial for neuroblastoma progression, we first analyzed the MYCN-bound promoter regions in SK-N-BE(2)-C cells (MYCN-amplified, MNA) using chromatin immunoprecipitation−sequencing (ChIP−seq). Additionally, we integrated several expression datasets from Gene Expression Omnibus (GEO) regarding patients with neuroblastoma and demonstrated some genes which were highly correlated with MYCN expression in MYCN-amplified neuroblastoma. Combining the results from ChIP-Seq and the integrated datasets, we selected PAICS as the candidate gene in the following experiment. Using the tetracycline-repressible (tet-off) system to inhibit MYCN transcription in Tet21n cells, we found that PAICS (phosphoribosyl-aminoimidazole carboxylase, phosphoribosyl-aminoimidazole succino-carboxamide synthetase), a metabolic enzyme in purine biosynthesis and therefore supports DNA and RNA synthesis, was significantly downregulated under MYCN knockdown detected by real-time PCR, suggesting that MYCN may directly activate this downstream gene. The positive correlation between MYCN and PAICS was also observed in cell lines and tissues with different MYCN expression levels. Furthermore, promoter luciferase assay revealed PAICS existing promoter region regulated by MYCN. Knockdown of PAICS also inhibited cell proliferation. Taken together, our results suggest that PAICS is a potential therapeutic target that links tumorigenesis and cancer biology in advanced neuroblastoma.