探討LIN28B在神經母細胞瘤中之角色

Abstract

神經母細胞瘤在兒童為最常見的顱外實質固態瘤，源自於交感神經系統中的胚胎神經脊細胞，為未分化的神經母細胞。病人呈現出的臨床表癥有很高的異質性, 可由自發性的痊癒到高度惡性轉移的腫瘤，在臨床上具有MYCN增殖其病人腫瘤常發生轉移，預後多屬於極差的一群。 Lin28a/Lin28b屬於核醣核酸結合蛋白，兩者的胺基酸序列以及功能類似，皆可透過抑制微核醣核酸Let-7的生合成來影響其他基因的表現，或透過與目標基因mRNA結合增加轉譯效率以提升表現量。過去的文獻指出Lin28a於線蟲至小鼠等動物中基因序列為高度保守，並皆在發育過程中扮演重要的角色；在神經發育中僅在早期為高表現，隨著神經進行分化其表現量則會下降。而在幹細胞的研究中，Lin28a/Lin28b於胚胎幹細胞中皆為高表現，可做為多能性基因用來重編組細胞成為誘導性多能幹細胞。在目前已有許多研究指出，Lin28a及Lin28b會高度表現於多種癌症中並與其惡性程度以及較差的預後度有很高的相關性。先前的研究報導在神經母細胞瘤細胞株中MYCN可以調控Lin28b使其表現量上昇，並推測可能與MYCN在神經母細胞瘤中致癌的情形有關。 本研究分析台大醫院38位神經母細胞瘤病人腫瘤中Lin28b與MYCN的表現，並發現兩者之間有正相關，並從開放的神經母細胞瘤微陣列基因表現資料庫中分析得到相同的結果；在神經母細胞瘤病人預後的分析中，大於1.5歲的病人中高表現Lin28b在傾向預後較差。於TH-MYCN轉基因小鼠的腫瘤中，也同樣觀察到Lin28b的高度表現，表示Lin28b的確會受到MYCN調控，並可能於MYCN-driven的神經母細胞瘤中執行致癌基因的功能。本研究欲了解於高表現MYCN的神經母細胞瘤致癌過程中Lin28b扮演的角色，選用兩株MYCN增殖且高表現Lin28b的神經母細胞瘤細胞株SK-N-DZ與SK-N-BE將Lin28b表現予以shRNAs抑制後，觀察到細胞形態改變走向神經分化，增生速率減緩、細胞週期停滯於G0/G1，且神經幹細胞標記Nestin與致癌基因MYCN表現量下降（表示細胞走向神經分化）、細胞非貼附性生長與侵襲能力等惡性度指標皆有減弱。使用Lin28b表現質體將Lin28b過度表現於低表現Lin28b與MYCN的SK-N-SH細胞株中，觀察到Nestin與MYCN的表現量大量上升，顯示Lin28b於神經母細胞瘤細胞中高表現可能會造成細胞去分化以及惡性度的提昇。綜合本研究的實驗結果，Lin28b於神經母細胞瘤中為一致癌基因，可能於MYCN增殖的神經母細胞瘤中促進腫瘤維持未分化的狀態以及惡性度，此外在非MYCN增殖的神經母細胞瘤中也可以透過正向調控MYCN表現量或其他基因的表現影響腫瘤的惡性度，因此Lin28b有潛力成為一新的藥物標的用於治療MYCN增殖或高表現Lin28b的神經母細胞瘤。

Neuroblastoma (NB), the most common extracranial childhood solid tumor and most common cancer in children younger than one year of age, is arising from neural crest cells of sympathetic nervous system. The clinical outcome of neuroblastoma patients present very high heterogeneity, ranging from spontaneous regression to rapid progression and metastasis. MYCN amplification in neuroblastoma is an unfavorable prognosis marker, and the tumors are highly malignant which often occurs to metastasis and hard to be cured. Lin28a and Lin28b are RNA-binding proteins, sharing similar amino acid sequence and function, Lin28a and Lin28b inhibit the maturation process of the microRNA family of tumor suppressor, Let-7, and subsequently hinder the inhibition of Let-7 targets. Lin28a/b can also to target mRNAs and promote their translational efficiency. Previous studies showed that Lin28a is highly conserved from C. elegans to mouse, and plays an important role in development. Lin28a is highly express in early stage of nervous system development, but be inhibited during neuronal differentiation. Lin28a/b are found highly expressed in embryonic stem cells, and were used in reprogramming cell to induce pluripotent stem cell (iPS cell). Recently, Lin28a and Lin28b are found to be expressed in various types of human cancers, and their expression was associated with advanced disease stage and poor prognosis. In neuroblastoma, MYCN was known to up-regulate Lin28b expression, and we speculated that Lin28b may participate in pathological mechanism by MYCN-drived neuroblastoma. A positive correlation between the expression of Lin28b and MYCN that found in our screening of 38 neuroblastoma samples collected in National Taiwan University Hospital, and was confirmed in neuroblastoma data acquired from open resource databases, however, Lin28a expression is not found correlated to expression of MYCN. Higher expression of Lin28b was marginally associated with poor prognosis in those patients with age-of-onset older than 1.5 years. Tumors developed from TH-MYCN transgenic mouse, also present high expression of Lin28b. All these results suggest that Lin28b could be up-regulated by MYCN, and play an oncogenic role in MYCN-driven neuroblastoma. Neuroblastoma cell lines, SK-N-DZ and SK-N-BE are MYCN-amplified and express high level of Lin28b. Knocking down Lin28b expression by lentiviral-delivered shRNAs results in morphological changes, the cell proliferation inhibition, and cell cycle arresting at G0/G1 phase. Moreover, Nestin and MYCN are down-regulated, which suggest that cells are skewing to neuronal differentiation. Reduced anchorage-independent cell growth and matrigel invasion also indicate that Lin28b reduction attenuates malignant characteristics of the cells. Overexpressing Lin28b in SK-N-SH cell line, conversely, upregulates Nestin and MYCN, which suggests that the ectopic expression of Lin28b promote cell de-differentiation and may enhance malignancy. These results indicate that Lin28b is an oncogene in neuroblastoma, and may have a role in promoting MYCN-amplified neuroblastoma. Of notice, our findings also point out that Lin28b up-regulates MYCN or regulates other gene expression to promote malignancy in non-MYCN-amplified neuroblastoma cells. Accordingly, Lin28b can be a new therapeutic target in neuroblastoma.