GPR137基因沉默对人脑胶质瘤细胞增殖，克隆和周期的实验研究

发布时间：2019-06-25 10:37

【摘要】：目的利用RNA干扰（RNAi）技术沉默GPR137基因在多种恶性胶质瘤细胞中的表达，，阐明GPR137对恶性胶质瘤细胞增殖、克隆形成能力等方面的影响。方法利用免疫组化技术对29例不同级别人胶质瘤组织中GPR137的表达及与病理分级相关性进行分析。利用半定量反转录－聚合酶链反应（semi-quantitative RT-PCR）检测GPR137基因在五种恶性胶质瘤细胞（U251,U-87MG, U-118MG, A172,U373）中的表达。构建针对GPR137基因的短发卡(shRNA)慢病毒表达载体，包装成病毒颗粒并感染人胶质瘤U251和A172细胞，采用实时定量聚合酶链反应和Western印迹法验证GPR137基因的mRNA和蛋白的表达，确定其抑制效率。采用噻唑蓝(MTT)比色法和克隆形成实验检测细胞增殖和克隆形成能力情况，应用流式细胞分析法检测细胞周期分布情况。观察沉默GPR137基因对U251和A172细胞增殖、克隆形成和周期分布的影响。结果不同级别人胶质瘤组织中GPR137表达均异常上调，且与胶质瘤临床病理分级呈正相关。通过半定量反转录－聚合酶链反应证实，GPR137基因在五种胶质瘤细胞中均显著表达。实时定量聚合酶链反应和Western印迹法显示，GPR137-shRNA能有效抑制U251和A172细胞中GPR137基因的mRNA和蛋白的表达。MTT和克隆形成实验显示，感染后的胶质瘤细胞的增殖和克隆形成能力明显受到抑制。流式细胞分析法显示，感染后的胶质瘤细胞周期停滞在S期。上述差异均具有统计学意义龴P＜0.05㖞。讨论以往的文献已经证实信号转导通路和相关的膜蛋白，特别是G蛋白偶联受体(GPCRs)家族在胶质瘤的形成中发挥着重要的作用。肿瘤细胞通过G蛋白偶联受体得以存活、增殖、侵犯周围组织、并躲避免疫机制。人体内已知的800多种G蛋白偶联受体大部分已经明确其结构和功能，并成为药物作用靶标。然而，目前仍有100多种被称为孤儿受体的G蛋白偶联受体，其结构和作用仍然不清楚，GPR137正是其中一种孤儿受体。GPR137位于人第11号染色体的R56区，在中枢神经系统内广泛表达。已经有文献报道GPR137基因参与体内一些肿瘤的形成过程，如肝癌、乳腺癌、前列腺癌等。但在脑胶质瘤中的作用国内外尚未有报道。本研究中，GPR137在多种脑胶质瘤细胞中高表达。敲减GPR137后，U251和A172细胞增殖、克隆形成受到显著抑制，而且细胞周期停滞在S期。说明GPR137在胶质瘤的增殖，克隆形成等方面发挥着重要的作用。然而相关的分子机制有待后续研究证实。综上所述，GPR137基因在体外对胶质瘤细胞的增殖、克隆形成具有重要影响。同时，为GPR137成为胶质瘤靶向治疗基因提供了理论依据。
[Abstract]:Objective to silence the expression of GPR137 gene in various malignant glioma cells by RNA interference (RNAi), and to elucidate the effect of GPR137 on the proliferation and cloning ability of malignant glioma cells. Methods Immunohistochemical technique was used to analyze the expression of GPR137 in 29 cases of human gliomas of different grades and its correlation with pathological grade. Semi-quantitative reverse transcription-polymerase chain reaction (semi-quantitative RT-PCR) was used to detect the expression of GPR137 gene in five kinds of malignant glioma cells (U251, UX87MG, U18MG, A172, U373). A short hairpin (shRNA) lentivirus expression vector targeting GPR137 gene was constructed and packaged into virus particles and infected human glioma U251 and A172 cells. The expression of mRNA and protein of GPR137 gene was verified by real-time quantitative polymerase chain reaction and Western printing, and its inhibitory efficiency was determined. The cell proliferation and clone formation ability were detected by thiazolyl blue (MTT) colorimetric assay and clone formation assay, and the cell cycle distribution was detected by flow cytometry. To observe the effect of silencing GPR137 gene on proliferation, clone formation and cycle distribution of U251 and A172 cells. Results the expression of GPR137 in different grades of human gliomas was abnormally up-regulated, which was positively correlated with the clinicopathological grade of gliomas. The expression of GPR137 gene in five kinds of glioma cells was confirmed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Real-time quantitative polymerase chain reaction and Western staining showed that GPR137-shRNA could effectively inhibit the expression of GPR137 gene mRNA and protein in U251 and A172 cells. GPR137-shRNA and clone formation assay showed that the proliferation and clone formation ability of infected glioma cells were significantly inhibited. Flow cytometry showed that the infected glioma cell cycle stagnated in S phase. The above differences were statistically significant (P < 0.05). It has been confirmed that signal transduction pathway and related membrane proteins, especially G protein-coupled receptor (GPCRs) family, play an important role in the formation of glioma. Tumor cells survive, proliferate, invade surrounding tissues and avoid immune mechanisms through G-protein-coupled receptors. Most of the more than 800 kinds of G protein coupling receptors known in human body have been identified in their structure and function, and have become the target of drug action. However, there are still more than 100 kinds of G protein-coupled receptors called orphan receptors, and their structure and function are still unclear. GPR137 is one of the orphan receptors. GPR137 is located in the R56 region of human chromosome 11 and is widely expressed in the central nervous system. It has been reported that GPR137 gene is involved in the formation of some tumors in vivo, such as liver cancer, breast cancer, prostate cancer and so on. However, the role of glioma has not been reported at home and abroad. In this study, GPR137 was highly expressed in a variety of glioma cells. After knockout of GPR137, U251 and A172 cells proliferated and clone formation was significantly inhibited, and the cell cycle stagnated in S phase. It is suggested that GPR137 plays an important role in the proliferation and clone formation of gliomas. However, the related molecular mechanisms need to be confirmed by follow-up studies. In conclusion, GPR137 gene plays an important role in the proliferation and cloning of glioma cells in vitro. At the same time, it provides a theoretical basis for GPR137 to become a targeted therapeutic gene for glioma.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41

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