MicroRNA-148a與其標靶基因14-3-3β在胃癌轉移機制之探討

The Metastatic Mechanism of MicroRNA-148a and Its Target Gene 14-3-3β in Gastric Cancer

曾建偉(Chien-Wei Tseng)

指導教授 ： 阮雪芬

國立臺灣大學/生命科學院/分子與細胞生物學研究所/博士(2011年)

https://doi.org/10.6342/NTU.2011.02424

摘要

胃癌在全世界中為癌症致死率的第二名 (世界衛生組織2009年報導)。被診斷晚末期的胃癌病人五年存活率不到35%。較差的預後能力主要與腫瘤轉移有關。此外，有些微型核醣核酸被報導與腫瘤相關，其功能可能是致癌基因或腫瘤抑制基因，並且涉及了腫瘤形成及癌症發展。在本研究中，我們研究胃癌轉移的機制並鑑定出一個抗轉移的微型核醣核酸-148a，它在腫瘤組織中的表現較低。Kaplan–Meier存活方法顯示，相較於含有較低微型核醣核酸-148a含量的病人(32.1%)，其含有較高微型核醣核酸-148a含量的病人具有較高的五年整體存活率 (71.4%) (P = 0.03)。臨床資料指出微型核醣核酸-148a表現量增加與腫瘤遠處轉移(P = 0.043)、器官侵犯 (P = 0.013) 及腹膜侵犯 (P = 0.04) 有高度相關性。大量表現微型核醣核酸-148a減少腫瘤細胞侵犯、轉移及附著能力。此外，大量表現微型核醣核酸-148a抑制細胞生長並誘導細胞凋亡。我們利用了同位素標記相對和絕對定量方法分析微型核醣核酸-148a所調控的蛋白質體表現。微型核醣核酸-148a所調控的蛋白質體與腫瘤發展有高度相關性，其包括了細胞移動、生長與增生以及細胞凋亡。這些結果與我們之前的結果一致，微型核醣核酸-148a抑制胃癌細胞轉移相關的功能。另一方面，進一步的利用了冷光酶分析方法，我們確認了微型核醣核酸-148a可以直接調控14-3-3β表現。14-3-3β在腫瘤組織中的表現增加 (N = 40, P < 0.01)，而且血液14-3-3β在胃癌病人的含量也顯著的比正常人高(N = 63) (P < 0.0001)。具有較高血液14-3-3β含量的病人有較差的整體存活率(P = 0.038)。大量表現14-3-3β增加了腫瘤細胞生長、侵犯與移動能力。綜合上述結果，14-3-3β涉及了胃癌的轉移。微型核醣核酸-148a也許功能上是個腫瘤抑制基因，並且透過調控一個有潛力作為胃癌偵測與預後的14-3-3β生物標記表現以抑制了腫瘤細胞的轉移。

關鍵字

微型核醣核酸-148a ； 14-3-3β ； 胃癌 ； 轉移 ； 生物標記

並列摘要

Gastric cancer is the second leading cause of cancer deaths worldwide (WHO 2009 report). Patients diagnosed with advanced stages have a survival rate of less than 35% beyond 5 years. The poor prognosis is mainly related to tumor metastasis. In addition, some microRNAs (miRNAs) are reported as oncomirs which function as either oncogenes or tumor suppressors and involved in tumorigenesis and cancer progression. Here, we studied the mechanisms of gastric cancer metastasis and identified an antimetastatic miRNA, miR-148a, that was down-regulated in tumor tissues. Kaplan–Meier survival method revealed that patients with higher miR-148a expression levels had higher 5-year overall survival rates (71.4%) compared with patients with low miR-148a levels (32.1%, P = 0.03). Clinical data indicated that elevated miR-148a levels highly correlated with distant metastasis (P = 0.043), organ (P = 0.013) and peritoneal invasion (P = 0.04). Over-expression of miR-148a could decrease invasiveness, migration and adhesion of tumor cells. Moreover, Over-expression of miR-148a could repress cell growth and induce cell apoptosis. We further used isobaric tag for relative and absolute quantitation (iTRAQ) method to analyze miR-148a-regulated proteome. The results showed that miR-148a-regulated proteome was closely related with tumor progression, including cell movement, growth and proliferation as well as cell death. These results are consistent with our previous data that miR-148a suppresses metastasis-related functions of gastric cancer cells. On the other hand, we verified that miR-148a could directly regulate 14-3-3β expression using luciferase assay. 14-3-3β levels were elevated in tumor tissues (N = 40, P < 0.01), and serum 14-3-3β levels in cancer patients (N = 145) were also significantly higher than healthy controls (N = 63) (P < 0.0001). Patients with higher serum 14-3-3β levels had worse overall survival (P = 0.038). Over-expression of 14-3-3β enhanced the growth, invasiveness and migration of tumor cells. In conclusion, 14-3-3β is involved in metastasis of gastric cancer. miR-148a may function as a tumor suppressor in gastric cancer, suppressing cell metastasis through targeting 14-3-3β, a potential detective and prognostic marker in gastric cancer.

並列關鍵字

miR-148a ； 14-3-3β ； gastric cancer ； metastasis ； biomarker

參考文獻

[1] Bartel, D. P., MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004, 116, 281-297.

[2] Guo, H., Ingolia, N. T., Weissman, J. S., Bartel, D. P., Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 2010, 466, 835-840.

[3] Doench, J. G., Sharp, P. A., Specificity of microRNA target selection in translational repression. Genes Dev 2004, 18, 504-511.

[4] Esquela-Kerscher, A., Slack, F. J., Oncomirs - microRNAs with a role in cancer. Nat Rev Cancer 2006, 6, 259-269.

[5] Asangani, I. A., Rasheed, S. A., Nikolova, D. A., Leupold, J. H., et al., MicroRNA-21 (miR-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer. Oncogene 2008, 27, 2128-2136.