宫颈肿瘤相关巨噬细胞中IL-10基因的转录调控表达机制研究

发布时间：2018-06-02 21:27

  本文选题：宫颈癌 + IL-10　； 参考：《华北理工大学》2017年硕士论文

【摘要】：目的宫颈癌的发病趋势已呈现低龄化。白介素-10(IL-10)作为一种具有免疫抑制作用的细胞因子对宫颈癌变的发生、发展具有重要作用。当其在人体内大量合成可抑制免疫系统的调控,引起HPV病毒入侵,导致宫颈癌的发生。在宫颈肿瘤生长的微环境中,肿瘤相关巨噬细胞(tumor associated macrophages,TAMs)通过分泌多种细胞因子参与肿瘤的生长、侵袭和迁移。TAMs作为表达IL-10的主要来源之一,其在宫颈癌变中调控IL-10表达的分子机制未曾报道。课题组前期研究已证实,宫颈肿瘤局部组织IL-10表达明显升高,且该基因启动子的低甲基化修饰促进其表达,这提示DNA甲基化修饰在应对病毒入侵和局部炎症环境的变化过程中具有重要影响。生物信息学分析显示:E26转录因子-1(Ets1)和特异性蛋白1(Sp1)在IL-10基因启动子上结合的顺式作用元件含有CG位点,且有报道称Ets1和Sp1的表达与宫颈癌的发生相关,但其是否参与TAMs表达IL-10的调控机制及其在宫颈癌中的作用研究未见报道。因此,本研究利用人单核巨噬细胞、人宫颈癌细胞和人不同病理宫颈组织,探讨TAMs中DNA甲基化参与Ets1和或Sp1介导IL-10基因转录调控的分子机制及其在宫颈癌变中的作用,旨在为以TAMs作为靶点的宫颈癌靶向治疗提供新思路。方法选取2014年9月至2016年3月就诊于唐山市工人医院并行宫颈活检及手术治疗患者160例,其中正常宫颈47例(对照组)、子宫颈上皮内瘤变Ⅰ级27例(CINⅠ组)、子宫颈上皮内瘤变Ⅱ-Ⅲ级46例(CINⅡ-Ⅲ组)和宫颈鳞癌40例(CSCC组)。在线查看所有患者的电子病例并了解患者的基本信息情况。采用免疫双荧光染色检测各组人宫颈组织中IL-10、Ets1和Sp1分别同CD68的共表达;临床分析IL-10+TAMs、Ets1+TAMs和Sp1+TAMs水平与宫颈鳞癌临床病理特征的关系;采用人单核细胞THP-1体外诱导形成宫颈TAMs;利用si RNA干扰以及q RT-PCR和Western blot技术分析TAMs中转录因子Ets1和(或)Sp1对IL-10基因表达水平的影响;生物信息学分析转录因子Sp1和Ets1在IL-10基因启动子近端2000bp范围内可能的结合位点,并通过对TAMs甲基化干预、荧光素酶报告基因、染色质免疫沉淀(CHIP)和甲基化特异性PCR(MSP)等分子生物学方法分析在宫颈TAMs中DNA甲基化对转录因子Sp1、Ets1与IL-10基因启动子的结合及其转录活性的影响。结果1对照组、CINⅠ组、CINⅡ-Ⅲ组和CSCC组患者的基本信息(包括:年龄、初潮年龄、月经周期、经期天数、怀孕次数、流产次数、吸烟情况、饮酒情况)比较,差异均无统计学意义(P0.05)。2免疫双荧光染色检测不同病理宫颈组织中IL-10、Ets1和Sp1与TAMs的共表达结果显示,伴随宫颈病变的进展,宫颈组织中TAMs以及IL-10+TAMs、Ets1+TAMs和Sp1+TAMs的表达百分比均逐渐增加,且与正常组相比,CINⅠ组、CINⅡ-Ⅲ组和CSCC组中的表达逐渐升高,差异有统计学意义(P0.05)。3在CSCC组织中IL-10+TAMs、Ets1+TAMs和Sp1+TAMs的表达水平与年龄、肿瘤大小无关(P0.05);与分化程度、FIGO分期和淋巴结转移有关(P0.05)。4在TAMs中采用干扰si RNA敲低Ets1、Sp1或同时敲低Ets1和Sp1基因表达,q RT-PCR和Western blot实验结果显示:敲低Sp1或同时敲低Ets1和Sp1基因均可下调IL-10的表达,且双敲抑制其表达更为明显,而仅敲低Ets1对IL-10的表达影响较小。5在甲基化干预实验以及MSP测序和Ch IP实验结果显示,在TAMs中IL-10基因启动子区的去甲基化促使转录因子Sp1与其结合并上调IL-10的表达。6进一步CHIP和免疫荧光染色实验结果表明,Ets1在Sp1调控IL-10基因启动子活性过程中起协同作用。结论1 TAMs源IL-10的表达与宫颈癌变呈正相关性。2在宫颈癌中转录因子Ets1和Sp1的表达上调且与TAMs源IL-10的表达相关。3在TAMs中Sp1结合位点DNA去甲基化在调控IL-10基因启动子活性及其在宫颈癌变发生中具有重要促进作用,而Ets1表达促进Sp1调控IL-10基因启动子的转录活性。
[Abstract]:Objective the incidence of cervical cancer has been reduced. Interleukins -10 (IL-10), as a cytokine with immunosuppressive effects, plays an important role in the development of cervical cancer. It is an important role in the development of cervical cancer. When it is widely synthesized in human body, the regulation of immunosuppressive immune system, the invasion of HPV virus, the occurrence of cervical cancer and the growth of cervical cancer. In the microenvironment, tumor associated macrophages (TAMs) is involved in the growth of tumor by secreting a variety of cytokines, invasion and migration of.TAMs as one of the main sources of IL-10 expression. The molecular mechanism of the regulation of IL-10 expression in cervical cancer has not been reported. The expression of IL-10 in the tissue is significantly elevated, and the gene promoter's low methylation modification promotes its expression. This suggests that DNA methylation modification plays an important role in response to changes in virus invasion and local inflammatory environment. Bioinformatics analysis shows that the E26 transcription factor -1 (Ets1) and specific protein 1 (Sp1) are on the IL-10 gene promoter. The combination of cis acting elements contains CG loci, and it is reported that the expression of Ets1 and Sp1 is related to the occurrence of cervical cancer, but it is not reported whether it participates in the regulatory mechanism of TAMs expression IL-10 and its role in cervical cancer. Therefore, this study used human mononuclear macrophages, human cervical cancer cells and different pathological cervical tissues. To discuss the molecular mechanism of DNA methylation involved in Ets1 and or Sp1 mediated transcription regulation of IL-10 gene in TAMs and its role in cervical cancer. The aim is to provide a new idea for the targeting therapy of cervical cancer with TAMs as a target. Methods 160 cases of cervical biopsy and surgical treatment in Tangshan City workers' hospital from September 2014 to March 2016 were selected. 47 cases of normal cervix (control group), 27 cases of cervical intraepithelial neoplasia (group CIN I), 46 cases of cervical intraepithelial neoplasia II - III (group CIN II - III) and 40 cases of cervical squamous cell carcinoma (group CSCC). The electronic cases of all patients were examined online and the basic information of the patients was understood. The cervical tissue of each group was detected by immunofluorescence staining. IL-10, Ets1 and Sp1 were co expressed with CD68, and the relationship between IL-10+TAMs, Ets1+TAMs and Sp1+TAMs levels with the clinicopathological features of cervical squamous cell carcinoma was analyzed. Human mononuclear cell THP-1 was induced to form cervical TAMs in vitro, and Si RNA interference and the transcriptional factor and / or transcriptional factors were analyzed. The effect of expression level; bioinformatics analysis of the possible binding sites of transcription factors Sp1 and Ets1 in the near end 2000bp of IL-10 gene promoter, and the analysis of DNA methyl in cervical TAMs by molecular biological methods such as TAMs methylation intervention, luciferase reporter gene, chromatin immunoprecipitation (CHIP) and methylation specific PCR (MSP). The effect of the combination of transcription factor Sp1, Ets1 and IL-10 gene promoter and its transcriptional activity. Results the basic information of 1 control groups, CIN I group, CIN II - III group and CSCC group (including age, menarche age, menstrual cycle, menstrual period, pregnancy times, abortion number, smoking, drinking) were not statistically significant (P 0.05) the co expression of IL-10, Ets1, Sp1 and TAMs in different pathological cervix tissues by.2 immunofluorescence staining showed that the percentage of TAMs, IL-10+TAMs, Ets1+TAMs and Sp1+TAMs in cervical tissues increased gradually with the progression of cervical lesions, and the expression of TAMs in the CIN I group, CIN II - III group and CSCC group compared with the normal group. The difference was statistically significant (P0.05), the expression level of IL-10+TAMs, Ets1+TAMs and Sp1+TAMs in the CSCC tissues was not related to the size of the tumor (P0.05) and the degree of differentiation, FIGO staging and lymph node metastasis (P0.05).4 in TAMs. Blot experimental results showed that the expression of IL-10 was downregulated by knocking low Sp1 or at the same time knocking at low Ets1 and Sp1 genes, and the expression of double knockdown inhibition was more obvious, but only the expression of low Ets1 on the expression of IL-10 had less influence on.5 in the methylation intervention experiment and the results of MSP sequencing and Ch IP experiment showed that the demethylation of the promoter region of the gene promoted the transfer in TAMs. Transcription factor Sp1 is combined with and up regulation of IL-10 expression.6 further CHIP and immunofluorescence staining experiments show that Ets1 plays a synergistic role in the Sp1 regulation of IL-10 gene promoter activity. Conclusion the expression of IL-10 in 1 TAMs source is positively correlated with cervical cancer,.2 is up regulation of Ets1 and Sp1 expressions in cervical cancer and is up to the source. 0 the expression of.3 related to the Sp1 binding site DNA demethylation in TAMs plays an important role in regulating IL-10 gene promoter activity and in cervical carcinogenesis, and Ets1 expression promotes the transcriptional activity of the IL-10 gene promoter by Sp1.
【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33

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