微核醣核酸-X在神經母細胞瘤中之抑癌角色

Abstract

神經母細胞瘤(Neuroblastoma, NB)為兒童癌症中最常見之顱外固態腫瘤，其佔兒童癌症死亡率約12%，而發生轉錄因子MYCN增幅的NB病人存活率較差。微核醣核酸(microRNA or miRNA)為一種非轉譯之小核醣核酸分子，透過轉錄後機制抑制標的基因的表現。過往miRNA研究中發現MYCN增幅的NB腫瘤與MYCN轉殖小鼠中皆有一群miRNA表現量顯著下降，包括miR-X。已知miR-X在非小細胞肺癌、腎臟惡性腫瘤與神經纖維肉瘤中表現量顯著減低，並具抑癌作用。初步分析腫瘤中miR-X表現量較高的NB病人，其存活率顯著較好。顯示miR-X在NB中可能有抑癌作用。然而其確實調控角色則尚不清楚。 分析台大兒童醫院40位NB病人於進行化療前收集的腫瘤檢體內miR-X表現量，發現臨床分期上風險較高的病人miR-X表現量顯著較低，而miR-X表現量高的病人的整體存活率與無事故存活率都較高。在六株人類NB細胞株中，有MYCN增幅的細胞株miR-X表現量有較低之趨勢。在有MYCN增幅的兩株NB細胞(SK-N-DZ，SK-N-BE)中以用慢病毒轉導使過度表現miR-X後細胞生長速度明顯減緩，分析細胞週期時發現有G0/G1停滯的現象；細胞非貼附生長能力、侵襲能力與遷移能力也顯著下降。顯示miR-X有抑制NB細胞腫瘤惡性程度的功能。分析過度表現miR-X後細胞的ATP產量與葡萄糖吸收量都有顯著上升，說明miR-X可調控細胞之糖類代謝。以微陣列(microarray)分析發現共151個基因在過表現miR-X後基因表現量顯著下降，其中15個基因為可能直接受miR-X調控表現之分子，我們鎖定在R2網路基因資料庫分析中顯示與NB臨床預後有關的PPARGC1B作後續研究。 PPARGC1B為一參與粒腺體新生(mitochondrial biogenesis)的基因，同時亦可調控細胞能量代謝。已知PPARGC1B在大腸癌及乳癌中扮演致癌角色，腫瘤細胞中PPARGC1B高表現已證實與增進癌細胞非貼附生長能力、降低細胞ATP產量生成相關。而進一步以RT-qPCR方法發現過度表現miR-X的兩NB細胞株，其PPARGC1B表現量確實下降，與微陣列分析結果符合。在人類NB細胞株中，具有MYCN增幅之SK-N-DZ與SK-N-BE細胞中PPARGC1B表現量較高，我們利用shRNA將SK-N-DZ細胞中PPARGC1B基因默化。而SK-N-DZ細胞在默化PPARGC1B後生長速度顯著下降，與過表現miR-X後的結果相符。 未來我們將通過reporter assay分析miR-X是否確實能直接調降PPARGC1B基因表現，同時探討PPARGC1B通過何種影響何種下游分子從而抑制NB細胞腫瘤惡性度；另外亦會建立miR-X剔除/默化的NB細胞株，從而確實其在NB中扮演抑癌角色的假設。

Neuroblastoma (NB) is an extracranial solid tumor with aggressive and metastatic features affecting young children. Patients with MYCN proto-oncogene amplification will have poor prognosis and survival. MicroRNA belongs to the short non-coding RNA family known to post-transcriptionally regulate the expression of their target genes, and play important roles in tumorigenesis. Recent miRNA profiling studies revealed a group of miRNAs were down-regulated in MYCN-amplified (MNA) NB, including miR-X, which was known as a tumor suppressor in non-small cell lung cancer and renal cell carcinoma. This suggests a tumor suppressor role of miR-X in NB. Here we evaluated tumor miR-X expression in 40 primary NB tumors obtained from untreated patients, it was noted that patients with INSS low risk stages (stages 1, 2) or without MNA had a higher miR-X expression in the tumors. Patients with higher tumor miR-X expression had better survival. Human NB cell lines bearing MYCN amplification (SK-N-DZ and SK-N-BE) showed a relatively lower miR-X expression as compared to cells without MNA (SK-N-SH, SH-SY-5Y). Overexpression of miR-X in MNA-NB cells by lentiviral transduction downregulated cell proliferation, anchorage-independent colony formation, migration ability and matrigel invasiveness. Furthermore, overexpression of miR-X increased glucose uptake and ATP production in MNA NB cell lines, indicating its effect on regulating energy metabolism. Microarray analysis identified 151 genes were significantly downregulated in miR-X-overexpressed compared to the control SK-N-DZ cells. Of particular interest, PPARGC1B, which encodes PGC-1 that is involved in mitochondria biogenesis and regulating glucose metabolism, and has shown significant clinical impact in the public NB database R2, was found to be decreased in the miR-X-overexpressed cells. We then generated PPARGC1B knock down SK-N-DZ cell using shRNA. Knocking down PPARGC1B significantly inhibited SK-N-DZ cell growth and anchorage-independent clonogenic formation. We will further investigate whether miR-X can inhibit PPARGC1B expression through direct targeting to its 3’-UTR using reporter assay. Secondary, the possible PPARGC1B down-stream targets will be analyzed using Western blot and RT-qPCR, to clarify the miR-X-PPARGC1B and its down-stream regulating pathway that is responsible to the phenotypic changes in the miR-X overexpressed NB cells. MiR-X knock out/down assay will be also necessary for validation of the tumor suppressive effects of miR-X in NB cell lines.