胃癌间质干细胞中YAP信号在胃癌发展过程中的作用

发布时间：2018-06-15 19:38

本文选题：YAP + 间质干细胞　；参考：《江苏大学》2017年硕士论文

【摘要】：目的:间质干细胞(mesenchymal stem cell,MSC)作为肿瘤微环境中的重要组成成分调节着肿瘤的生物学行为。在过去的研究中我们成功从胃癌组织中分离得到胃癌间质干细胞(gastric cancer-derived MSC,GC-MSC)并发现GC-MSC促进胃癌的发展。但GC-MSC中YAP分子作用仍不清楚。本研究旨在阐明GC-MSC中YAP分子在胃癌发展过程中的作用。方法:Western blot检测比较骨髓间质干细胞(bone marrow mesenchymal stem cell,BM-MSC)与GC-MSC中YAP分子的表达差异。通过慢病毒YAP-ShRNA对GC-MSC中YAP分子进行敲减,应用qRT-PCR和Western blot检测YAP的敲减效率。平板克隆实验、Western blot、免疫荧光实验、Transwell迁移实验和侵袭实验检测敲减后的GC-MSC细胞增殖、迁移、侵袭、上皮间质转化(epithelial-mesenchymal transition,EMT)以及干性能力的改变。平板克隆实验,免疫荧光实验、流式细胞术、qRT-PCR和Western blot分别检测Blank组SGC-7901细胞(Blank SGC-7901),Control组GC-MSC培养上清(conditioned medium,CM)处理的SGC-7901细胞[GC-MSC(Control)-CM SGC-7901]以及ShYAP组GC-MSC培养上清处理的SGC-7901细胞[GC-MSC(ShYAP)-CM SGC-7901]的增殖、凋亡、迁移、侵袭能力及EMT指标的变化。qRT-PCR检测3种不同培养上清处理的SGC-7901细胞中促血管形成相关基因IL-6、PDGF和VEGF的变化。采用血管形成试验和迁移实验检测敲减YAP的GC-MSC培养上清处理SGC-7901细胞72h后SGC-7901上清促血管形成能力的改变。qRT-PCR和Western blot检测三种不同处理的SGC-7901中β-catenin及其下游靶基因CD44和CyclinD的表达变化。分别将3种不同培养上清处理的SGC-7901细胞接种裸鼠,构建裸鼠5致瘤模型,测定3组模型致瘤瘤体大小和重量。免疫组化和免疫荧光检测瘤组织中Ki67、β-catenin、E-cadherin、Vimentin及CD31的表达。结果:与BM-MSC相比,YAP在GC-MSC中高表达。YAP敲减可抑制GC-MSC中增殖相关蛋白PCNA及Ki67的表达,EMT相关蛋白N-cadherin、Vimentin的下调和E-cadherin的上调,干性蛋白CD44、Sall4和Nanog的下调。同时,GC-MSC增殖、迁移和侵袭能力也明显下降。GC-MSC(ShYAP)-CM SGC-7901中PCNA及Ki67的表达降低,EMT相关蛋白N-cadherin、Vimentin的下调和E-cadherin的上调,血管形成相关基因IL-8、PDGF和VEGF表达下调,凋亡相关蛋白Bcl2和Bax没有明显改变。其增殖、迁移、侵袭能力及促血管形成能力也明显下降。研究显示YAP敲减的GC-MSC培养上清可以抑制SGC-7901中对胃癌发展有重要作用的β-catenin及其下游靶基因CD44和CyclinD的表达。裸鼠皮下致瘤实验表明,YAP敲减的GC-MSC培养上清处理的胃癌细胞体内致瘤能力明显下降。免疫组化、免疫荧光结果显示GC-MSC(ShYAP)-CM SGC-7901形成的瘤组织中E-cadherin表达升高,Ki67、β-catenin、Vimentin及CD31表达明显下降。结论:YAP敲减不仅可以抑制GC-MSC增殖、迁移、侵袭、EMT及干性分子的表达,还能抑制体外胃癌细胞的增殖,迁移,侵袭及促血管形成能力,抑制体内胃癌的发展。YAP敲减的GC-MSC培养上清可能通过调控胃癌细胞中β-catenin的表达来影响胃癌的发展。
[Abstract]:Aim: mesenchymal stem cells (MSCs), as an important component of tumor microenvironment, regulate the biological behavior of tumor. In previous studies, we successfully isolated gastric cancer mesenchymal stem cells from gastric cancer tissues and found that GC-MSC promoted the development of gastric cancer. However, the interaction of YAP molecules in GC-MSC is still unclear. The purpose of this study was to elucidate the role of YAP molecules in the development of gastric cancer. Methods the expression of YAP in bone marrow mesenchymal stem cells (BM-MSC) and GC-MSC was detected by Western blot. Yap molecules in GC-MSC were knocked down by lentivirus YAP-ShRNA, and the knockout efficiency of YAP was detected by qRT-PCR and Western blot. Western blot, Transwell migration assay and invasion assay were used to detect the proliferation, migration, invasion, epithelial-mesenchymal transitionof epithelial mesenchymal transition (EMTT) and the change of dry ability. The proliferation, apoptosis and migration of SGC-7901 cells treated with SGC-7901 cell line Blank SGC-7901 and SGC-7901 treated with GC-MSC culture supernatant were detected by plate cloning assay, immunofluorescence assay, flow cytometry qRT-PCR and Western blot, respectively, and the proliferation, apoptosis and migration of SGC-7901 cells treated with SGC-7901 and ShYAP GC-MSC culture supernatant were detected. The changes of invasion ability and EMT index. QRT-PCR was used to detect the changes of angiogenic genes IL-6, PDGF and VEGF in SGC-7901 cells treated with three different supernatants. Angiogenesis test and migration assay were used to detect the expression of 尾 -catenin and its downstream target genes CD44 and CyclinD in SGC-7901 cells treated with SC-MSC culture supernatant of knockdown Yap for 72 h. The expression of 尾 -catenin and its downstream target genes CD44 and CyclinD in SGC-7901 were detected by qRT-PCR and Western blot. SGC-7901 cells treated with three different culture supernatants were inoculated into nude mice, and the tumorigenic model of nude mice was established. The tumor size and weight of the three groups were measured. The expression of Ki67, 尾 -cateninine E-cadherinn and CD31 were detected by immunohistochemistry and immunofluorescence. Results: compared with BM-MSC, the expression of proliferation-associated protein PCNA and Ki67 in GC-MSC was inhibited by high expression of YAP. YAP knockout inhibited the down-regulation of N-cadherin and E-cadherin, and down-regulation of CD44-Sall4 and Nanog in GC-MSC. At the same time, the proliferation, migration and invasion ability of GC-MSC were also significantly decreased. The expression of PCNA and Ki67 in GC-MSCC ShYAP-CM SGC-7901 was significantly decreased, the expression of N-cadherin Vimentin and E-cadherin was down-regulated, and the expression of IL-8, PDGF and VEGF was down-regulated, while the apoptosis-related proteins Bcl2 and Bax were not significantly changed. Proliferation, migration, invasion and angiogenesis were also significantly decreased. The results showed that the supernatant of GC-MSC with YAP knockout could inhibit the expression of 尾 -catenin and its downstream target genes CD44 and CyclinD in SGC-7901. The results of subcutaneous tumorigenesis in nude mice showed that the tumorigenic ability of gastric cancer cells treated with the supernatant of GC-MSC by YAP knockout was significantly decreased. Immunohistochemistry and immunofluorescence showed that the expression of E-cadherin increased, 尾 -catenin Vimentin and CD31 decreased significantly in the tumor tissue formed by GC-MSC-ShYAP-CM SGC-7901. Conclusion WYAP knockout can not only inhibit the proliferation, migration, invasion of EMT and the expression of dry molecules in GC-MSC, but also inhibit the proliferation, migration, invasion and angiogenesis of gastric cancer cells in vitro. Inhibiting the development of gastric cancer in vivo. The culture supernatant of GC-MSC with YAP knockout may affect the development of gastric cancer by regulating the expression of 尾 -catenin in gastric cancer cells.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

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