SPANXA1經由表皮間葉細胞轉型過程抑制肺癌腫瘤轉移

Abstract

SPANX (Sperm protein associated with the nucleus, X-linked) 此基因家族在睪丸生殖細胞瘤、黑色素瘤、膠質母細胞瘤、惡性血液病中都有高度表現。然而在我們先前Affymetrix基因晶片分析研究中顯示，SPANX family member A1 (SPANXA1) 表現量在低侵襲能力的肺癌細胞株CL 1-0中的表現量較高侵襲能力的細胞株CL 1-5高出97倍，進而以即時定量聚合酶連鎖反應確認SPANXA1信使核醣核酸(mRNA)表現量在CL 1-0比CL 1-5有八倍上升。體外細胞實驗中發現，CL 1-5 SPANXA1穩定表現細胞株和載體穩定表現細胞株相比，其細胞侵襲和移動能力下降跟SPANXA1表現量呈現劑量依賴關係。並且CL 1-5 SPANXA1穩定表現細胞株的細胞形態由本來的間質形態變成表皮形態，呈現典型的反轉表皮間葉細胞轉型過程，而此現象和其侵襲能力下降相吻合。因此我們使用CL 1-5 SPANXA1穩定表現細胞株和載體穩定表現細胞株以鼠尾注射技術來進行體內小鼠腫瘤轉移試驗，結果發現SPANXA1會抑制腫瘤細胞在體內轉移的能力。進一步，我們建立CL 1-0 SPANXA1小髮夾核醣核酸抑制株，在體外實驗中，也可以增加細胞侵襲和移動能力。由於SPANXA1會造成細胞形態改變，我們使用免疫墨點法確認表皮間葉細胞轉型過程標誌的表現，發現在有SPANXA1表現時，表皮型標誌E-cadherin表現上升且間質型標誌N-cadherin和vimentin下降。最後，為了釐清SPANXA1下游可能訊息傳導機制，我們使用Affymetrix 基因晶片來比較CL 1-5 SPANXA1穩定表現細胞株和載體穩定表現細胞株的基因表現差異。實驗結果顯示相關性最高的前十名訊息傳導路徑中，三個路徑都與表皮間葉細胞轉型過程相關，證實表皮間葉細胞轉型過程可能在SPANXA1下游分子機制扮演重要的角色。

SPANX (Sperm protein associated with the nucleus, X-linked) gene family has been found higher expression in many cancers, such as testicular germ tumor, melanoma, glioblastoma and hematologic malignancies. However, in our previous study, the gene expression array analysis showed that the expression of SPANX family member A1 (SPANXA1) was up-regulated in lower invasive lung cancer cells, CL 1-0, than in highly invasive cells, CL 1-5. Real-time PCR was used to confirm that mRNA expression of SPANXA1 was 8 times higher in CL 1-0 than CL 1-5. In vitro assays showed that migration and invasion abilities of stably SPANXA1-expressing CL 1-5 transfectants were decreased in a dose-depended manner. Interestingly, cell morphology was dramatically changed from mesenchymal-like to epithelial-like in stably SPANXA1-expressing CL 1-5 transfectants, which was consistent to its decreased invasive ability in vitro. Therefore, we demonstrated metastatic ability of stably SPANXA1-expressing CL 1-5 transfectants would be down-regulated in vivo, which was performed in a mouse metastasis model by tail-vein injection. Next, we set up CL 1-0 lentiviral-base shRNA knockdown clones, and the cell mobility and invasion ability were increased. Because SPANXA1 could induce the cell morphology change, we evaluate the effect of SPANXA1 on the expression of epithelial-mesenchymal transition (EMT) markers. We found up-regulation of E-cadherin, an epithelial marker, and down-regulation of vimentin and N-cadherin, which were known as mesenchymal markers in presence of SPANXA1. Finally, to identify the underlying molecular mechanism of SPANXA1, we carried out gene expression array and 3 of top 10 pathways are EMT-related indicated that EMT pathway might play a major role in SPANXA1-repressed metastasis.