抑制NEAT1表現量對癌轉移及癌侵襲能力之影響

Abstract

胰臟癌是高度惡性的疾病，大多數患者出現明顯症狀時已屬晚期，往往伴隨著淋巴血管侵襲 (lymphovascular invasion) 以及轉移 (metastasis)，其存活率並不樂觀。本實驗室從惡性胰管腺癌 (ductal adenocarcinoma) 病患中建立了兩株胰臟癌細胞株，分別是 PK1 (derived from primary tumor)及PK2 (derived from metastatic site)。透過核醣核酸定序 (RNA Sequencing) 做廣泛的RNA level分析，而此研究探討的是其中“長鏈非編碼RNA (long non-coding RNA, LncRNA)”的部份。我們發現 NEAT1 在定序結果中不僅相對上讀值較高，且在兩者之間的表現量有顯著差異；利用 RT-qPCR 證實 NEAT1 在 PK2 的表現量確實高於 PK1。因此，為了了解 NEAT1 在胰臟癌轉移中的功能，初步使用 siRNA 的方式 knockdown NEAT1 在 PK2細胞中的表現量，並逐步檢測是否影響 EMT marker 的mRNA及蛋白質的表現量。在 NEAT1 表現量受到抑制的 PK2 細胞中，發現 Slug, Snail, Vimentin 的mRNA及蛋白表現量明顯減少，而 E-cadherin 表現量增加。進一步進行功能檢測：與控制組相比，當 siRNA降低 NEAT1 表現時，PK2 細胞在 wound healing assay 以及single cell tracking中的移動能力有顯著的下降；從invasion assay也可觀察到NEAT1 表現降低時會使得細胞侵襲能力下降。而且不只在PK2 knockdown NEAT1會影響其細胞移動及侵襲，我們也在肺癌細胞株CL1-5驗證，不論是phenotype change或是分子層級的表現都得到同樣的結果。除此之外，抑制NEAT1在胰臟癌細胞株AsPC-1/PL45及大腸癌細胞株HCT-15也會看到相同的phenotype change。綜合本研究結果，NEAT1 確實在癌移動與侵襲過程中扮演某些角色，但其中分子作用機制仍有待更進一步之研究證實。

Pancreatic cancer is a lethal disease needing vigorous bio-medical research. We established pancreatic cancer cell lines, PK1 and PK2, from a patient with poorly-differentiated ductal adenocarcinoma (PDAC). In order to dig out novel genetic alterations and potential treatment targets, genome-wide studies were carried out. The RNA-seq study showed that, NEAT1 long non-coding RNA was upregulated in PK2 cells as compared to PK1 cells. The RNA-seq results were verified by quantitative real-time RT-PCR (RT-qPCR). We employed small interfering/hairpin RNA knockdown of NEAT1 in PK2 cells. The results showed that the migration and invasion abilities of PK2 cells were reduced with NEAT1 knockdown. These findings were supported by the RT-qPCR and immunoblotting results in that the expression of epithelial-mesenchymal transition (EMT)-related markers, such as SNAIL, SLUG, and Vimentin, were suppressed while the expression of E-cadherin was increased in both transcript and protein levels with NEAT1 knocked down. Furthermore, NEAT1 knockdown also resulted in similar EMT marker alterations as well as reduced cell migration and invasion abilities in CL1-5 lung cancer cells. In addition, the same phenotypicale changes were observed in AsPC-1, PL45 and HCT-15 cells. These results suggest that NEAT1 may play a certain role in cancer migration and invasion. Detail mechanisms and involving signaling pathways deserved further studies.