基于放射敏感性的分子标签及放射增敏新策略的研究

发布时间：2018-07-05 00:50

本文选题：肿瘤放射治疗 + 放疗增敏剂　；参考：《南昌大学》2016年博士论文

【摘要】：第一部分:新型WNT通路抑制剂LGK974放射增敏作用和机制研究目的:探讨WNT信号传导通路抑制剂LGK974在肿瘤细胞中的放疗增敏作用和机制。方法:1.通过克隆形成实验分别观察LGK974合并放疗对MLH1表达完整和MLH1表达缺失的细胞株的放疗增敏作用。2.通过克隆形成实验分别观察LGK974合并放疗对瞬时导入MLH1结肠癌细胞株HCT116(MLH1表达是缺失的)的放疗增敏作用。3.通过比较结肠癌HCT116细胞株(MLH1表达缺失)分别经对照组DMSO和实验组LGK974处理后合并放疗,用免疫荧光检测细胞中γH2AX的变化来反映LGK974通过抑制DNA损伤修复增强MLH1缺失肿瘤细胞的放疗敏感性。4.通过蛋白激酶组学Kinomic Profiling实验检测LGK974放疗增敏的可能磷酸化靶点。结果:1.LGK974在MLH1表达完整的宫颈癌HeLa细胞株中无放疗增敏作用,而在MLH1表达缺失的结直肠癌HCT116细胞株、子宫内膜癌AN3CA细胞株、卵巢癌顺铂耐药A2789/cp70细胞株中则有放疗增敏作用。2.野生型MLH1重新导入HCT116细胞株后LGK974的放疗增敏作用明显减弱。3.LGK974相对对照组DMSO而言,合并放疗后HCT116中检测到的γH2AX数目明显减少,细胞DNA损伤修复能力降低,说明LGK974有放疗增敏作用;而把MLH1导入HCT116后发现LGK974相对对照组DMSO而言,合并放疗后HCT116中检测到的γH2AX数目明显增多,细胞DNA损伤修复能力增强,LGK974放疗增敏作用减弱。4.蛋白激酶组学实验(Kinomic Profiling)发现LGK974合并放疗后PRKACα和PRKACβ磷酸化作用减弱,提示它们可能为LGK974放疗增敏的潜在靶点。结论:作为一种新型Wnt信号通路抑制剂,LGK974在MLH-1表达缺失的肿瘤中通过抑制DNA损伤修复和作用于PRKACα和PRKACβ磷酸化靶点产生放疗增敏作用。这些发现说明LGK974可能会成为一种新型的针对MLH1表达缺失肿瘤的放疗增敏剂,PRKACα和PRKACβ磷酸化靶点有可能是潜在的放疗增敏靶点。第二部分:预测头颈部鳞癌放疗敏感性MiRNA的筛选与验证目的:头颈部鳞癌中预测放疗敏感性MiRNA的筛选与验证方法:1.通过选取一对分别取自共济失调-毛细血管扩张症ATM患者(患者)及其直系亲属(正常)的人淋巴母细胞样细胞系来分析miRNA差异表达与电离放射的关系,两组分别经放疗和未放疗处理后对比进行完整的miRNA芯片实验,得到28个表达有差异的miRNA。2.在目前最大的癌症数据库TCGA数据库中通过找寻病理类型为鳞状细胞癌病例来评估这28个miRNA的临床相关性。3.利用MDA反向阶段蛋白质芯片技术检测TCGA数据库中头颈鳞癌样本中的蛋白表达水平,分别计算ATM在放射敏感和放射抗拒的患者中的平均表达水平。4.通过检测头颈鳞癌细胞系Cal27,验证放射治疗前后在抑制或不抑制ATM情况下的miRNA表达谱与最初的miRNA筛选结果及TCGA临床数据一致。5.利用STRING数据库(一个搜寻已知蛋白质之间和预测蛋白质之间相互作用的系统)(STRING9.1),对这5个可预测头颈部鳞癌放射敏感性的miRNA分子的调控目标进行通路分析(pathway analysis)。结果:共有28个miRNA在放射治疗后出现不同的差异表达,但都依赖于共济失调-毛细血管扩张症突变(Ataxia-TelangiectasiaMutated,ATM)激酶。通过验证这些miRNA最终确认有5个miRNA分子标志物可预测头颈部鳞癌放射敏感性,且这些患者中的ATM表达水平与放射敏感性相关。结论:miRNA分子标志物可用于临床预测头颈鳞癌放射治疗的敏感性。第三部分:RAS蛋白促进人脑膜瘤细胞增殖且抑制其凋亡目的:探讨RAS蛋白对人脑膜瘤细胞生长的影响。方法:将人脑膜瘤IOMM-LEE细胞分为空白对照组(细胞未进行任何药物处理)、阴性对照组(细胞经等体积生理盐水代替药物)和FTS处理组(细胞经FTS处理),采用四甲基偶氮唑盐微量酶反应比色法(MTT法),FTS(75μmol/L)处理48h后采用流式细胞技术检测降低RAS活性后IOMM-LEE细胞的增殖和凋亡情况,用Western Blot检测ERK和AKT信号通路;按肾包膜下移植法建立人脑膜瘤动物模型,小鼠随机分为实验组(50mg/kg组、75mg/kg组和100mg/kg,皮下注射FTS)和对照组,免疫组化增殖细胞检测核抗原(PCNA),用Western Blot检测ERK和AKT信号通路。结果:实验发现在75 μmol/LFTS浓度下,IOMM-LEE细胞的存活率随时间的推移而明显下降(P0.05)。75 μmol/LFTS浓度处理48h后,处理组细胞凋亡较空白组及阴性对照组均明显增加(均P0.05);且细胞周期检测结果显示:与空白组及阴性对照组相比,处理组细胞生长阻滞在G1期,差异有统计学意义(P0.05)。75 μmol/LFTS浓度处理48h后,相对于空白组和阴性对照组,处理组ERK和AKT的磷酸化水平显著降低(P0.05)。体内实验结果显示:FTS处理后,与对照组和50mg/kg组相比,75mg/kg组和100mg/kg组肿瘤体积均明显减小,PCNALI均明显降低,p-ERK和p-AKT水平明显降低(均P0.05)。结论:RAS蛋白在脑膜瘤细胞中高表达,抑制其活性可能通过下调ERK和AKT信号通路,进而调控细胞的生长。放疗疗效的本质是射线导致细胞凋亡,RAS在肿瘤放射治疗中可能是一个可调控的重要靶点。
[Abstract]:Part one: a new WNT pathway inhibitor LGK974 radiosensitizing effect and Mechanism Study: To explore the effect and mechanism of radiation sensitization of WNT signal transduction pathway inhibitor LGK974 in tumor cells. Method: 1. through cloning and forming experiment, the radiation enhancement of LGK974 combined with radiotherapy to complete and MLH1 deletion of MLH1 surface was observed. The sensitivity of.2. through the cloning and formation test to observe the radiation sensitization effect of LGK974 combined with radiotherapy on the transient introduction of MLH1 colon cancer cell line HCT116 (MLH1 expression is missing).3. by comparing the colon cancer HCT116 cell line (MLH1 expression deletion) by the control group DMSO and the experimental group LGK974 after the combined radiotherapy, using immunofluorescence detection finely. The changes in the intracellular gamma H2AX reflect the radiation sensitivity of LGK974 by inhibiting DNA damage and repairing the radiation sensitivity of MLH1 missing tumor cells. The possible phosphorylation targets of LGK974 radiation sensitization by the protein kinase group Kinomic Profiling test were detected by the protein kinase group Kinomic Profiling experiment. Colorectal cancer HCT116 cell lines with LH1 deletion, endometrial carcinoma AN3CA cell lines, ovarian cancer cisplatin resistant A2789/cp70 cell lines, the radiation sensitization effect of.2. wild type MLH1 re introduced to HCT116 cell lines after.2. was significantly weakened by.3.LGK974 relative to the control group DMSO, and the combined radiotherapy was detected in HCT116 after radiotherapy. The number of gamma H2AX decreased significantly and the ability of DNA injury and repair decreased, indicating that LGK974 had radiation sensitization. After MLH1 was introduced into HCT116, LGK974 relative to the control group DMSO, the number of gamma H2AX detected in HCT116 was significantly increased after the combined radiotherapy, and the ability to repair the DNA injury was enhanced and the LGK974 radiotherapy sensitization weakened the.4. protein kinase group. The study (Kinomic Profiling) found that PRKAC alpha and PRKAC beta phosphorylation in LGK974 combined with radiotherapy weakened, suggesting that they may be potential targets for sensitizing LGK974 radiation. Conclusion: as a new type of Wnt signal pathway inhibitor, LGK974 can inhibit DNA damage repair and act on PRKAC alpha and PRKAC beta phosphoric acid in the tumor of MLH-1 expression deletion. These findings suggest that LGK974 may become a new type of radiation sensitizer for MLH1 expression deletion tumors. PRKAC alpha and PRKAC beta phosphorylation targets may be potential targets for radiation sensitization. Second: the screening and validation of MiRNA for the prediction of radiation sensitivity of head and neck squamous cell carcinoma: head and neck scales Screening and validation methods for predicting radiation sensitivity MiRNA in cancer: 1. the relationship between differential expression of miRNA and ionizing radiation was analyzed by selecting a pair of ATM patients and their direct relative (normal) lymphoblastoid cell lines from patients with ataxia - telangiectasia (patients) and their direct relatives (normal). The two groups were compared after radiotherapy and no radiotherapy respectively. A complete miRNA chip experiment was carried out to obtain 28 differentially expressed miRNA.2. in the current largest cancer database TCGA database to evaluate the clinical relevance of the 28 miRNA by finding the pathological type of squamous cell carcinoma..3. was used to detect the head and neck squamous cell carcinoma in the TCGA database by the MDA reverse phase protein chip technology. The level of protein expression, the average expression level of ATM in patients with radiosensitivity and radiological resistance,.4. was detected by the detection of Cal27 in the head and neck cancer cell line, which verified that the miRNA expression profiles under the condition of inhibition or non inhibition of ATM before and after radiotherapy were consistent with the initial miRNA screening results and TCGA clinical data and.5. using the STRING database. A system for searching for the interaction between known proteins and predicting proteins (STRING9.1), pathway analysis (pathway analysis) for these 5 miRNA molecules that predict radiosensitivity of the head and neck squamous cell carcinoma (pathway analysis). Results: a total of 28 miRNA had different differential expressions after radiation therapy, but they all depended on ataxia - Ataxia-TelangiectasiaMutated (ATM) kinase. By verifying these miRNA, 5 miRNA markers can be confirmed to predict radiosensitivity in the head and neck squamous cell carcinoma, and the level of ATM expression in these patients is associated with radiosensitivity. Conclusion: the miRNA molecular marker can be used to predict the radiation of head and neck squamous cell carcinoma The sensitivity of treatment. The third part: RAS protein promotes the proliferation of meningioma cells and inhibits its apoptosis: the effect of RAS protein on the growth of human meningioma cells. Methods: the human meningioma IOMM-LEE cells are divided into blank control group (the cells are not treated with any drug treatment), and the negative control group (cell via equal volume of physiological saline instead of the drug) ) and FTS treatment group (cells treated by FTS), the proliferation and apoptosis of IOMM-LEE cells were detected by flow cytometry with four methylazazolazolide Microenzyme reaction colorimetric assay (MTT) and FTS (75 mol/L), and ERK and AKT signal pathways were detected by Western Blot, and human meningioma was established by subcapsular transplantation. The mice were randomly divided into experimental group (group 50mg/kg, group 75mg/kg and 100mg/kg, subcutaneous injection of FTS) and control group. Immuno proliferating cells were used to detect nuclear antigen (PCNA), and ERK and AKT signal pathways were detected by Western Blot. Results: the experimental results showed that the survival rate of IOMM-LEE cells decreased significantly at the concentration of 75 micron mol/LFTS (P0.05) (P0.05) significantly decreased with time (P0.05). When the concentration of.75 mu mol/LFTS was treated with 48h, the cell apoptosis in the treatment group was significantly higher than that in the blank group and the negative control group (all P0.05). And the cell cycle detection results showed that the cell growth block in the treatment group was compared with the blank group and the negative control group, and the difference was statistically significant (P0.05).75 mu mol/LFTS concentration treatment 48h, compared with the blank space of the blank group and the negative control group. In the group and the negative control group, the phosphorylation level of ERK and AKT in the treatment group was significantly lower (P0.05). In vivo experiment results showed that after FTS treatment, the tumor volume of 75mg/kg and 100mg/kg groups decreased significantly compared with the control group and 50mg/kg group, PCNALI decreased obviously, p-ERK and p-AKT water level obviously decreased (P0.05). Conclusion: RAS protein is in meningioma cells. The expression of middle height, which inhibits its activity, may regulate the growth of cells by downregulating the ERK and AKT signaling pathways. The essence of the radiotherapy effect is that radiation leads to cell apoptosis. RAS may be an important regulatory target in tumor radiotherapy.
【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.55

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