藉由二維差異電泳分析乳腺細胞缺乏PTEN之影響

Effect of PTEN-deficiency on protein profile of mammary cell via two-dimensional differential gel electrophoresis

王筱婷(Hsiao-Ting Wang)

指導教授 ： 林立元

國立清華大學/生命科學院/分子與細胞生物研究所/碩士(2010年)

摘要

在癌細胞中經常觀察到腫瘤抑制基因或蛋白的缺失，並伴隨其他基因或蛋白質調控情形的改變。因此，在本篇實驗中，我們利用siRNA的方式在正常乳腺細胞 (ME16C)降低一種乳癌中常見缺失的腫瘤抑制蛋白質—PTEN，並藉由二維差異電泳及基質輔助雷射-飛行時間式質譜儀分析蛋白質體的改變，鑑定出十個在PTEN缺失時具差異表現的蛋白質。分析發現，它們大部分具有致癌或腫瘤抑制的能力，例如調控細胞生長 (alpha-enolase)、增生 (NF2)、細胞骨架重組 (Hsp27)、凋亡 (ST20，suppressor of tumorigenicity 20)等功能，其中也有未知功能的蛋白質 (CCDC90A)。我們更進一步的利用西方墨點法和即時定量聚合酶連鎖反應，來觀察這些蛋白質是在基因轉錄或蛋白質轉譯層級受到PTEN表現量改變的調控；最後比較這些蛋白質在正常乳腺細胞 (MCF10A)、低侵略性 (MCF7)和高侵略性 (MDA231)乳癌細胞中，基因及蛋白質的表現情形。綜合分析之後，我們提出這些蛋白質可能受PTEN調控的機制及其在乳癌進程中的意義和關聯性；其中CCDC90A這個未知功能的蛋白質更首次被我們發現受PTEN調控，且在模式細胞株中具有隨癌化程度增加表現的趨勢。期待透過本篇研究，對於往後瞭解這些蛋白質受調控的機制及其與癌症的關係具有新的方向及幫助。

關鍵字

二維差異電泳 ； 乳腺細胞 ； 蛋白質體

並列摘要

Loss of tumor suppressor is a common phenomenon in tumor cell. The defect accompanies with alterations of gene transcription and protein expression profiles. In this study, a frequently lost tumor suppressor in breast cancer, PTEN, is knockdown by siRNA in human mammary cell. Protein profile of the PTEN depleted cells was analyzed by two-dimension differential gel electrophoresis combined with matrix-assisted laser desorption ionization/time of flight mass spectrometry. Ten of the protein spots were identified, and most of them play roles in oncogenesis or tumor suppression. These proteins participate in cell growth (alpha-enolase), proliferation (NF2), cytoskeleton rearrangement (Hsp27), and apoptosis (suppressor of tumorigenicity 20, ST20). Additionally, a functional unknown protein, CCDC90A, is also found. We subsequently used Western blotting and real-time quantitative polymerase chain reaction to examine whether the differential expressions are regulated at gene transcriptional or protein translational level. The gene expression and quantity of the identified proteins were compared among normal mammary cell (MCF10A), non-invasive and invasive breast cancer cell (MCF7 and MDA231, respectively). In summary, we investigated the proteins that differentially expressed in PTEN knock-down cells and analyzed the correlations to breast tumor progression. We identified that CCDC90A is negatively regulated by PTEN expression and up-regulated through tumor progression; a phenomenon that has not been reported. This study offers extended information for future research on those proteins to explore the occurrence and progression of breast cancer.

並列關鍵字

PTEN ； 2D-DIGE ； MALDI-TOF

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