微型核糖核酸逆調控LCN2在乳癌進程所扮演的角色

Downregulation of LCN2 through microRNA to suppress breast cancer progression

林吟庭(Ying-Ting Lin)

指導教授 ： 鄭鈞文

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2015年)

摘要

研究指出Lipocalin 2(LCN2)與乳癌細胞增生、分化、形態、移動及侵襲能力等有關。臨床上發現乳癌預後不良患者組織中有高度表達LCN2的情形。LCN2會與MMP-9結合形成異源二聚體 (heterodimer)，保護MMP9不被降解 (degradation)，LCN2/MMP-9會促進血管新生因子 (VEGF) 生成，使腫瘤得到更多養分，另外，LCN2會活化AKT訊號傳遞路徑使乳癌細胞轉移及侵襲。微型核糖核酸 (microRNA, miRNA)會尋找特定目標基因，抑制核糖體轉譯標的基因蛋白之表現，能調節癌細胞增生、抗凋亡、血管新生、轉移及侵襲，本篇研究想找到調控LCN2的miRNA，以此miRNA來抑制乳癌進程。先以Targetscan預測調節LCN2的miRNA，並將找出的結果與乳癌病患組織之microarray比對，發現病患預後不良組織中某個miRNA表現量較低，且也被預測可結合LCN2 3’-UTR，因此以此作為本論文研究重點。首先，以冷光素酶報導實驗 (luciferase reporter assay) 分析發現此miRNA確實會結合LCN2 3’-UTR，並經由西方墨點法 (Western blot)證實此miRNA能抑制LCN2的蛋白表現，由功能性試驗分析結果得知，穩定表達此miRNA的乳癌細胞株能降低細胞遷移及侵襲能力，進而降低LCN2表現、ERK及AKT訊息傳遞路徑之活化，以抑制乳癌細胞的進程。

關鍵字

LCN2 ； 乳癌 ； 微型核醣核酸

並列摘要

Lipocalin 2 (LCN2) is about cell proliferation, differentiation, morphology, movement and invasion in breast cancer. And late-stage breast cancer cells content higher level of LCN2. Clinically, poor prognosis for patients in breast cancer tissue was found contain highly expressed LCN2. LCN2 and MMP-9 are to combine to form a heterodimer, then MMP9 will be protected and won't autodegradation. LCN2 / MMP-9 promote angiogenesis factor (VEGF), and the tumor can get more nutrients. In addition, LCN2 activates AKT signal pathway resulted in breast cancer cell metastasis and invasion. microRNA regulate cancer cell proliferation, apoptosis, angiogenesis, metastasis and invasion in human cancers. microRNA (miRNA) will be looking for a specific target gene. miRNA can inhibit the translation of ribosomal of target gene. We want to find which miRNA can regulate LCN2. In our research, we want to find the miRNA which can regulate LCN2. We take the predicted result to match with patients’ tissue. This study predicted the candidate miRNA which is able to adjust LCN2 by Targetscan. The candidate miRNA is compared with previous in-vivo study on micrroarry of breast cancer patients’ tissue. It comes out the result of lower expression in tissues of poor prognosis patients’. So we use this miRNA to do our key point. First, we used Targetscan to predict which miRNA may bind on LCN2 3'UTR. And we analyse this miRNA and LCN2 3'UTR combination by luciferase reporter assay. At the same time, we prove this miRNA inhibits protein expression of LCN2 by Western blot. And we found this miRNA not only reduce expression of ERK and AKT phosphorylation, but also MMP-9 and VEGF. The result of the functional assay, this miRNA can downregulate LCN2 and suppress migration and invasion in breast cancer. This miRNA will bind on LCN2 3’-UTR, and suppress translation of LCN2. Further, reducing LCN2 effect on active AKT and ERK signal pathway, and inhibit migration and invasion in breast cancer.

並列關鍵字

LCN2 ； breast cancer ； miRNA

參考文獻

1. Hsieh Kun-Pin, C.L.-C., Cheung Kwok-Leung and Y.-H. Yang, Risks of nonadherence to hormone therapy in Asian women with breast cancer. The Kaohsiung Journal of Medical Sciences, 2015. 31(6): p. 328-334.

2. Society, A.C., Breast Cancer Staging. American Joint Committee on Cancer, 2009(7th EDITION).

3. Morgan Jenna L., B.M., Collins Karen, et al., The balance of clinician and patient input into treatment decision-making in older women with operable breast cancer. Psycho-Oncology, 2015: p. n/a-n/a.

4. Taipaleenmaki, H., et al., Targeting of Runx2 by miR-135 and miR-203 Impairs Progression of Breast Cancer and Metastatic Bone Disease. Cancer Research, 2015. 75(7): p. 1433-1444.

5. Ying Hua, J.X., Ying Yanga, et al., Lipocalin 2 Upregulation Protects Hepatocytes from IL1-β-Induced Stress. Cellular Physiology and Biochemistry, 2015. 36: p. 753-762.