MiR-1271透過抑制標的CAMKK2降低多型性膠質母細胞瘤的增生與侵犯

MiR-1271 attenuates proliferation and invasion in glioblastoma multiforme by inhibiting the expression of CAMKK2

黃裕凱(Yu-Kai Huang)

指導教授 ： 林志隆

高雄醫學大學/醫學院/臨床醫學研究所碩士班/碩士(2019年)

摘要

神經膠質瘤的特徵是增生不受控制，血管生成和正常周圍腦組織浸潤，約佔成人腦腫瘤的50％，膠質母細胞瘤（Glioblastoma, GBM）是最常見的惡性膠質瘤。根據世界衛生組織（World Health Organization, WHO）的分類，GBM被分類為IV級膠質瘤。 膠質母細胞瘤患者的平均生存時間約為14個月。雖然相關研究不斷的進步，但膠質母細胞瘤增生(Proliferation)和侵犯(Invasion)的調控機制尚不明確，目前還沒有有效的治療方法。目前的主要研究模式是以膠質瘤細胞株來探討膠質母細胞瘤的病理及分子機制。因此，本研究以膠質瘤細胞株探討增生和侵犯的相關機制，此研究對於今後的臨床治療具有重要意義。 miRNA （microRNA或miR）對癌症訊號傳導途徑的抑制作用已運用於前瞻性癌症治療。 MiR-1271在胃癌和前列腺癌中具有腫瘤抑制作用，但其在腦癌中的功能尚不清楚。細胞內過高的鈣離子濃度會導致多種的腫瘤轉移(Metastasis)和增生。細胞內的鈣離子會活化CaMKK2並誘導AKT活化，這個訊息在膠質母細胞瘤細胞增生(Proliferation)和侵犯(Invasion)中具有重要作用。本研究旨在探討miR-1271在調節細胞內鈣離子所誘導的CaMKK2/AKT活化路徑，以及促使膠質母細胞瘤細胞增生(Proliferation)和侵犯(Invasion)中所扮演的角色。
本研究使用RNA-seq來分析人類高惡性度膠質細胞瘤和膠質母細胞瘤的組織之間基因表現差異。我們的研究結果顯示，CACNA1G，RYR3和CAMKK2基因在膠質母細胞瘤患者中高度表現。CACNA1G及RYR3是鈣離子通道蛋白，會提高細胞內鈣離子濃度，CaMKK2是接受鈣離子後活化的蛋白，並進行訊息傳遞導致膠質母細胞瘤細胞增生(Proliferation)和侵犯(Invasion)。此外本研究也發現，與低惡性度膠質細胞瘤相比，高惡性度膠質細胞瘤患者中miR-1271的表現顯著降低。透過real-time PCR和western blot證實miR-1271能夠降低膠質母細胞瘤細胞中的CaMKK2表現。
我們的結果顯示miR-1271透過調控細胞內CaMKK2途徑抑制了膠質母細胞瘤細胞增生和侵犯。這些證據使我們推測miR-1271可能對膠質母細胞瘤具有潛在抑制作用。因此，我們得出結論miR-1271透過標的CaMKK2進而調節膠質母細胞瘤細胞的增生和侵犯。

關鍵字

多型性膠質母細胞瘤 ； 轉移 ； 增殖

並列摘要

Gliomas are characterized by uncontrolled proliferation, angiogenesis and infiltration of normal surrounding brain tissue, accounting for approximately 50% of adult brain tumors Glioblastoma (GBM) is the most common malignant glioma and has been classified into four grades according to the World Health Organization (WHO) classification. The median survival time of patient with GBM is approximately 14 months. Although the relevant researches continue to improve, the regulatory mechanism GBM on invasion and proliferation is not clear up to now; there is no effective treatment for GBM therapy. The current main research model is to investigate the pathological and molecular mechanisms of glioblastoma by glioma cell lines. Therefore, this study explores the mechanisms of invasion and proliferation in glioma cell lines, and this study is one of great significance for future clinical treatment. The inhibition role of miRNA (microRNA or miR) on cancer signaling pathways has been used to prospective cancer treatment. miR-1271 plays a tumor suppressor role in gastric and prostate cancer, but its function in brain cancer is not clear.
Excessive intracellular calcium concentration leads to a variety of tumor invasion and proliferation. Intracellular calcium ions activate CaMKK2 and induce AKT activation, a message that plays an important role in GBM cells invasion and activation. This study was designed to investigate the role of miR-1271 in regulating CaMKK2/AKT activation pathway induced by intracellular calcium and in promoting GBM cells invasion and proliferation.
The RNA-seq assay was used to analyze the differences in gene expression between human high grade glioma and GBM tissues. Our results showed that CAMKK2, CRYR3 and CACNA1G genes (calcium channels) were highly expressed in patients with GBM. CACNA1G and RYR3 are calcium channel proteins that increase intracellular calcium ion concentration. CaMKK2 is a kinase protein that is activated by calcium ions and transmits signals to cause proliferative and invasion of GBM cells.
We have also found that the expression of miR-1271 was significantly reduced in patients with high- grade glioma compared to the low grade glioma. MiR-1271 was experimentally confirmed to knock down the expression of CaMKK2 in GBM cells by real-time PCR and western blot.
Our results showed that miR-1271 decreased GBM cells invasion and proliferation through intracellular calcium induced pathway. In addition, we also found that the cacna1g, ryr3 and camkk2 genes were highly expressed in patients with grade IV glioma. CACNA1G and RYR3 are calcium channels that increase intracellular calcium concentration. CaMKK1 is a protein that is activated by calcium ions and transmits signals, leading GBM cells invasion and activation. Moreover, our experimental results also showed that miR-1271 was significantly decreased in patients with high-grade gliomas compared with low-grade gliomas. It was confirmed by real time PCR and western blot that miR-1271 reduced the expression of CaMKK2 in GBM cell lines (U87 and 8401). Our results showed that miR-1271 reduced GBM cells invasion and proliferation through regulation of intracellular CaMKK2 pathway. These evidences suggest that miR-1271 may have inhibitory effect on glioma development, which regulates GBM proliferation and invasion through the targeting of CaMKK2.

並列關鍵字

glioblastoma ； miR-1271 ； metastasis ； proliferation ； RYR3 ； CACNA1G

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