| Abstract: | 乳癌是台灣地區婦女好發癌症的第二位，臨床研究指出，導致乳癌患者疾病惡化和死亡的主要成因，是基於癌細胞發生腋下淋巴或是遠端組織器官的腫瘤轉移，以致對生命產生嚴重的威脅性。因此，對婦女乳癌而言，探討乳癌癌化進程和淋巴發生侵襲轉移的機制，已是公共衛生的重要課題。腫瘤轉移侵襲的微環境改變是複雜動態、而且是多重基因異常表現。文獻指出，在上皮細胞/間質細胞轉化作用(epithelial-mesenchymal transition, EMT)是關聯於胚胎發育、纖維化及上皮癌症轉移時的型態轉變。癌細胞移動(migration)和侵襲(invasion)的過程中，上皮細胞會形成游離且呈現類鞘狀(sheath-like)型態的纖維組織母細胞(fibroblast)。此時，上皮細胞經由EMT，促使間質細胞增加vimentin和 fibronectin的表現，並降低E-cadherin。以往對EMT相關基因的調控，著眼於探討轉錄因子負責特定基因表現的開啟和關閉(turn on and off)。近年來，研究指出腫瘤細胞於缺氧條件下，會緊密關聯到EMT的發展，而缺氧誘導因子(Hypoxia-Inducible Factor-1α, HIF-1α) 在缺氧微環境下表現程度增加，影響EMT調節蛋白的過度表現，促使癌細胞的進程發展惡化。 微型核糖核酸為一群未轉譯的遺傳序列，會影響幹細胞的分化和胚胎發育的過程。研究指出miRNA表現程度的差異與腫瘤的生成發展有密切的關係。然而，有關乳癌發生進程之miRNAs標記及其作用機轉的研究尚未清楚；而以miRNA的表現視為乳癌進程、甚至是臨床預後評估更是有限。有鑑於此，本研究構想的緣起，則是就外顯基因體學(epigenetics)來找尋攸關婦女乳癌腫瘤進程發展的miRNA作用機制及其生物標記。已知在缺氧微環境下，HIF-1α會加強CXCR4蛋白表現以活化EMT的下游蛋白，促使癌細胞發生侵襲和轉移。假設存在特定的miRNA序列會結合上HIF-1α 和CXCR4 基因 3’-UTR序列，抑制HIF-1α和CXCR4的蛋白表現，進而逆調節EMT路徑下游蛋白的表達，而活化MET路徑，可以促進乳房上皮細胞進行再分化的訊息傳導，這將有利於乳癌患者預後的發展。為了驗證此一假說，我們先前試驗以miRNA microarray為基礎，探討miRNA標記在婦女乳癌進程中所扮演的角色。相較於未發生淋巴轉移的乳癌患者，在淋巴轉移的乳癌患者組織中，我們發現miR-622和miR-139呈現低度表現。進一步，經由miRanda、Pictar和Targetscan等資料庫進行生物資訊的比對分析後，推測HIF-1α和CXCR 4有可能分別是miR-622和miR-139作用的下游標的基因(putative target gene)。因此，我們擬訂為期三年的研究計畫，以釐清miR-622和miR-139分別抑制HIF-1α和CXCR4蛋白表現的分子機制，並嘗試連結miR-622和miR-139和乳癌預後評估的轉譯醫學研究。於是，我們將以乳癌細胞模式建構重組miR-622和miR-139之質體表現，以luciferase reporter assay、Western blotting、wound healing、invasion/migration assays證實miRNAs和HIF-1α、CXCR4的作用關係。再者，我們將攜帶有miR-622和miR-139穩定表現之乳癌細胞株殖入小鼠，透過動物模式，觀察miR-622和miR-139轉殖小鼠的腫瘤大小、血管分佈情況、血管增生和糖解作用等機制為標的，探討是否會因為miRNAs的注入，來抑制小鼠腫瘤細胞的發展。在臨床檢體的分析實驗中，我們擬定收集450個乳癌案例進行乳癌之轉譯醫學研究，來支持miR-622和miR-139做為乳癌預後評估之標記。實驗中，我們將以雷射捕獲微擷取(laser capture microdissection)將乳癌切片之癌細胞和相鄰正常細胞部分加以分離(共計900個均質化的細胞檢體)，將其miRNA分離純化後，以qRT-PCR定量miR-622和miR-139的表現程度，分析特異性標的miRNAs群組和乳癌病理特性，依據(a)腫瘤大小(b)分化程度(c)臨床分期和(d)乳癌是否發生腋下淋巴轉移(e)存活分析[disease-free survival (DFS)和overall survival(OS)]等預後指標進行關聯性分析，以釐清複合性miR-622和miR-139表現程度差異對腫瘤預後發展的影響。本研究計劃將從細胞、動物模式到乳癌轉譯醫學研究，其研究成果將對我們了解epigenomic regulation的相關機制提供關鍵訊息；並可以針對miRNAs在臨床上對乳癌生成、進展和預後提供重要的生物標記外；在未來，亦可以就miR-622和miR-139為標的，發展成為有效的乳癌治療策略。
Tumor recurrence and metastasis result in an unfavorable prognosis for cancer patients. Interconversion between epithelial and mesenchymal cell phenotypes, processes termed epithelial-mesenchymal transition (EMT) has been reported in mammary cell development. Beyond tumor formation, EMT programs have been suggested to facilitate tumor cell spread, metastasis and invasion. Under hypoxia condition, overexpression of HIF-1a was reported to activate a variety of downstream target genes which involve in angiogenesis, cell proliferation, metastasis/invasion, and cell survival in cancer cells. Further, accumulating evidences has been indicated that CXCR4 expression level is dominant in metastatic tumor cells toward HIF- 1a-overexpressing organs when these cells stabilized by hypoxia environments. Besides, HIF-1a was suggested to bind the hypoxia-response element (HRE) of the promoter of Twist gene and mRNA transcription of several genes activated by Twist was enhanced by examination of the Twist null mice model, suggesting that increased HIF-1a level involves in EMT pathway regarding metastatic phenotype of cancers. A growing body of evidence reveals that epigenetic events of EMT was regulated by microRNAs (miRNAs); a novel class of short non-coding RNA molecules consisting of 19-25 nucleotides with the potential to inhibit gene expression by binding to complementary sequences in the 35-untranslated region (35-UTR) of target mRNA transcripts. In more recent, we performed a global testing of epigenomic miRNA study, the result showed that expression levels of miR-622 and miR-139 were significantly reduced in breast tumors whose phenotypes were diagnosed as positivity of lymph node metastasis. On the basis of the software programs (miRanda, Pictar, and Targetscan) to search mRNAs for these miRNA target prediction, we found that the 35-UTR seed sequences of HIF-1a and CXCR4 might be targeted by miR-622 and miR-139, respectively. Hypothesis that of expression levels of HIF-1a and CXCR4 can be down-modulated by these two miRNAs, ectopically expressed of miR-622 and miR-139 in breast cancer may endorse inhibitory effects on tumor cell migration and invasiveness. Further, repression of HIF-1a and CXCR4 may correlate decreased Slug and Snail expression during EMT, and therefore, miR-622 and -139 can be considered as miRNA biomarkers in predicting breast cancer prognosis. With our preliminary findings in bioinformatics study, in this proposed project, we will investigate the molecular mechanism underlying the miR-622 and miR-139 down-modulate HIF-1a and CXCR4 expression by which results in inhibiting migration and invasion of breast cancer in vitro and in experiments under hypoxia condition. In addition, application of the biomarkers of the miR-622 and miR-139 are being subjected to translational approach from lab side to clinical samples, a sample size of 450 female patients with invasive ductal carcinoma (IDC) will be enrolled to collect paired tumor and corresponding non-neoplastic counterparts using laser capture microdissection (LCM) in each tumor tissue specimen. Expression levels of miR-622 and -139 in these LCM-treated specimens will be analyzed using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) to validate prognostic evaluations of both miRNA biomarkers. Moreover, the trade-off for extended period of more than 5-year follow-up is especially importance in clinical benefits in our breast cancer study cohort. In conclusion, this proposed project will conduct a key concept regarding multiple miRNAs to understand the cross-talk between miR-622 and -139 in hypoxia- and EMT-related pathways in breast cancer progression. Our study may provide support to account for the preferential role of novel miRNA biomarkers, miR-622 and -139, in prognostic evaluation and potential targets for the development of more effective treatment strategies for breast cancer. |
