核黄素转运基因2（RFT2）在胶质瘤中表达及作用研究

发布时间：2018-12-12 13:59

【摘要】：目的:原发的成人脑肿瘤中胶质瘤是最常见的类型。它侵袭性强,与周围组织界限不清晰。因此,常规的治疗手段如手术治疗和放射治疗对它作用都很有限。不但如此,胶质瘤的耐药性也导致其化学治疗收获甚微。因此,胶质瘤患者需要更有效的治疗方案,而这些治疗方案的建立需要我们找到新的分子靶点。核黄素转运基因2(RFT2)是SLC52家族的一员,它在核黄素的代谢中起着重要的作用。最近,一些研究已经证实,RFT2在许多人类肿瘤(食管鳞状细胞癌、胃癌、宫颈癌)的发生发展中十分重要。本研究旨在探究RFT2在人类胶质瘤中的表达情况及其对胶质瘤细胞生物学特性的作用,进而为胶质瘤的分子治疗提供新的靶点。方法:为了检测RFT2在正常脑组织和人类脑胶质瘤组织中的表达水平,我们对120例不同级别的胶质瘤组织、24例正常脑组织进行了免疫组织化学染色。利用RFT2-siRNA-3抑制胶质瘤细胞系LN308和LN229中RFT2的表达。使用MTT、平板克隆形成实验、流式细胞术、Transwell实验、Western blot和裸鼠皮下荷瘤模型从体内和体外两方面来进行胶质瘤细胞增殖、迁移、侵袭等表型的实验评估。结果:1、与正常脑组织相比RFT2在胶质瘤组织中是高表达的。而且,胶质瘤中RFT2的表达水平与WHO分级呈正相关。2、与对照组和无义序列转染组相比,敲低LN308和LN229细胞RFT2的表达后细胞增殖被明显抑制(P0.01),细胞集落形成率明显下降(P0.01)。敲低RFT2可有效抑制移植瘤的体内生长(P0.001)。3、敲低RFT2后LN308和LN229细胞阻滞在G1/G0期(P0.05)。在LN308和LN229细胞中敲低RFT2可以明显升高其凋亡细胞的比例(P0.05)。各组中细胞周期和凋亡相关调节蛋白(p21、p27、Bcl-2、Bax)的表达都明显改变。4、敲低RFT2后LN308和LN229细胞的迁移和侵袭能力受到抑制,其相关调节蛋白MMP-2和MMP-9的表达水平也相应下降。结论:本研究发现,胶质瘤组织中RFT2呈高表达,它的表达水平与胶质瘤WHO分级呈正相关。敲低RFT2促进了LN308和LN229细胞的细胞周期阻滞、细胞凋亡,抑制了细胞增殖、迁移和侵袭的能力。不但如此,裸鼠荷瘤模型也进一步更好地证明了敲低RFT2后胶质瘤细胞的增殖受到抑制。这些结果提示,RFT2可以成为胶质瘤分子治疗的新靶点。
[Abstract]:Objective: gliomas are the most common type of primary adult brain tumors. It is aggressive, and its boundaries with surrounding tissues are not clear. As a result, conventional treatments such as surgery and radiotherapy have limited effects on it. Moreover, the drug resistance of gliomas leads to little success in chemotherapy. Therefore, glioma patients need more effective treatment protocols, and the establishment of these protocols requires us to find new molecular targets. Riboflavin transporter gene 2 (RFT2) is a member of SLC52 family and plays an important role in riboflavin metabolism. Recently, some studies have confirmed that RFT2 plays an important role in the development of many human tumors (esophageal squamous cell carcinoma, gastric cancer, cervical cancer). The purpose of this study was to investigate the expression of RFT2 in human glioma and its effect on the biological characteristics of glioma cells, and to provide a new target for the molecular therapy of glioma. Methods: in order to detect the expression of RFT2 in normal brain tissues and human glioma tissues, immunohistochemical staining was performed in 120 gliomas of different grades and 24 normal brain tissues. The expression of RFT2 in glioma cell line LN308 and LN229 was inhibited by RFT2-siRNA-3. The phenotypes of glioma cell proliferation, migration and invasion were evaluated in vivo and in vitro by MTT, plate clone formation assay, flow cytometry, Transwell assay, Western blot and nude mice subcutaneous tumor model. Results: 1. The expression of RFT2 in glioma was higher than that in normal brain tissue. Moreover, the expression of RFT2 in gliomas was positively correlated with the WHO grade. 2Compared with the control group and the nonsense sequence transfection group, the proliferation of LN308 and LN229 cells was significantly inhibited after the expression of RFT2 was lowered (P0.01). The colony formation rate decreased significantly (P0.01). Knockout of RFT2 could effectively inhibit the growth of transplanted tumor in vivo (P0.001). 3. LN308 and LN229 cells were blocked in G1/G0 phase after knock down RFT2 (P0.05). Knockout of RFT2 in LN308 and LN229 cells significantly increased the percentage of apoptotic cells (P0.05). The cell cycle and the expression of apoptosis-related regulatory protein (p21, p27, Bcl-2, Bax) were significantly changed in each group. 4. The migration and invasion of LN308 and LN229 cells were inhibited after knockout with RFT2. The expression levels of MMP-2 and MMP-9 were also decreased. Conclusion: high expression of RFT2 was found in glioma tissues, and its expression level was positively correlated with WHO grade of gliomas. Knockout of RFT2 promoted cell cycle arrest, apoptosis and cell proliferation, migration and invasion of LN308 and LN229 cells. In addition, the nude mice tumor-bearing model further demonstrated that the proliferation of glioma cells was inhibited after knockout of RFT2. These results suggest that RFT2 may be a new target for glioma molecular therapy.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41

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