miR-99a-5p调控骨髓MSC向胃癌MSC转分化作用及机制

发布时间：2018-07-08 21:33

本文选题：骨髓间质干细胞 + 胃癌间质干细胞　；参考：《江苏大学》2015年博士论文

【摘要】：目的：肿瘤微环境在肿瘤发生、发展中发挥着重要的调控作用,非编码微小RNA(microRNAs/miRNAs)参与介导微环境对肿瘤细胞的作用及微环境重塑。癌相关间质干细胞(mesenchymal stem cell,MSC)作为一种重要的肿瘤微环境细胞被相继分离与鉴定,但是目前有关癌相关MSC的来源及其异常表达的miRNAs在其活化过程中的作用与分子机制尚不清楚。骨髓MSC是微环境细胞的重要来源,研究发现胃癌MSC在表面标记和多向分化潜能与骨髓MSC相似,但较骨髓MSC具有较强的增殖能力和促胃癌作用。本研究旨在建立胃癌细胞诱导骨髓MSC转分化模型,筛选胃癌MSC异常表达的miRNAs并以此为切入点,明确调控骨髓MSC向胃癌MSC转分化的关键miRNAs,阐明miRNAs再编程骨髓MSC的分子机制,方法：分离培养胃癌MSC、癌旁MSC、人骨髓MSC和615小鼠骨髓MSC。采用胃癌细胞上清处理骨髓MSC,建立体外胃癌细胞诱导骨髓MSC转分化模型。采用Agilent human miRNA芯片筛选检测胃癌MSC和癌旁MSC差异表达miRNAs、定量PCR检测验证、结合体外胃癌细胞上清诱导前后骨髓MSC中miRNAs的检测分析,确定胃癌MSC差异表达的miRNAs。模拟胃癌MSC中miRNAs低表达,采用miRNAs inhibitor转染骨髓MSC抑制miRNA的表达。采用免疫荧光染色检测α平滑肌肌动蛋白(a-smooth muscle actin,α-SMA)和成纤维细胞活化蛋白(Fibroblast Activation Protein, FAP)的表达,luminex检测细胞培养上清中细胞因子的分泌量进行表型分析。收集骨髓MSC的培养上清并作用胃癌细胞,细胞克隆形成试验、Transwell细胞迁移和侵袭试验、体内裸鼠致瘤分析转染后骨髓MSC对胃癌细胞的功能。采用miRNAs mimics转染胃癌MSC过表达miRNAs,检测转染前后胃癌MSC的表型和功能,分析miRNAs对胃癌MSC逆分化作用。采用靶基因预测、3’UTR报告基因载体构建和western blot检测并确定miRNAs调控的靶基因。在此基础之上,干预胃癌细胞上清诱导骨髓MSC、miRNAs转染骨髓MSC转分化模型和胃癌MSC中靶基因,检测处理前后MSC的表型、功能及相关信号通路活化情况,分析靶基因在转分化过程中的作用。结果：miRNAs芯片筛选检测胃癌MSC中差异倍数大于2且P0.01的miRNA共有61个,其中高表达miRNAs有38个,低表达miRNAs有23个。随机选择部分表达差异显著且与肿瘤调控密切相关的miRNAs,进一步在5对胃癌MSC和癌旁MSC进行检测验证,结果显示miR-99a-5p在胃癌MSC表达显著低于癌旁MSC。体外成功建立胃癌细胞上清诱导骨髓MSC向胃癌MSC样细胞转分化模型,定量PCR检测发现诱导后骨髓MSC中miR-99a-5p显著低表达。抑制骨髓MSC中miR-99a-5p的表达,可显著促进骨髓MSC中a-SMA和FAP的表达,luminex检测显示转染后骨髓MSC细胞培养上清中IL-6、IL-8、MCP-1、RANTES等细胞因子含量增加。将转染后骨髓MSC细胞上清作用胃癌细胞,可显著促进胃癌细胞克隆形成、迁移、侵袭和裸鼠体内致瘤力。过表达miR-99a-5p可显著阻断胃癌细胞上清对骨髓MSC的诱导作用。将胃癌MSC中miR-99a-5p高表达,可显著抑制其a-SMA和FAP的表达、细胞因子的分泌、阻断其对胃癌细胞增殖、迁移和侵袭的促进作用。靶基因预测和报告基因的检测分析确定FGFR3和IGF1R为miR-99a-5p调控的靶点。Western blot检测结果显示FGFR3和IGF1R的蛋白及其下游信号通路分子AKT和ERK在胃癌细胞上清诱导后的骨髓MSC、低表达miR-99a-5p的骨髓MSC和胃癌MSC中表达显著增加和活化。采用FGFR3抑制剂AZD4547口IGF1R抑制剂OSI-906分别预处理骨髓MSC,检测结果显示AZD4547可抑制胃癌细胞上清或miRNAs inhibitor对骨髓MSC中a-SMA和FAP的表达和下游信号通路活化的促进作用,阻断作用后的骨髓MSC对胃癌细胞的促进作用。AZD4547处理胃癌MSC可直接抑制其a-SMA和FAP的表达,阻断其对胃癌细胞迁移的促进作用。OSI-906的预处理不影响胃癌细胞上清和低表达的miR-99a-5p对骨髓MSC的转分化作用。结论：体外成功建立胃癌细胞上清诱导骨髓MSC向胃癌MSC转分化模型,确定胃癌MSC低表达miR-99a-5p。miR-99a-5p低表达可促进骨髓MSC向胃癌MSC转分化。高表达miR-99a-5p可促进胃癌MSC向骨髓MSC样细胞逆分化。FGFR3是介导胃癌细胞上清和miR-99a-5p再编程骨髓MSC的关键分子。本研究工作的完成将阐明骨髓MSC向胃癌MSC转分化作用及分子机制,为胃癌微环境细胞胃癌MSC的来源与重塑提供新的试验依据,为胃癌的治疗提供靶向微环境新的靶点和治疗策略,具有重要的科学研究意义和临床应用前景。
[Abstract]:Objective: tumor microenvironment plays an important role in the development of tumor, and it plays an important role in the development of tumor cells. Non coding micro RNA (microRNAs/miRNAs) is involved in mediating the effect of microenvironment on tumor cells and the remodeling of microenvironment. Cancer related mesenchymal stem cells (mesenchymal stem cell, MSC) are isolated and identified as an important tumor microenvironment cell. However, the function and molecular mechanism of the origin of cancer related MSC and its abnormal expression of miRNAs in its activation process is not clear. Bone marrow MSC is an important source of microenvironmental cells. The study found that the surface marker and multidirectional differentiation potential of gastric cancer MSC are similar to that of bone marrow MSC, but it has stronger proliferation and gastric cancer than bone marrow MSC. The purpose of this study is to establish a MSC transdifferentiation model of bone marrow induced by gastric cancer cells, to screen the miRNAs of abnormal expression of MSC in gastric cancer and take this as a breakthrough point, to clearly regulate the key miRNAs to MSC transdifferentiation of bone marrow MSC to gastric cancer, and to clarify the molecular mechanism of miRNAs reprogramming of bone marrow MSC, methods: the separation and cultivation of gastric cancer MSC, paracancerous MSC, human bone marrow MSC and 615 mice Bone marrow MSC. was treated with gastric cancer cell supernatant to treat bone marrow MSC, and to establish MSC transdifferentiation model of bone marrow induced by gastric cancer cells in vitro. Agilent human miRNA chip was used to screen and detect the differential expression of gastric cancer MSC and paracancerous MSC, and the quantitative PCR test was verified. SC differentially expressed miRNAs. simulated low expression of miRNAs in gastric cancer MSC, and miRNAs inhibitor was used to transfect bone marrow MSC to inhibit the expression of miRNA. Immunofluorescence staining was used to detect the expression of alpha smooth muscle actin (a-Smooth muscle actin, alpha -SMA) and fibroblast activation protein. The secretory quantity of cytokine in the supernatant was analyzed by phenotypic analysis. The culture supernatant of bone marrow MSC was collected and the gastric cancer cell, cell clone formation test, Transwell cell migration and invasion test, the function of bone marrow MSC on the gastric cancer cells after tumor analysis in nude mice. MiRNAs mimics transfection of gastric cancer MSC over expression miRNAs was used to detect the transfection. The phenotype and function of gastric cancer MSC were analyzed by miRNAs, and the target gene was predicted by target gene, 3 'UTR reporter gene vector construction and Western blot detection and miRNAs regulation target genes. On this basis, intervention of bone marrow MSC in gastric carcinoma cell supernatant, miRNAs transfection of bone marrow MSC transdifferentiation model and target base of gastric carcinoma MSC on this basis. Because of the phenotype, function and activation of the related signal pathway of MSC before and after detection, the role of target gene in the process of transdifferentiation was analyzed. Results: miRNAs chip screening detection of gastric cancer MSC was more than 2 and P0.01 miRNA had 61, of which there were 38 high expression miRNAs, 23 low expression miRNAs, and random selection of partial expression differences. MiRNAs, which was closely related to tumor regulation, was further tested in 5 gastric cancer MSC and paracancerous MSC. The results showed that the expression of miR-99a-5p in gastric cancer was significantly lower than that of MSC. in the side of the carcinoma, and the transdifferentiation of the bone marrow MSC to the MSC like cells was induced by the supernatant of gastric cancer cells, and the quantitative PCR detection was found in the miR-9 of the bone marrow MSC. The expression of miR-99a-5p in bone marrow MSC could significantly promote the expression of a-SMA and FAP in bone marrow MSC. Luminex detection showed that the content of IL-6, IL-8, MCP-1, RANTES and other cytokines increased in the culture supernatant of bone marrow MSC cells after transfection. The gastric cancer cells could be significantly promoted by the clearance of gastric cancer cells on the transfected bone marrow cells after transfection. Formation, migration, invasion and tumorigenicity of nude mice. Overexpression of miR-99a-5p can significantly block the induction of MSC in gastric cancer cell supernatant. High expression of miR-99a-5p in gastric cancer MSC can significantly inhibit the expression of a-SMA and FAP, the secretion of cytokines, and block the promotion of cell proliferation, migration and invasion of gastric cancer. The detection and analysis of the detected and reported genes identified the target of FGFR3 and IGF1R as the target of miR-99a-5p regulation and.Western blot detection results showed that the protein of FGFR3 and IGF1R and the downstream signal pathway molecules AKT and ERK were expressed in the bone marrow MSC after the induction of the supernatant of gastric cancer cells, and the expression of the low expression miR-99a-5p bone marrow MSC and gastric cancer was significantly increased and activated. 3 inhibitor AZD4547 IGF1R inhibitor OSI-906 pretreated bone marrow MSC respectively. The results showed that AZD4547 could inhibit the expression of a-SMA and FAP in the bone marrow MSC and the activation of the downstream signal pathway in the bone marrow MSC, and the promoting effect of the bone marrow MSC on the gastric cancer cells after the blocking action.AZD4547 treated gastric cancer Direct inhibition of the expression of a-SMA and FAP, blocking the promotion of gastric cancer cell migration,.OSI-906 preconditioning does not affect the transdifferentiation of gastric cancer cell supernatant and low expression miR-99a-5p on bone marrow MSC. Conclusion: in vitro successfully established gastric carcinoma cell supernatant induced MSC transdifferentiation model of bone marrow MSC to gastric cancer, and determined the low expression of miR in gastric cancer. The low expression of -99a-5p.miR-99a-5p can promote the MSC transdifferentiation of bone marrow MSC to gastric cancer. High expression of miR-99a-5p can promote the reverse differentiation of MSC to bone marrow MSC like cells, which is the key molecule to mediate gastric cancer cell supernatant and miR-99a-5p reprogramming bone marrow MSC. The completion of this work will clarify the MSC transdifferentiation and molecular mechanism of bone marrow MSC to gastric cancer. It provides new experimental basis for the origin and reshaping of gastric cancer microenvironment cell gastric cancer MSC, and provides new target and treatment strategy for the target microenvironment for the treatment of gastric cancer. It has important scientific research significance and clinical application prospect.
【学位授予单位】：江苏大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.2

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