評估細胞轉移相關基因群作為口腔鱗狀上皮細胞癌之腫瘤標記

Assessment of metastasis related genes as tumor markers of oral squamous cell carcinoma

鄭宇珊(Yu-Shan Cheng)

指導教授 ： 張學偉

高雄醫學大學/生命科學院/生物醫學暨環境生物學研究所/碩士(2010年)

https://doi.org/10.6832/KMU.2010.00087

摘要

口腔癌已列入十大癌症死因之一。本研究目的為尋找出口腔鱗狀上皮細胞癌(oral squamous cell carcinoma, OSCC)的腫瘤標記。我們使用bioinformatics工具及先前microarray數據，來分析與細胞轉移和細胞外基質（ECM）途徑相關基因的蛋白質相互關係，候選基因如 matrix metalloproteinase-2 (MMP2)、matrix metalloproteinase-9 (MMP9)、TIMP metallopeptidase inhibitor 1 (TIMP1)、met proto-oncogene (MET)、syndecan 4 (SDC4) 和laminin alpha 4 (LAMA4)，之後以Real-time PCR實驗驗證檢體中各個基因表現結果。為了驗證其表現量，我們收集了53組成對的OSCC組織和周邊安全邊緣的非腫瘤組織以 Real time-PCR分析，各候選基因的比較表現量如下: MMP2 (51.71±2.37 vs. 51.02±1.78; p=0.048; paired-samples t-test)、MMP9 (48.85±3.17 vs. 43.90±5.77, p<0.0001)、TIMP1 (51.76±2.72 vs. 50.47±3.54, p=0.006)、MET (41.12±2.87 vs.. 38.99±3.34, p<0.001)、SDC4 (51.60±1.94 vs. 49.65±5.5651; p<0.008)及 LAMA4 (49.32±3.45 vs. 49.04±3.13; p=0.593)。而後使用Receiver Operation Characteristic (ROC)分析，評估候選基因其敏感性和專一性。各個基因的曲線下面積(Area Under Curves)分別為 0.614 (MMP2)、0.822 (MMP9)、0.595 (TIMP1)、0.712 (MET)、0.676 (SDC4)和0.534 (LAMA4)。此外，我們依照年齡、性別、癌症分期和吸煙、飲酒、嚼檳榔的習慣與否等分群作適度調整，而後進行相關統計分析。以Kruskal-Wallis test可得到LAMA4基因相對表現與有無抽菸習慣有顯著差異(p=0.01)；以??2 test可得頰黏膜/臼齒後區的腫瘤發生高表現量群與MET表現具有顯著差異(p=0.04)，高表現量群有無喝酒菸習慣與SDC4具有顯著差異(p=0.03)，在LAMA4上與有無抽菸習慣高表現量群有顯著性的差異存在(p=0.002 )。結論，本研究驗證有四個基因具有顯著差異，其中MMP9為最有潛力作為口腔癌的分子標記，其次分別為MET、 SDC4及 MMP2。

關鍵字

口腔鱗狀上皮細胞癌 ； 即時定量聚合連鎖反應敏 ； 敏感性和專一性 ； 腫瘤標記

並列摘要

Oral cancer has been included among the top ten causes of death among cancer. However, it still challenged to find out more tumor markers in oral squamous cell carcinoma (OSCC). We performed protein interaction analysis for some candidate genes related to cell metastasis and extracelluar matrix (ECM) pathway by using bioinformatics tools to mine on our previous microarray data. Candidate genes including matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-9 (MMP9), TIMP metallopeptidase inhibitor 1 (TIMP1), met proto-oncogene (MET), syndecan 4 (SDC4) and laminin alpha 4 (LAMA4). To validate the performance for detection OSCC, we had collected 53 paired OSCC and margin controls for real-time PCR analysis. Currently, gene expression of these candidates in OSCC are compare with that of in control for MMP2 (51.71±2.37 vs. 51.02±1.78; p=0.048; paired-samples t-test), MMP9 (48.85±3.17 vs. 43.90±5.77, p<0.0001), TIMP1 (51.76±2.72 vs. 50.47±3.54, p=0.006), MET (41.12±2.87 vs. 38.99±3.34, p<0.001), SDC4 (51.60±1.94 vs. 49.65±5.5651; p<0.008) and LAMA4 (49.32±3.45 vs. 49.04±3.13; p=0.593) genes. Using the receiver operation characteristic (ROC) analysis to evaluate the sensitivity and specificity for all these candidate genes tested, we found that the areas under curve (AUC) are 0.614, 0.822, and 0.595, 0.712, 0.676 and 0.534 for MMP2, MMP9, TIMP1, MET, SDC4, and LAMA4, respectively. Besides, we will perform the statistics analysis by adjusting with age, gender, cancer stage/grade, smoking, drinking and betel chewing. There are significant difference of LAMA4 gene in smoking(p=0.01) by Kruskal-Wallis test. The result suggested the gene up-expression for MET gene in BM/retromolar area(p=0.04,?n??2?ntest), SDC4 in drinking (p=0.03)and LAMA4 in smoking(p<0.002) significantly detected the OSCC. In conclusion, this study demonstrated significant differences in four genes, including MMP9 for the most promising molecular marker for oral cancer, followed by the MET, SDC4 and MMP2.

並列關鍵字

oral squamous cell carcinoma ； Real-time PCR ； Receiver Operation Characteristic (ROC) ； matrix metalloproteinase-2 (MMP2) ； matrix metalloproteinase-9 (MMP9) ； TIMP metallopeptidase inhibitor 1 (TIMP1) ； met proto-oncogene (MET) ； syndecan 4 (SDC4) ； laminin alpha 4 (LAMA4)

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