炎症因子TNFα与大肠和乳腺癌患病风险的相关性及Dexamethasone诱导乳腺癌耐药的机制

发布时间：2018-04-27 06:46

本文选题：CRC + BRC　；参考：《昆明医科大学》2017年博士论文

【摘要】：第一部分TNF-α-308 G/A多态性与结直肠癌和乳腺癌患病风险的研究[目的]TNFα是一个炎症相关的细胞因子,它参与多种癌症的发生发展过程。目前TNF-α-308G/A的多态性与结直肠癌(CRC)和乳腺癌(BRC)的相关性尚不完全清楚,尤其是与CRC、BRC的临床特征、实验室指标的关联性尚未有深入的研究。本实验中,综合CRC和BRC各自的特点,把疾病分为不同的亚型、将传统组织病理学分型与基因分型相结合,研究TNFα基因多态性与CRC和BRC发生与转移的风险从而指导个体化的诊断与治疗。[方法]研究收录昆明医科大学第一附属医院和云南省肿瘤医院570例CRC、490例BRC患者及相应的无病对照。收集相应的人口学资料和临床资料;对照组收集人口学资料。采用高通量时间飞行质谱生物芯片系统检测实验对象TNFα的单核苷酸多态性(SNP)。通过比较病例组与对照组人口学特征、CRC和BRC各亚型间的临床特征和实验室指标及各等位基因分布频率的差异,计算OR及95%CI,从而比较不同基因型与CRC和BRC发生及转移的相对危险度。[结果]TNFα基因rs1800629位点检测到GG、AA及GA三种基因型。结直肠癌中,年龄和性别在病例组和对照组中满足频率匹配(P=0.460.77),TNF-α-308 G和A的等位基因频率符合哈迪温伯格平衡定律(PHWE=0.850.67)。BRC中病例组和对照组之间的年龄分布无显著差异(P=0.065),G和A的等位基因频率符合哈迪温伯格平衡定律(PHWE=0.870.96)。在所有的CRC患者中,TNF-α-308 GA 增加了 CRC 患者远处转移的风险(OR=2.911,95%CI:1.697-4.992,P= 0.00017),经多因素Logistic回归分析,该数值仍具有显著的差异(OR=2.947,95%CI:1.515-5.572,P=0.00121)。有趣的是,将CRC分组后,我们发现GA和AA仅仅是直肠癌远处转移的危险因素(OR=4.481,95%CI:2.072-9.693,P=0.00025)而和结肠癌的远处转移发生没有确切相关性(OR=1.685,95%CI:0.592-4.796),校正所有临床资料后该数值仍具有显著的差异(OR =7.099,95%CI:2.482-20.301,P= 0.000256)。GA和AA基因型是CRC患者发生肥胖、肿瘤体积增大的危险因素,携带GA和AA基因型的患者的肥胖风险较携带GG基因型增加(OR = 2.056,95%CI:1.197-3.530,P=0.01);携带 GA 和 AA 型的 CRC 具有更大的肿瘤体积(OR=2.225,95%CI:1.049-4.719,P=0.044)。TNF-α-308 GA是CRC患者中性粒细胞增高的危险因素(OR= 4.764,95%CI:2.39-9.495,P=0.000012)。BRC患者中,GA和AA基因型是肿瘤体积的保护因素,携带GA和AA基因型的BRC患者肿瘤体积相对较小(OR = 0.137,95%CI:0.019-1.013,P=0.023),经多因素Logistic回归分析后,结果仍然有意义(OR = 0.115,95%CI:0.013-0.991,P=0.023)。在TNBC中,GA和AA基因型更频繁的出现于初潮年龄大于15岁的患者(OR =11.75,95%CI:1.761-78.416,P=0.003)及罹患高血压的患者中(OR=8,95%CI:1.02-62.737,P=0.025)。在 Luminal-A 型患者中,右侧乳腺癌的GA和AA基因型频率较高(OR=5.073,95%CI:1.022-25.194,P=0.031),HER-2患者中,具有GA和AA基因型的患者初次诊断乳腺癌的年龄偏大(OR= 1.211,95%CI:1.004-1.46,P=0.029)。通过比较实验室指标基因型的分布差异,揭示了 GA和AA基因型与白蛋白和胆固醇水平的相关性,TNF-α-308 GA 是 BRC 低蛋白血症(OR= 2.571,95%CI:1.194-5.535,P=0.013)及 TNBC高胆固醇血症(OR= 7.75,95%CI:0.842-69.708,P=0.041)的危险因素。[结论]TNF-α-308 GA多态性是CRC发生远处转移的危险因素,尤其增加了直肠癌患者发生远处转移的风险,而和乳腺癌的复发转移没有明显相关性,GA和AA基因型是CRC患者发生肥胖、肿瘤体积增大的危险因素,提示TNFα基因rs 1800629位点是影响CRC预后的重要因素之一。GA和AA基因型增加了BRC低蛋白血症和TNBC高胆固醇血症、TNBC罹患高血压及Luminal-A患者右乳癌的患病风险,具有GA和AA基因型的患者有较小的肿瘤体积,提示TNF-α-308 GA的多态可以预测肿瘤的原发部位、大小及评估BRC的营养状况。第二部分Dexamethasone诱导三阴性乳腺癌耐药的机制研究[目的]研究地塞米松(Dex)在促进三阴性乳腺癌(TNBC)耐药中的机制[方法]用SRB实验检测Dex对TNBC细胞经化疗药物顺铂(Cpt)、多西他赛(DTX)处理后对生长、增殖的影响;用免疫印迹实验检测糖皮质激素(GCs)对KLF5蛋白的诱导,以及相关凋亡蛋白的检测;用real-time PCR、双荧光素酶报告基因实验检测地塞米松对KLF5的转录活性的影响;用ChIP实验验证糖皮质激素受体(GR)对KLF5启动子调控的具体位点;用流式细胞仪检测Dex通过KLF5介导的TNBC细胞凋亡的影响;用裸鼠成瘤实验在体内证明Dex在TNBC对DTX敏感性中的影响。[结果]Dex可以促进TNBC细胞HCC1937和HCC1806的生长和增殖,同时我们发现其诱导KLF5在乳腺癌细胞系中的表达;在TNBC细胞中,Dex可以促进KLF5的转录活性,活化GR并促其结合至KLF5启动子区域的Sp1位点,增强KLF5的启动子的活性,进而促进KLF5的转录表达;敲低KLF5能够抑制三阴性乳腺癌细胞的存活;KLF5介导了 Dex对DDTX和Cpt引起的细胞凋亡的抑制作用;Dex降低了乳腺肿瘤在动物体内对化疗药物的敏感性,而沉默KLF5表达则回复了肿瘤对DTX的敏感性;这些结果说明Dex至少部分通过KLF5调控三阴性乳腺癌细胞对DTX和Cpt的耐药。[结论]我们证实了 Dex通过GR调控下游靶基因KLF5的转录,进而促进TNBC细胞的增殖;Dex通过KLF5的表达,进而促进了 TNBC对化疗药物的耐药。说明,KLF5是三阴性乳腺癌治疗和预测疗效潜在的靶点和生物标记物。
[Abstract]:Part one TNF- alpha -308 G/A polymorphism and the risk of colorectal and breast cancer [Objective]TNF alpha is an inflammatory cytokine that is involved in the development of a variety of cancers. The association of TNF- alpha -308G/A with colorectal cancer (CRC) and breast cancer (BRC) is not completely clear, especially with CRC, BRC. The correlation of clinical features and laboratory indicators has not been studied in depth. In this experiment, the characteristics of CRC and BRC were divided into different subtypes, and the traditional histopathological and genotyping were combined to study the risk of TNF alpha gene polymorphism and the occurrence and transfer of CRC and BRC and to guide the individualized diagnosis and treatment. Methods: the study included 570 cases of CRC, 490 Cases of BRC patients and the corresponding disease-free control in the First Affiliated Hospital of Kunming Medical University and Yunnan cancer hospital. The corresponding demographic data and clinical data were collected. The control group collected human data. The high throughput time flight mass spectrometry biochip system was used to detect the single nucleotide polymorphisms of TNF alpha. SNP. By comparing the demographic characteristics of the case group with the control group, the differences in the clinical features, the laboratory indexes and the frequency of the distribution of the alleles in the CRC and BRC subtypes, the OR and 95%CI were calculated, and the relative risk of the occurrence and metastasis of CRC and BRC was compared. [results] the rs1800629 loci of]TNF a gene detected GG, AA and GA. Three genotypes. In colorectal cancer, age and sex meet frequency matching (P=0.460.77) in case group and control group. The allele frequency of TNF- alpha -308 G and A is consistent with Hardy Weinberg equilibrium law (PHWE=0.850.67).BRC between case group and control group, there is no significant difference in age distribution (P=0.065), G and A allele frequency conforms to ha. Di Weinberg's law of equilibrium (PHWE=0.870.96). In all CRC patients, TNF- alpha -308 GA increases the risk of distant metastasis of CRC patients (OR=2.911,95%CI:1.697-4.992, P= 0.00017). The value of this value is still significant (OR=2.947,95% CI:1.515-5.572, P=0.00121). We found that GA and AA were only a risk factor for distant metastasis of rectal cancer (OR=4.481,95%CI:2.072-9.693, P=0.00025) and had no definite correlation with distant metastasis of colon cancer (OR=1.685,95%CI:0.592-4.796). After correcting all clinical data, the value still had significant difference (OR =7.099,95%CI:2.482-20.301, P= 0.000256).GA and AA base. The risk factors for obesity and tumor volume increased in CRC patients. The risk of obesity carrying GA and AA genotype was higher than that of GG genotype (OR = 2.056,95%CI:1.197-3.530, P=0.01), and CRC with GA and AA type CRC had greater tumor volume (OR=2.225,95%CI: 1.049-4.719,) In OR= 4.764,95%CI:2.39-9.495 (P=0.000012).BRC patients, the GA and AA genotypes are the protective factors of tumor volume. The volume of the tumor carrying GA and AA genotype is relatively small (OR = 0.137,95%CI:0.019-1.013, P=0.023). CI:0.013-0.991, P=0.023). In TNBC, the GA and AA genotypes appear more frequently in patients aged more than 15 years old (OR =11.75,95%CI:1.761-78.416, P=0.003) and patients with hypertension (OR=8,95%CI:1.02-62.737, P=0.025). In Luminal-A type patients, the frequency of GA and genotypes in right side breast cancer is higher. -25.194, P=0.031), in patients with HER-2, the age of the first diagnosis of breast cancer with GA and AA genotypes was larger (OR= 1.211,95%CI:1.004-1.46, P=0.029). The correlation between the genotype of GA and AA was revealed by comparing the distribution differences in the laboratory index genotypes. 2.571,95%CI:1.194-5.535, P=0.013) and the risk factors of TNBC hypercholesterolemia (OR= 7.75,95%CI:0.842-69.708, P=0.041). [conclusion]TNF- alpha -308 GA polymorphism is a risk factor for distant metastasis of CRC, especially increasing the risk of distant metastasis in rectal cancer patients, but not associated with recurrence and metastasis of breast cancer, GA and AA. Genotype is a risk factor for obesity and tumor volume in CRC patients. It is suggested that the RS 1800629 locus of TNF a gene is one of the important factors affecting the prognosis of CRC. The.GA and AA genotypes increase BRC hypoproteinemia and TNBC hypercholesterolemia, and the risk of high blood pressure and Luminal-A patients with right breast cancer with GA and AA genotypes The patient has a small tumor volume, suggesting that TNF- alpha -308 GA polymorphism can predict the primary site of the tumor, size and evaluation of the nutritional status of BRC. Second the mechanism of Dexamethasone induced resistance to three negative breast cancer [Objective] to study the mechanism of dexamethasone (Dex) in promoting the resistance of three negative breast cancer (TNBC) [method] with SRB test The effects of Dex on the growth and proliferation of TNBC cells treated with chemotherapeutic drugs cisplatin (Cpt) and docetaxel (DTX) were detected. The induction of KLF5 protein by Glucocorticoid (GCs) and the detection of related apoptotic proteins were detected by immunoblot test, and the transcriptional activity of dexamethasone was detected by real-time PCR and double fluoroenzyme reporter gene test. Influence; the specific site of glucocorticoid receptor (GR) Regulation of KLF5 promoter was verified by ChIP test; the effect of Dex on the apoptosis of TNBC cells mediated by KLF5 was detected by flow cytometry; the effect of Dex in TNBC on DTX sensitivity was demonstrated in nude mice in vivo. [results] Dex could promote the growth of TNBC cell HCC1937 and the growth of TNBC. Proliferation, and we found that it induces the expression of KLF5 in the breast cancer cell lines; in TNBC cells, Dex can promote the transcriptional activity of KLF5, activate GR and bind to the Sp1 loci of the KLF5 promoter region, enhance the activity of the promoter of KLF5, and promote the transcriptional table of KLF5; knockdown KLF5 can inhibit the survival of three negative breast cancer cells. Live; KLF5 mediates the inhibitory effect of Dex on cell apoptosis induced by DDTX and Cpt; Dex reduces the sensitivity of breast tumors to chemotherapeutic drugs in animals, while silent KLF5 expression revert to the tumor's sensitivity to DTX; these results indicate that Dex at least partly controls the resistance of three negative breast cancer cells to DTX and Cpt. [Conclusion] We confirm that Dex regulates the transcription of the downstream target gene KLF5 by GR and thus promotes the proliferation of TNBC cells. Dex promotes the resistance of TNBC to chemotherapeutic drugs through KLF5 expression, indicating that KLF5 is a potential target and biomarker for the treatment and prediction of three negative breast cancer.

【学位授予单位】：昆明医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.9;R735.34

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