腦膠質瘤(glioma) 是大腦中常見的惡性腫瘤，目前較普遍的治療方法是利用手術切除配合化學治療與(或)放射性治療來殺死腫瘤細胞，然而預後之存活率的提升仍然有限。B-cell leukimia/lymophoma 11B(Bcl11b,或稱CTIP2)為一種轉錄因子，表現在許多不同組織，包含中樞神經系統、胸腺、皮膚、胃上皮組織等。Bcl11b也被視為腫瘤抑制基因，在小鼠實驗研究發現，若缺乏Bcl11b基因，將會導致淋巴癌或白血症等病變。近期有越來越多的文獻指出，Bcl11b若表現在腫瘤細胞中，反而會扮演支持與促進腫瘤生長的角色，如在Ewing sarcoma細胞中，有大量Bcl11b基因表現，並使其更加惡化，本實驗室先前研究發現在生長較快速的腦膠質瘤細胞株C6中也發現高表現量的Bcl11b基因；並且，在多株人類神經膠質瘤細胞株中，也有高量的Bcl11b表現；因此，本研究欲進一步探討Bcl11b基因對於腦膠質瘤細胞之影響。本實驗以大鼠腦膠質瘤C6細胞株與人類腦膠質瘤細胞株U87為細胞模式，並利用帶有sh-Bcl11b基因之慢病毒感染細胞株，實驗結果顯示，在大鼠與人類腦膠質瘤細胞中，若降低Bcl11b基因表現，會抑制腫瘤細胞的增長速度和形成細胞群落之能力。另外，研究指出，細胞內代謝失調，導致過多的ROS累積，進而造成正常細胞的受損；但在腫瘤細胞中，過多的ROS累積卻會促使腫瘤細胞的生長。本研究結果指出，抑制Bcl11b基因之腫瘤細胞，其細胞內之ROS程度也大幅降低。而另一方面，在C6細胞與U87細胞觀察到，抑制Bcl11b基因後，造成週期蛋白依賴性激酶抑制分子(CDKi, p21)表現量上升。Bcl11b基因表現下降促使腫瘤細胞之細胞週期停滯，並且導致腫瘤細胞趨向老化之現象。基於本篇研究結果，我們指出表現於腦膠質瘤細胞的Bcl11b分子藉由作用於細胞週期抑制分子調控細胞的生長機制。

INTRODUCTION
Glioma is the most malignant brain tumor in adult. The most common treatments to glioma include a combination of surgery, radiotherapy, and chemotherapy. However, due to the high recurrence and invasiveness of glioma, the low survival rate of the patients with glioma after prognosis remains. B-cell leukimia/lymophoma 11B (Bcl11b), also named as CTIP2, is a transcription factor that is expressed predominantly in the central nervous system, thymocytes, skin, olfactory epithelium, and epithelia of oral and gut. The studies from others have demonstrated that Bcl11b acts as a tumor suppressor. Mutations or deletions of Bcl11b have been found in T-cell acute lymphoblastic leukemia. In contrast, the deficiency of Bcl11b can induce DNA damage and result in cell apoptosis. Recently, researchers have reported that Bcl11b was highly expressed in some tumor cells, such as Ewing sarcoma cells. In our previous study, the expression of Bcl11b was enriched in tumorigenic C6 glioma cell line. Thus, we were to determine the biological function of Bcl11b in glioma cells.

MATERIALS AND METHODS
Tumorigenic rat glioma cell line (C6) and human malignant glioblastoma (MG) cell line (U87) were used in this study to determine the functional role of Bcl11b in tumor cells. Lentivirus-mediated gene knockdown approach was conducted to inhibit the expression of Bcl11b in the two cell lines. Quantitative reverse transcription polymerase chain reaction (Q-PCR) and western blotting were used to measure mRNA expression and protein levels of Bcl11b in glioma cells. Immunofluorescence was used to examine the cellular localization of Bcl11b. MTT assay and colony formation assay were used to investigate cell proliferation and colony formation ability. Propidium iodide (PI) staining was used to detect the cell cycle of glioma cells. Senescence detection kit was used to investigate the activity of β-galactosidase which is highly accumulated in the lysosomes of senescent cells. 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) was used to detect ROS levels in glioma cells.

RESULTS
Two cell strains with stably suppression of Bcl11b expression in C6 cells were generated by lentivirus-mediated Bcl11b gene knockdown system (C6-KD1 and C6-KD2). The inhibition of Bcl11b expression caused significant reduction in cell proliferation and colony formation ability of C6-KD1 and C6-KD2 cells compared with C6-mock cells. We also noticed that the cell morphology of the C6-KD1 and C6-KD2 cells changed from a bipolar shape toward a flattened form. In addition, Bcl11b was highly expressed in several human malignant glioma cell lines, including U373MG, U87MG, A172, U251 cells. Among these human malignant glioma cell lines, U87MG cells expressed the highest level of Bcl11b. Similar to the findings observed in C6 cells, the inhibition of Bcl11b expression not only caused the morphological change of U87 cells to the flatten shape, but also effectively suppressed their cell proliferation and decreased the colony formation ability. The result from ROS generation assay showed that the ROS level in Bcl11b-KD cells is lower the control cells. Furthermore, p21, a cyclin-dependent kinase inhibitor (CDKi), was significantly upregulated in C6 and U87 cells after Bcl11b gene knockdown. Propidium iodide (PI) staining combined with flow cytometry analysis provided the evidence showing that Bcl11b downregulation resulted in cell cycle arrest. Interestingly, after the downregulation of Bcl11b, glioma cells underwent senescence.

CONCLUSIONS
Bcl11b plays the essential role in the cell proliferation of glioma cells. The deficiency of Bcl11b can cause arrested growth and promoted cellular senescence in glioma cells. This could be due to the upregulation of p21 expression after Bcl11b gene knockdown.

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