探討人工微型核醣核酸p-27-5p於乳癌細胞株T-47D基因調控網路之影響

Gene Regulatory Networks of Artificial MicroRNA p-27-5p-Treated Human Breast Cancer Cell Line T-47D

10.6342/NTU.2010.00849

許仲誠

微型核醣核酸 ； 乳癌 ； 細胞週期 ； 外顯子微陣列 ； 基因調控網路 ； miRNAs ； breast cancer ； cell cycle ； exon array ； regulatory network

臺灣大學分子與細胞生物學研究所學位論文

2010年

碩士

阮雪芬

繁體中文

微型核醣核酸microRNA（miRNA）是一群長度約20-23個鹼基之間的短片段內生型核酸，本身並不會轉譯出蛋白質，其功能和基因表現的調控有關，對於其目標基因能夠藉著與其訊息RNA (mRNA) 的3’-非轉譯區 (3’-UTR) 結合來進行負向調控，以抑制蛋白質的合成。最近的研究中發現微型核醣核酸除了與細胞的生長發育有密切關係之外，與癌症的發生與發展有密切的關係。此外，微型核醣核酸在細胞訊息傳遞路徑中所扮演的角色也逐漸被發現，但其確切的功能尚未完全解開。李文雄院士、施純傑博士與我們組成的團隊於2008年利用電腦預測的方法發現一個可能的微型核醣核酸p-27-5p，我們以人類乳癌細胞株MDA-MB-231及T-47D為材料進行研究，發現p-27-5p在這些細胞株中並不會有內生性的表現。以人工方式讓此兩株細胞表現p-27-5p後，在細胞生長曲線實驗中發現p-27-5p會導致細胞株T-47D生長減緩，而在細胞週期分析中發現p-27-5p會造成乳癌細胞株T-47D的細胞週期停滯，進一步用外顯子微陣列 (exon array) 及網路分析軟體Ingenuity Pathway Analysis分析p-27-5p對乳癌細胞株T-47D造成的基因調控網路影響，發現細胞表現p-27-5p後，細胞週期、細胞凋亡及癌症相關基因網路明顯受到改變。我們經由冷光酶活性測定分析驗證週期素依賴激酶第四型(cyclin-dependent kinase 4, CDK4) 為p-27-5p的目標基因，並利用西方墨點法證實p-27-5p會抑制T-47D細胞株CDK4的蛋白質表現以及RB1蛋白質之磷酸化程度，藉此抑制E2F1的活化，使細胞無法通過G1/S檢查點，進而抑制細胞增生。總而言之，本項研究發現以人工方法將p-27-5p送入乳癌細胞中，會抑制CDK4蛋白質的表現量，進而抑制其下游的基因調控網路，導致細胞週期受到影響，最終抑制癌細胞的生長。

MicroRNAs (miRNAs) are small, endogenous non-coding RNAs of 20–23 nucleotides in length that negatively regulate the expression of target genes at the post-transcriptional level. miRNAs have recently emerged as important regulators that play significant roles in tumorigenesis. The present study attempted to elucidate the functions of p-27-5p, a novel possible miRNA we recently discovered (Chang et al. 2008). In our study, we found that cell growth exhibited a decrease and showed that the breast cancer cell line T-47D were induced to arrest at G0/G1 phase after treatment with p-27-5p mimic. To understand the regulatory mechanism of p-27-5p, we performed exon array and Ingenuity Pathway Analysis to construct gene expression networks of p-27-5p-treated breast cancer cell line T-47D. The results demonstrated that several genes associated with cancer cell apoptosis or proliferation experienced an expression change after p-27-5p treatment. We demonstrated that cyclin-dependent kinase 4 (CDK4) is the target of p-27-5p by using luciferase reporter assay. Previous studies indicated that CDK4 can stimulate cell cycle progression and is often overexpressed in cancer cells. We also demonstrated that p-27-5p downregulates CDK4 protein expression and RB1 phosphorylation by using western blotting. Taken together, our data suggest this artificial miRNA, p-27-5p, plays an important role in cancer progression and has the potential to be applied in breast cancer therapy.