2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38636Cancer is believed to result from a series of genetic alterations leading to progressive disorder of the normal mechanisms controlling growth, differentiation, cell death, or genomic instability. Alterations of protein-coding oncogenes and tumor suppressor genes (TSGs) have been thought to be the causes of tumorigenesis and driving for the metastatic process.With the investigations of genes that non-coding RNA transcripts with no significant coding can lead to silencing of the target genes through the RNA interference pathway, such a class of small cellular RNAs, named microRNAs (miRNAs). More importantly, recent evidences indicated that some miRNAs may effect on either oncogenes or TSGs in contributing to cell tumorigenesis. Thus, the analyses of expression profiles can yield characteristic of miRNA signatures in human cancers. The tumor-suppressor phosphatase with tensin homology (PTEN) is the most important negative regulator of the cell-survival signaling pathway that is initiated by PI3K. In the face of DNA strand break, the role of PTEN is being an inhibitor of PI3K pathway to result in the sequestration of cell cycle checkpoints, being a collaborator and an effector of p27kip1 tumor suppressor to cause G1/S cell arrest, being a transcriptional regulator of DNA damage repair proteins, and being a modulator of constitutively regulatingWnt/b-catenin-dependent signaling. This proposed study therefore speculates whether PTEN collaborating PI3K, p27kip1 and E-cad/b-catenin may associate with the development of breast cancer. To understand the mechanism linked to PTEN-regulated expression in contribution to tumor progression in breast cancer, a multigenic investigation of miRNAs is to be performed. We will strive to establish a methodological framework involved in such multistep process by examining the multiple gene expressions, including hsa-miR-21, hsa-miR-221and hsa-miR-200c, are respectively responsible for regulation of cell proliferation (PTEN), cell cycle (p27kip1), and components of cellular architectures (E-cadherin). Moreover, the effects of heterogeneity within a tumor cell were minimized by ensuring that genetic and phenotypic examinations are being carried out on the same tumor tissues using laser capture microdissection (LCM), allowing a more precise evaluation of specific associations between genetic alteration and pathological manifestation of breast cancer in Taiwan. Specific Aims. (1) To understand tumorigenic contribution associated with PTEN’s participation in the development of breast cancer, and the network communications of PI3K/Akt and Wnt/b-catenin signaling; (2) To provide important clues using multigenic relationship of differential expression of miRNAs that linked to PTEN/p27kip1/E-cadherin during tumor development of breast cancer; (3) To provide comprehensive picture of targeted miRNAs, miR-21, miR-200c and miR-221, acting as suitable molecular markers of prognostic evaluation in breast cancer. Materials and Methods. During a three-year execution period, there are two principal purposes in this proposed project to explore the role of miRNAs in association with PTEN/p27kip1/E-cadherin in breast cancer. Part I. Molecular epidemiological study of the first year: Methylation-specific PCR (MSP) (i) We will recruit 150 female breast cancer patients. The tumors were diagnosed as histologically confirmed invasive ductal carcinomas. Patients enrolled will contain paired primary tumors and adjacent non-tumors on the same patients of the breast and that will be carefully collected by laser capture microdissection (300 samples); (ii) Sodium bisulfite modification of genomic DNA followed by methylation-specific PCR (MSP) and sequencing are being used to determine hypermethylated statuses of miRNAs; (iii)Identification of the clinical significance between novel CpG island of miRNAs and poor progression of breast cancer by using multivariate logistic regression analysis. Part II. Molecular epidemiological study of the second year: Real-time quantitative PCR technique by online LightCycler detection (i) RNAwas extracted from LCM-collected tumor and normal tissues; (ii) mRNA expression of these three regions, miR-21, miR-200c and miR-221 will be analyzed using LightCycler-based quantitative RT-PCR (qRT-PCR); (iii)Possible correlations between each miRNAmarker and PTEN-related signaling will be evaluated for prediction of the pathological and clinical outcomes of breast cancer. Part III. Cancer genetics study of the second and the third years: (i) Gene transfection is being conducted of anti-miRNAs, antagomiR-21 and antagomiR-221 in different cells, including HBL100, MCF-7 (ER(+)), Hs578T (ER(-)), HeLa and HEK293T cells; (ii) All transfectants will be subjected to examining the expression level of their downstream regulated proteins, consisting PTEN/PI3K, Wnt/b-cat, and p27kip1/Cdk/cyclin; (iii)The physiological response of the anti-miRNA-treated cells will be examined by using chemotaxis for evaluating the capabilities of tumor cells with invasion and migration. Anticipated Results and Importance. To expand our understanding of the role of PTEN-related signaling pathway, linking to breast tumor progression, including cell invasion and migration, this proposed study will explore the miRNA expression of PTEN, p27kip1, Pdcd4 and E-cadherin, by which these genes directly or indirectly correlate cell oncogoing of breast. This should allow a more comprehensive insight into the role of PTEN-associated mechanism in related to breast tumorigenesis. In conclusion, this kind of miRNA investigation may shed light on the markers of poor prognostic evaluation, but it also helps to develop new anticancer strategy for therapeutic approaches.(1) 引發此研究之計畫及構想： 乳癌是台灣地區婦女好發癌症的第二位，每年約有近2000 名新發生的個案，尤 其是發生率及死亡率在逐年上昇中，更值得我們加以重視。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者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。文獻指出，對於腫瘤侵襲的過程是複雜動態、而且 是透過多重基因的異常表現，以致使其癌細胞發生程序性的轉移。因此，對婦女乳 癌而言，探討腫瘤癌化進程以至於發生淋巴轉移的機轉，便有其特殊意義的急迫性。 微RNA(microRNA, miRNA)為一群未轉譯的遺傳序列。研究發現，miRNA 的表 現程度差異被指出與致癌基因和腫瘤抑制基因所在位置的調控有關，是攸關於腫瘤 組織的發展。更有研究指出，miRNA 的表現程度可做為癌症分期診斷和臨床預後的 評估標記。儘管如此，有關miRNA 之分子遺傳標記在乳癌發生和癌轉移的分子機 轉至今仍不清楚。本研究的緣起，則是就基因剖面來探討miRNA 遺傳序列的表現 在婦女乳癌腫瘤進程發展所扮演的角色。建立於我們實驗室以往對婦女乳癌的研究 經驗，在腫瘤細胞的發展過程中，關於細胞分裂的調控、腫瘤抑制基因和細胞間基 質蛋白(extracellular matrix)的表現等，關連到細胞的增生、分化和維繫細胞間接合 的訊息傳導等作用，是影響腫瘤細胞增生以致於惡化的關鍵因子。因此，本研究計 畫則是收集乳癌患者的乳癌病灶和相鄰正常組織的部分，探究標的miRNAs: hsa-miR-21、hsa-miR-200c 和hsa-miR-221/miR222 的表現與乳癌惡化進程的關聯。 進一步，以乳癌細胞株建構miRNA 之分子作用模式，來釐清其調控乳癌細胞發展 進程的可能機制。針對多重基因調控的複合性研究，將對台灣地區婦女乳癌發生提 供重要關鍵的研究訊息。 (2)目的： 藉由分子遺傳流行病學和細胞遺傳學實驗分析miRNAs: miR-21 (PTEN; Pdcd4)、miR-200c (E-cadherin)和miR-221 (p27kip1)與乳癌致癌危險的關聯。我們以 雷射捕獲微分離技術(Laser Capture Microdissection, LCM)將患者乳部癌組織和相鄰 的正常組織加以分離後，進行其組織DNA 和RNA 的萃取，分析miRNA 遺傳序列 的甲基化作用和miRNAs 表現的程度差異和乳癌分期的關聯。進一步，以乳癌細胞 株建構miRNA 之分子作用模式，來探討miRNA 於乳癌生成和癌侵襲轉移過程中所 扮演的角色。 (3)方法： 分子遺傳流行病學部分： 第一年計畫： (i) 擬定收集150 例婦女乳癌病患，根據乳癌分期標準(TNM staging)，將所收集的 乳癌病患依據(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 轉移加以分組。每一病例具有配對的乳癌組織及其相鄰的正常(非癌)組織(共計 300 個組織檢體)； (ii) 以雷射顯微擷取技術分離乳部癌組織和相鄰正常組織，純化各組織中的 DNA，以重亞硫酸鹽試驗(sodium bisulfite)、結合甲基化特異性PCR (methylation-specific PCR)，以分析這三區miRNA 基因序列在乳癌組織中發生 甲基化的位置和表現程度； (iii)關連於miRNA 多重基因的甲基化剖面進行多變相統計分析，探討與乳癌發生、 分期和腫瘤轉移的危險性評估。 第二年計畫： (i) 將所收集到具有配對之乳癌檢體，以雷射顯微擷取分離癌組織和相鄰正常組織 後，再抽取各組織中的RNA； (ii) 利用TaqMan 螢光探針( probe)為引子，以real-time RT-PCR (qRT-PCR)技術針對 hsa-miR-21、hsa-miR-200c 和hsa-miR-221/miR222 加以比較這三處miRNA 在乳 癌組織和其相鄰正常組織其miRNA 表現程度的差異； (iii)協同miRNA 基因甲基化作用連結於RNA 的表現程度差異，輔以乳癌病患臨床 分期之統計相關分析，將可以對乳癌發展以至於癌侵襲轉移找出適切的miRNA 遺傳標記。 細胞遺傳學的研究部分： 第二、三年計畫： (i) 培養正常乳房細胞株HBL100、乳癌細胞株MCF-7 (ER(+))、Hs578T (ER(+))、 HeLa (human cervical cancer cells)和HEK293T (human embryo kidney cell)，以 探討miRNA 調控PTEN、Pdcd4 和p27kip1 表現對乳癌發生的影響。 (ii) 將遺傳分子anti-miRNAs: antagomir-21 (PTEN、Pdcd4)和antagomir-221/222 (p27Kip1)，轉殖入各細胞株內；探討anti-miRNA 對下游E-cadherin、b-catenin 和MMPs 等基因表現的調控作用； (iii)以侵襲和轉移實驗(invasion and migration assay)，評估antagomiR-21 和 antagomiR-221 於植入細胞後，影響癌細胞侵襲和轉移惡化發展的分子機制。 (4)實施進度： 本研究預計以三個年度來完成整個計畫。 (5)預期效果及其重要性： 總結而言，本研究針對microRNA 多基因複合性表現的研究，能夠全面性的釐清 攸關於致癌基因和腫瘤抑制基因受到miRNA 調控下對乳癌發生和侵襲、轉移的分 子機制，並同時找出適切的乳癌臨床分子遺傳標記。再者，透過miRNA 在乳癌組 織和正常乳房上皮細胞的表現差異，經由DNA 和RNA 的分析結果關聯於乳癌分期 的分析，其研究成果亦將有利於清楚的呈現標的miRNA 對乳癌細胞生成以致於癌 細胞惡化、侵襲和轉移的預後評估，來提供重要關鍵的資料外；更有助於以miRNA 為標的，在未來發展有效的乳癌治療策略。Project