【作者】 李春涛；

【导师】 刘运生；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【摘要】 目的：胶质瘤是最常见的原发性脑部肿瘤,且多数切除后有复发可能。目前,针对恶性肿瘤细胞增殖、凋亡等方面的研究十分普遍。临床上使用的抗肿瘤药物大都是通过抑制肿瘤细胞的增殖、促进肿瘤细胞的凋亡来发挥作用的。PI3K/Akt/mTOR信号通路在多种恶性肿瘤中过度激活,调控恶性肿瘤的发生和发展。mTOR是PI3K/Akt信号通路下游重要的激酶,参与调控包括神经胶质瘤在内的多种肿瘤细胞的生存和增殖。雷帕霉素是mTOR的抑制剂,能通过抑制mTOR的活性,参与调控PI3K/Akt/mTOR信号通路在恶性肿瘤细胞中的作用。然而,关于雷帕霉素治疗神经胶质瘤的作用及相关分子机制尚未研究清楚。在恶性肿瘤细胞中,糖酵解水平异常升高,多种糖酵解酶的表达和活性被过度激活,提示糖酵解途径可能为抗癌治疗提供丰富的靶点。最近有研究报道,microRNA-143受mTOR通路调控,且在多种恶性肿瘤中表达异常下调,其靶基因包括糖酵解关键限速酶己糖激酶2(HK2)。然而,关于mTOR通路、miRNA-143以及糖酵解途径在神经胶质瘤中的调控作用及三者之间的关系尚不清楚。本研究利用人神经胶质瘤细胞系U251和CHG-5,探索雷帕霉素在增殖、凋亡、糖酵解等多个方面对神经胶质瘤的调控作用,并探讨相关的分子调控机制。方法：1)用100mM的雷帕霉素分别处理人胶质瘤U251和CHG-5细胞0、6、12、24h,MTT法检测U251和CHG-5细胞的增殖能力。2)用100mM的雷帕霉素处理人胶质瘤U251和CHG-5细胞0、6、12、24h, FCM检测U251和CHG-5细胞凋亡水平的改变。3)用100mM的雷帕霉素处理人胶质瘤U251和CHG-5细胞0、6、12、24h, Western Blotting检测磷酸化的S6激酶蛋白分子的表达水平。4)用100mM的雷帕霉素分别处理人胶质瘤U251和CHG-5细胞0、6、12、24h,检测葡萄糖摄取和乳酸分泌的变化。5)用100mM的雷帕霉素分别处理人胶质瘤U251和CHG-5细胞0、6、12、24h,分别检测己糖激酶、丙酮酸激酶和乳酸脱氢酶的活性。6)用100mM的雷帕霉素分别处理人胶质瘤U251和CHG-5细胞0、6、12、24h, Western Blotting分别检测己糖激酶、丙酮酸激酶和乳酸脱氢酶的蛋白表达水平。7)用100mM的雷帕霉素分别处理人神经胶质瘤U251和CHG-5细胞0、6、12、24h, Realtime qRT-PCR检测miR-143的表达变化。结果：1)用100mM的雷帕霉素分别处理神经胶质瘤U251和CHG-5细胞0、6、12、24h,随着处理时间的延长,细胞的增殖呈下降的趋势。2)用100mM的雷帕霉素分别处理神经胶质瘤U251和CHG-5细胞0、6、12、24h,随着处理时间的延长,细胞凋亡呈上升的趋势。3)用100mM的雷帕霉素分别处理神经胶质瘤U251和CHG-5细胞0、6、12、24h,随着处理时间的延长,S6K的磷酸化水平呈下降的趋势,提示mTOR活性受抑制。4)用100mM的雷帕霉素分别处理神经胶质瘤U251和CHG-5细胞0、6、12、24h,随着处理时间的延长,细胞葡萄糖摄取和乳酸分泌量呈下降趋势。5)用100mM的雷帕霉素分别处理神经胶质瘤U251和CHG-5细胞0、6、12、24h,随着处理时间的延长,糖酵解关键酶已糖激酶、丙酮酸激酶和乳酸脱氢酶的活性明显下调。6)用100mM的雷帕霉素分别处理神经胶质瘤U251和CHG-5细胞0、6、12、24h,随着处理时间的延长,糖酵解关键酶已糖激酶(HK2)、丙酮酸激酶(PK)和乳酸脱氢酶(LDH)的蛋白表达明显下调。7)用100mM的雷帕霉素分别处理神经胶质瘤U251和CHG-5细胞0、6、12、24h,随着处理时间的延长,miR-143的表达水平显著上调。结论：1)雷帕霉素通过抑制PI3K/Akt/mTOR信号通路的活性,抑制了神经胶质瘤细胞的增殖,同时诱导了细胞凋亡。2)雷帕霉素通过抑制PI3K/Akt/mTOR信号通路的活性,进而抑制了神经胶质瘤细胞糖酵解水平,这可能是导致神经胶质瘤细胞的增殖变慢的重要原因。3)雷帕霉素通过抑制PI3K/Akt/mTOR信号通路的活性,上调了miR-143的表达,进而下调了糖酵解限速酶HK2的表达,从而下调了神经胶质瘤细胞糖酵解水平。更多还原

【Abstract】 Objective Glioma is the most common primary brain tumor, and may recur after the surgical removal of glioma. At present, the study of tumor cell proliferation and apoptosis is very common. In clinical, many widely used anticancer drugs are effective in treating tumors through inhibiting proliferation and inducing apoptosis of malignant tumor cells. PI3K/Akt/mTOR signaling pathway is frequently activated in a variety of tumors, and involves in tumor growth and development. MTOR, as a downstream molecule of the PI3K/Akt pathway, plays a central role in regulating the survival and proliferation of many malignant tumors cells including glioma. Rapamycin is an inhibitor of mTOR, and involves in the regulation of PI3K/Akt/mTOR signaling pathway in the malignant cells, via inhibiting mTOR activity. However, the anti-cancer effect of rapamycin on glioma as well as the involved molecular mechanism has not been thoroughly studied.In various malignant tumor cells, glycolysis is commonly at a high level, mainly due to the abnormally elevated expression and activity of glycolytic enzymes, suggesting that the glycolytic pathway may provide sourceful targets for anti-cancer treatment. MicroRNA-143has been recently reported to be regulated by mTOR signal pathway, and its expression is abnormally reduced in a variety of malignant tumors, and its targets include glycolytic rate-limiting enzyme hexokinase2(HK2). However, the roles of mTOR pathway, miRNA-143, and glycolysis, as well as their relationship in the regulation of glioma remain unclear.In this study, human glioma cell lines U251and CHG-5were used to explore the regulatory effects of rapamycin on proliferation, apoptosis, and glycolysis of glioma cells, and we also invesitgated the involved molecular mechanism.MethodRapamycin (100mM) was used to treat human glioma cell lines U251and CHG-5. MTT was performed to analyze the proliferation capacity of U251and CHG-5cells treated by100mM rapamycin with time. FCM was used to analyze the apoptosis levels of U251and CHG-5cells treated by100mM rapamycin with time. Western Blotting was used to examine the protein expression of p-S6K, on behalf of the activity of mTOR, in U251and CHG-5cells treated by100mM rapamycin with time. Glucose uptake and lactic acid secretion were assayed in U251and CHG-5cells treated by100mM rapamycin with time. The activity and protein expression levels of hexokinase2(HK2), pyruvate kinase (PK) and lactate dehydrogenase (LDH), all of which were key enzymes in glycolysis, were determined in U251and CHG-5cells treated by100mM rapamycin with time. The expression levels of miR-143, which directly negatively regulates HK2, were assayed in U251and CHG-5cells treated by100mM rapamycin with time.ResultIn U251and CHG-5cells treated by100mM rapamycin at0,6,12,24h, respectively, with the treatment time, the cell proliferation rate was gradually decreased, while the apoptosis levels showed an increasing trend. Western Blotting result showed that with the treatment time, the protein expression of p-S6k was gradually downregulated, indicating that the activity of mTOR was inhibited. Moreover, with the treatment time, glucose uptake and lactic acid secretion were decreased in U251and CHG-5cells, and the activity and protein expression levels of HK2, PK and LDH were all downregulated, suggesting that the glycolysis level was repressed. Furthermore, the miR-143expression level was gradually decreased with the treatment time of rapamycin in U251and CHG-5cells.ConclusionRapamycin can inhibite cell proliferation and induce cell apoptosis in human glioma U251and CHG-5cells via downregulating the activity of PI3K/Akt/mTOR signaling pathway. Moreover, Rapamycin can also inhibiting the glycolysis in U251and CHG-5cells, which may contribute to the inhibition of cell proliferation. More importantly, rapamycin can upregulate the expression of miR-143through downregulate the acivity of PI3K/Akt/mTOR signaling, which can further inhibit the protein expression of the glycolysis rate-limiting enzyme HK2, the target of miR-143, and hence decrease the glycolysis level in U251and CHG-5cells.更多还原