探討微型核醣核酸miR-148a於胃癌細胞的蛋白質調控網路

Abstract

微型核醣核酸是一種長約22鹼基的小片段內生型核醣核酸，其本身不會轉譯出蛋白質，但可藉由直接結合標靶訊息核醣核酸 (mRNA)的 3’-非轉譯區 (3‘UTR)，抑制蛋白質的轉譯或是造成mRNA的降解，進而負調控基因的表現。近年來發現微型核醣核酸不只對生物的發育有重要的影響，同時在基因表現的平衡中扮演重要的角色。最近的研究更指出微型核醣核酸對於癌症的發生、發展與遷移能力有密切的關係。我們將微型核醣核酸-148a (miR-148a)轉染至胃癌細胞株AGS並觀察其生長狀態以及細胞遷移能力的表現，結果顯示有轉染miR-148a的細胞其生長有明顯的減緩，遷移能力也相對低於對照組。進一步使用同位素標記絕對與相對定量 (iTRAQ)、網路分析軟體 (Ingenuity Pathway Analysis)、網路預測微型核醣核酸標靶基因軟體 (Targetscan 5.1)以及蛋白質網路建構資料庫STRING 8.3分析miR-148a對AGS細胞蛋白質層次的調控，並建構出可能的調控網路。結果發現，有四個受到負調控的蛋白質參與在AKT訊息傳遞路徑上。另外有一個預測為標靶基因的蛋白質FXR1其蛋白質表現量下降。根據前人的研究報導指出， FXR1表現量下降可導致TNF-α表現量上升，使得TNF-α促進細胞凋亡(apoptosis)。根據我們的實驗結果與建構之蛋白質調控網路認為，miR-148a可能主要透過抑制AKT訊息傳遞路徑上的蛋白質表現，而降低細胞的存活能力，另外也可能直接抑制FXR1的表現進而促進細胞的死亡。 另外，本實驗室以SAM分析GEO上胃癌組織與正常胃部細胞的資料，發現MTA2的RNA在胃癌組織中的表現為正常細胞中的3.67倍。實驗結果顯示TSGH細胞株內生miR-148a的量是AGS細胞株的2.91倍，大量轉染miR-148a入TSGH細胞株會導致MTA2的RNA表現量下降約20%，但是在冷光酶活性測定分析結果表示，MTA2並非miR-148a的標靶基因，最後以西方墨點法測得MTA2蛋白質表現量顯示下降了0.93倍。此實驗結果顯示，MTA2受到miR-148a的表現在RNA以及蛋白質層次上受到抑制，可能進而導致TSGH細胞的生長以及遷移能力。

MicroRNAs (miRNAs) are endogenous small non-coding RNA about 22 nucleotides in length. They can directly bind to target genes, causing down-regulation of their expression post-transcriptionally by suppressing protein translation or degrading mRNA. In this study, we found a tumor suppressor miRNA, miR-148a, could decrease cell growth and metastasis of gastric cancer cells. To further understand the regulatory mechanism of miR-148a in tumor progression of gastric cancer, we used isobaric tags for relative and absolute quantitation (iTRAQ) method to identify differentially expressed proteins in miR-148a-over-expressed AGS cells and construct miR-148a-regulated network. We further analyzed the enriched functions within its network using Ingenuity Pathway Analysis (IPA), STRING 8.3 and TargetScan 5.1 database. Our results revealed that the most enriched biological process was AKT signaling, known as a cell survival-related pathway. We also identified many down-regulated proteins involved in AKT signaling, including Bcl-2-like 1 protein (BCL2L1), Heme oxygenase-1(HMOX1), Integring-linked kinase (ILK) and Glycogen synthase kinase-3 alpha (GSK3A), based on iTRAQ analysis. On the other hand, we identified a predicted target of miR-148a, Fragile X-related protein (FXR1), was down-regulated in miR-148a over-expressed tumor cells. Study reports that down-regulates FXR1 could increase TNFα expression, and cause apoptosis through TNFα. Taken together, these results suggest that miR-148a may suppress cell survival through regulating FXR1-AKT network. We analyzed microarray data from GEO by using SAM. The RNA expression of MTA2 in tumor tissue is 3.67 times higher than normal tissue. Furthermore, the endogenesis RNA expression of miR-148a in TSGH is 2.91 times higher than in AGS. As we transfect miR-148a precursor into TSGH, the RNA expression of MTA2 is downregulated up to 20%, but luciferase assay data revealed that MTA2 RNA is not the direct target of miR-148a. For further examination, the protein level of MTA2 is decreased 0.93 times under overexpression of miR-148a. MiR-148a might effect the expression of MTA2 in RNA and protein level, and regulate growth and migration ability of TSGH.