分子選殖RGS11基因及探討其在肺癌中所扮演之角色

G蛋白信號傳導調節因子(regulator of G-protein signaling；RGS)為包含一個120-125個胺基酸之RGS domain保守序列的家族，其具有GAP(GTPase acceleration protein)之活性。至今在哺乳類中已發現有30多種RGS蛋白，共分為8個亞族，而RGS11屬於其中之R7亞族。在許多細胞外引發細胞內生理功能改變之G蛋白訊息傳遞路徑中，RGS扮演着負調控因子的角色，終止訊息之傳遞。根據先前文獻報導，GNB5可增加RGS蛋白之表現，而且至今尚無RGS11與肺癌轉移之相關報導。本實驗使用兩種不同侵襲能力之肺癌細胞株模型：一種是CL1-0細胞，屬於低侵襲能力肺腺癌細胞株；另一種是CL1-5細胞，屬於高侵襲能力肺腺癌細胞株。首先利用RT-PCR方法，確認CL1-5細胞之RGS11基因表現量明顯高於CL1-0細胞。再利用分子轉殖技術完成建構RGS11和GNB5質體，透過轉染技術過量表現RGS11和GNB5於CL1-0細胞，進一步利用Transwell migration assay，觀察細胞爬行(cell migration)之蛋白功能分析。其結果顯示，當單獨表現RGS11或GNB5蛋白於CL1-0細胞時，其造成之生物功能不具有顯著性。然而同時表現RGS11和GNB5蛋白時，造成CL1-0細胞之爬行數量顯著增加。Western blot之結果顯示，若單獨表現RGS11或GNB5，並無過量表現其蛋白。但同時轉染RGS11和GNB5於CL1-0細胞時，RGS11蛋白可明顯地過量表現。依據本實驗的結果已明確顯示，RGS11與肺癌轉移之間有著密切相關性，更進一步了解肺癌轉移之機轉，將可提供為直接作用於調節G蛋白之肺癌新藥開發的一個重要標靶。

關鍵字

G蛋白； G蛋白信號傳導調節因子11 ；蛋白穩定度；細胞爬行；肺癌

並列摘要

Regulatory of G-protein signaling (RGS) is a large family which contains a 120-125 amino acid-long conserved RGS domain and the activity of GTPase acceleration protein (GAP). At least 30 more RGS proteins have been discovered to date and divided into 8 sub- families. The majority of RGS proteins in cells play a negative regulatory role on G protein-mediated signaling pathways in respond to outside stimulations. RGS11 belongs to R7 subfamily. In previous reports, its protein level could be significantly increased when GNB5 was present. However, the association between RGS11 and tumor metastasis of lung is completely obscure. In the present study, two lung adenocarcinoma cell lines with different cell invasion capability were used. CL1-5 cells are highly invasive, and its counterpart cells, CL1-0, are weakly for cell invasion. The first of above, experiments using RT-PCR and Western blotting analyses confirmed that the expression levels of both RGS11 and GNB5 were much higher in CL1-5 cells than in CL1-0 cells. Both of genes were molecularly cloned into a eukaryotic expression vector, pcDNA3.1, and overexpressed into CL1-0 cells using Lipofectamine cell transfection agent. The function of RGS11, GNB5 or both proteins on cell migration was examined by in vitro transwell assays. The results indicate that overexpression of either RGS11 or GNB5 protein alone did not significantly altered the biological behavior of CL1-0 cells in cell migration. In contrast, co-expression of RGS11 and GNB5 indeed dramatically enhanced the migration capability of the cells. Intriguingly, this result is consistent with the protein levels of RGS11 and GNB5 in Western blot analysis, showing that RGS11 protein was significantly overexpressed in CL1-0 cells co-transfected with RGS11 and GNB5 but not found in cells transfected with RGS11 or GNB5 alone. Summarily, in the presence of GNB5, increased level of RGS11 can significantly lead to enhancement of cell migration, and may be associated with tumor metastasis of lung cancer. Thus, further study what the molecular mechanism(s) mediated by RGS11 and GNB5 result in tumor malignancy does is urgently required and also important to clinical targeting therapy of lung cancer for new drug development in future.

並列關鍵字

G-protein ； Regulatory of G-protein signaling 11(RGS11) ； protein stability ； cell migration ； lung cancer

參考文獻

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分子選殖RGS11基因及探討其在肺癌中所扮演之角色

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