| 摘要: | n-3、n-6多不飽和脂肪酸(unsaturated fatty acid, PUFA)雖具抗癌作用，但對預後再 復發的乳癌轉移調控機制仍不清楚。由預備實驗發現，docosahexaenoic acid (DHA)顯著 抑制MCF-7乳癌細胞integrin α6、β1、β4、EGFR、ErbB2蛋白質表現，並抑制integrin-、 ErbB-mediated相關訊號路徑活化。研究指出integrin及EGFR於細胞膜lipid raft的聚集和交 互作用是啟動integrin-、ErbB-mediated轉移訊號的關鍵。然而DHA及其它PUFAs是否可 能透過破壞integrin、ErbB家族蛋白於lipid raft的結合，進而抑制乳癌轉移之機制，仍不 清楚。microRNA (miRNA)為可由生物體自行合成的RNA片段，在特定基因轉錄後修飾 作用中扮演負向調節者。雖然已有多個與癌細胞轉移相關miRs如miR-96、miR-124和 miR-301被發現，但受n-3 PUFAs影響而調控乳癌細胞發展的也僅miR-21獲得證實。預先 以 miR 晶 片 及 Venn diagram 比 對 出 四 個 與 癌 細 胞 轉 移 較 相 關 miRs: miR-21-3p/-197-3p/-363-5p和miR-1290作探討，以釐清PUFAs是否透過此特定miRs影響 其標的基因，而調控乳癌細胞轉移。OKL38 (OSGIN1)被認為是腫瘤抑制蛋白。臨床研 究發現惡性程度較高的腫瘤組織OKL38表現量皆高於鄰近正常組織，在乳癌細胞也發現 相似情況。截至目前，PUFAs調控OKL38的研究相當少，值得深入研究。由預備實驗得 知，只有DHA顯著誘發OKL38表現。本計畫以Hs578T、MDA-MB231、SKBR3及MCF-7 乳癌細胞為實驗模式，分二部分探討。(一) DHA及其它PUFAs是否透過影響細胞膜lipid raft組成而改變integrin、ErbB家族蛋白表現與活化，調控癌細胞轉移之訊號傳遞路徑及 基因表現。另外也將探討PUFAs是否透過特定miRs影響標的基因表現，而調控乳癌細胞 轉移。(二) DHA誘發OKL38基因表現機制，並釐清OKL38是否在DHA抑制乳癌細胞轉 移、促進癌細胞凋亡扮演重要角色。
Metastasis is the leading cause of death from breast cancer. Previous studies showed n-3 polyunsaturated fatty acids (PUFAs) exhibit an anti-cancer effect, but the effect of n-3 and n-6 PUFAs on metastasis of breast cancer cells is not fully clarified. The evidence from human gene expression array indicated the expression of integrin and EGFR family protein were down-regulated by DHA in MCF-7 breast cancer cells. In our preliminary, we found the integrin α6, β1, β4, EGFR and ErbB2 protein level was decreased by DHA, resulted in the suppression of integrin-mediated and ErbB-mediated signaling pathways. It has been shown that the cooperation between integrin and epidermal growth factor receptor (EGFR) signaling in lipid raft regulates certain signaling functions that are important for breast cancer progression. However, whether DHA and other PUFAs alter integrin- and EGFR-related signaling by disrupting its lipid raft association is not fully understood. MicroRNAs (miRs) are small non-coding RNAs of ~22 nucleotides that function at post-transcriptional level as negative regulators of gene expression. In spite of several miRs, such as miR-96, miR-124 and miR-301, were found to regulate migration and invasion of breast cancer cells. Up to data miR-21 is only targeted by n-3 PUFAS to regulate breast tumor growth. Our preliminary data indicated 51 miRs expression were modulated by DHA in MCF-7 cells. Among these miRs, we choose miR-21-3p/-197-3p/-363-5p and miR-1290 associated with cell migration to investigate whether predicted target genes involved in the inhibition of migration by DHA. OKL38 (OSGIN1) may be identified as a tumor suppressor gene that inhibits tumor cell growth. Clinical study showed OKL38 level was downregulated or lost in various malignant tumors and cancer cell lines, especially breast cancer cells. Thus, we are interested to investigate the effect of PUFAs on OKL38 expression. Among n-3 and n-6 PUFAs, only DHA significantly up-regulated OKL38 protein and mRNA expression, suggests the anti-tumor role of OKL38 may be involved in DHA’s anti-cancer action. However, the effect of OKL38 on DHA inhibition of breast cell migration and apoptosis is not fully clarified. In this project, we’ll used Hs578T, MDA-MB231, SK-BR3, and MCF-7 human breast cancer cells to study the effect of AA, LA, GLA, LNA, EPA and DHA on integrin/EGFR interaction in lipid raft and further clarify whether the inhibition of cell migration by PUFAs through suppression of integrin-mediated and EGF-mediated signaling by disrupting its lipid raft. In addition, we’ll investigate whether OKL38 and specific miRs and their target genes involved in the inhibition of migration by DHA in breast cancer cells. |
