神經膠質瘤為常見於中樞神經系統(CNS)之惡性腫瘤，其中具高度惡性之神經膠質瘤常稱多型性神經膠質母細胞瘤 (Glioblastoma, GBM)，並常伴隨著高度復發情形及轉移的特性導致現今治療上的困難及不良預後。膠質細胞為中樞神經系統中主要的免疫細胞，在許多文獻被認為會協同發展腦膠質瘤生成的微環境促進其惡化程度，因此膠質細胞在腫瘤微環境所產生的細胞激素為現今研究的重要方向之一。Gata6為腫瘤抑制基因並被文獻證實能有效抑制神經膠質瘤的增生，但對於抑制復發及轉移能力仍缺乏相關研究。因此本篇論文探討Gata6與神經膠質瘤轉移之關係。因此我們首先分析高增生能力的C6-1與高度爬行能力的C6-2膠質瘤細胞株其原生性Gata6基因表現並無太大差異。但進一步利用shRNA干擾兩株細胞的Gata6基因表現後皆增強細胞爬行能力，於C6-2細胞中也同時促進細胞增生。綜合上述結果，Gata6確實抑制大鼠神經膠質瘤細胞的細胞增生及遷徙能力。本實驗室先前研究發現IL-33蛋白與MGP蛋白會促進神經膠質瘤的惡性程度及轉移能力，且於C6-1膠質瘤細胞中具高度原生性IL33基因與蛋白表現。本篇論文也證實抑制Gata6基因後會增強IL-33及MGP的基因表現，進而促進神經膠質瘤細胞的細胞增生及遷徙能力。因此本篇論文提供Gata6透過調控IL-33與MGP的訊息傳遞路徑可作為新穎的研究目標及未來治療標的方向。

Gata6 gene downregulation enhances rat glioma growth and invasion
Author: Cong-Kai Luo
Advisor: Shun-Fen Tzeng PhD
Co-Advisor: I-Chen Peng PhD
National Cheng Kung University Institute of Life Sciences

SUMMARY

Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor, and it has a poor prognosis and more recurrence by the metastasis ability and resistance to radiation and chemotherapy. Gata6 is a zinc-finger transcription factor, which involved in the development of the different organs. Gata6 was reported to regulate the development of the central nervous system (CNS). Previous studies found that Gata6 inhibited the EMT in pancreatic cancer, and Gata6 is potential as a tumor suppressor gene. According to those studies, we wonder that whether Gata6 regulate the cell growth and another physiological function of glioma. First, using two rat C6 glioma cells with different tumorigenesis. Our results showed that Gata6 gene expression in the two glioma cell lines C6-1 and C6-2 was not different. We observed that knockdown of Gata6 in rat glioma cells increased the cell proliferation of C6-2 cells, but no change in C6-1 cells. Moreover, inhibition of Gata6 enhanced the invasion and migration ability. Previous studies in our laboratory found that IL-33 promoted the tumorigenesis of glioma, and IL-33 is highly expressed in C6-1 cell lines. In addition, our previous study demonstrated that MGP can regulate the cell migration activity in C6 cells. Interestingly, the gene expression of IL-33 and MGP were upregulated after the inhibition of Gata6. Together, our findings indicate Gata6 is involved in the tumorigenesis of rat glioma possibly through the IL-33 and MGP regulatory pathway.

Key words: glioma, Gata6, IL-33, migration

INTRODUCTION

Glioblastoma multiform (GBM) is a common and the most malignant brain tumor with a poor prognosis because of the resistance of radiation and chemotherapy, and the median survival of the GBM patients has remained at less more 12 months. GBM was a grade Ⅳ glioma by histological classification of the World Health Organization (WTO). Gata6 is a zinc-finger transcription factor and involves in the development of CNS. Furthermore, Gata6 was reported to inhibit the EMT in pancreatic cancer and be a tumor suppressor gene in glioma. Recently, a study found that Gata6 expression was absent in the GBM but present in the LGA of the patient demonstrating secondary glioma. This finding suggested that the loss of Gata6 was involved in the glioma progression. C6 cell lines was a rat malignant tumor cells, has two type C6-1 and C6-2 with different tumorigenetic capacity. C6-1 got a better ability of cell proliferation, and C6-2 was more than invasive and migratory. In addition, our previous study indicated that IL-33 would regulate the tumorigenetic activity of C6 cells, and C6-1 has higher levels of IL-33 gene expression. Matrix gla protein (MGP) is vitamin-K2 dependent and gla containing protein. MGP has a high affinity to bind with calcium ions and role in inhibition of tissue calcification and vascular mineralization, but its mechanism is unknown. MGP was reported to involve in the progress of several cancer types, such as ovarian cancer, breast cancer and glioma. Moreover, C6-2 has higher MGP gene expression and MGP regulated its migratory ability. Thus, we wonder whether Gata6 would regulate the cell proliferation, migration and invasion ability, and may involve in which pathway to inhibit tumor growth and invasion.

METERIALS AND METHODS

Used two tumorigenetic rat glioma cell lines C6-1 and C6-2 to demonstrate the function of Gata6 in tumor growth and tumorigenesis. Gata6 was downregulated to observe the change of physiological functions in C6 cells by lentivirus-mediated shRNA. To detect the cell proliferation of depleted Gata6 in C6 cells, we used MTT assay, colony formation. Used migration assay, cell invasion assay to observe the migratory and invasive capacity of Gata6 downregulated C6 cells. To detect the change of gene expression, we used quantitative polymerase chain reaction (QPCR). Used t-test to analyze the significant difference between the control and sample groups.

RESULTS

Knockdown of Gata6 did not affect the cell morphology of C6 cells, and promoted the cell proliferation of C6-2 cells but not C6-1 cells by MTT assay. Interestingly, downregulated Gata6 did not increase the colony formation numbers, but increased the colony size in C6-1 cells and colony numbers in C6-2 cells. Next, the migration ability was enhanced in C6 cells significantly by the depletion of Gata6, but only Gata6-KD1 promoted the cell invasion ability of C6 cells. In addition, the IL-33 and MGP mRNA levels were induced by downregulated Gata6 in C6 cells.

CONCLUSIONS

Our data found that Gata6 inhibited the tumorigenetic capacity in the two different malignancy C6 cells. In addition, previous study demonstrated that IL-33 and MGP would promote the progress and malignancy of glioma. Our findings suggested that the IL-33 and MGP gene expression were increased by knockdown of Gata6 in C6-1 cells. Thus, we indicated that Gata6 inhibited the tumorigenesis of C6 cells possibly through the IL-33 and MGP pathways. In the future, we will confirm the relationship between the Gata6 and IL-33 and MGP by more powerful evidences.

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