YAP调节前列腺癌干细胞特性的机制研究

发布时间：2018-07-03 06:12

本文选题：前列腺癌 + YAP　；参考：《天津医科大学》2017年硕士论文

【摘要】：目的:检测YAP相关蛋白在不同前列腺癌细胞中具有干细胞特性的细胞的具体表达情况。利用基因干扰技术,通过对YAP相关基因进行调节,观察前列腺癌干细胞特性在体内及离体的表达的变化。进而探讨YAP通过调节下游干细胞特性的Sox-2、OCT-4等的表达,影响前列腺癌干细胞特性及自我分化作用能力。方法:实验包括体内实验和离体实验。离体实验是利用干细胞特有标记抗体蛋白的免疫磁珠,分选相应具有干细胞特性前列腺癌细胞的方法,用干细胞标记蛋白CD133抗体以及CD44抗体,分别在LNCaP细胞中筛选出具有干细胞特性前列腺癌细胞。再利用shRNA技术,分别在CD133高表达CD133正常表达CD133低表达的LNCaP细胞中抑制YAP的表达。利用不表达YAP的前列腺癌干细胞进行球形成实验,验证YAP与肿瘤干细胞特性之间的密切联系。同时检测YAP敲除的细胞系。比较OCT4,Nanog,Cmet,CD133,CD44和Sox2的mRNA表达水平。利用人工合成的YAP突变体质粒,即野生型YAP结合域突变体,YAP/TEAD结合域突变体,YAP/WW域突变体和SH3域突变体YAP分别转染至C42细胞及LNCaP细胞中当中,通过蛋白免疫印迹实验证明YAP与肿瘤干细胞的特有标记物OCT4,Nanog,CD133和Sox2之间的作用机制。进而进一步证实YAP的结合域TEAD对YAP调节细胞的自我更新能力的作用。而体内实验则是在去势组和非去势组TRAMP转基因鼠体内分别注射VP和DMSO并取瘤,研究YAP通过对OCT4,Nanog,CD133和Sox2这些肿瘤干细胞标记蛋白的调控,进而影响前列腺癌干细胞特性及自我更新能力。结果:1.通过检测YAP敲除的LNCaP细胞中干细胞相关分子标记物,可以发现YAP对在体内外维持前列腺癌细胞的自我更新和去分化是必要的。2.对前列腺癌细胞系进行插入YAP的各个结合域突变质粒后检测发现,YAP调节细胞的自我更新依赖于YAP定义域的TEAD交互区域。3.在TRAMP转基因鼠的体内模型上,我们发现,在阻遏YAP信号通路能够增加ADT的治疗作用。结论:YAP对在体内外维持前列腺癌细胞的自我更新和去分化是必要的,YAP调节细胞的自我更新依赖于YAP定义域的TEAD交互区域。YAP与其下OCT4,Nanog和Sox2等肿瘤干细胞标记物之间的相互作用,有望成为前列腺癌发展及转移的新的关键分子机制。阻遏YAP信号通路能够增加ADT的治疗作用。此外,YAP有可能作为一种新的肿瘤标记物,为临床诊治前列腺癌的新方法奠定基础,对判断预后有重要作用,也提出了治疗晚期转移性前列腺癌的新的思路。
[Abstract]:Aim: to detect the specific expression of YAP associated protein in different prostate cancer cells with stem cell characteristics. The expression of prostate cancer stem cells in vivo and in vitro was observed by regulating YAP related genes by gene interference technique. Furthermore, the effect of YAP on the characteristics of prostate cancer stem cells and the ability of self-differentiation was investigated by regulating the expression of Sox-2OCT-4 and so on. Methods: the experiments included in vivo and in vitro experiments. In vitro, the method of sorting prostate cancer cells with stem cell characteristic by using the immunomagnetic beads of stem cell specific labeled antibody protein was used to label CD133 antibody and CD44 antibody. Prostate cancer cells with stem cell characteristics were isolated from LNCaP cells. Then shRNA technique was used to inhibit the expression of YAP in LNCaP cells with high expression of CD133 and normal expression of CD133 and low expression of CD133. Prostate cancer stem cells without YAP expression were used to test the relationship between the characteristics of tumor stem cells and YAP. The cell lines of YAP knockout were also detected. The mRNA expression levels of CD133, CD44 and Sox2 in OCT4 were compared. YAP / WW and SH3 mutant were transfected into C42 cells and LNCaP cells, respectively, using synthetic YAP mutants, namely wild type YAP / TEAD domain mutants, YAP / WW domain mutants and SH3 domain mutants, respectively, which were transfected into C42 cells and LNCaP cells, respectively, by transfection of YAP / WW and SH3 mutants into C42 cells and LNCaP cells, respectively. The mechanism between YAP and OCT4CT-NanogCD133 and Sox2 was proved by Western blot. It is further confirmed that the binding domain of YAP, tea, plays an important role in the self-renewal of YAP regulatory cells. In vivo, VP and DMSO were injected into the ovariectomized and non-ovariectomized TRAMP transgenic mice, respectively, and the tumor was removed. The regulation of YAP on tumor stem cell marker proteins, such as OCT4, NanogCon CD133 and Sox2, was studied. In turn, the characteristics and self-renewal ability of prostate cancer stem cells are affected. The result is 1: 1. By detecting stem cell related molecular markers in LNCaP cells knocked out by YAP, it is found that YAP is necessary to maintain self-renewal and dedifferentiation of prostate cancer cells in vivo and in vitro. After inserting YAP binding domain mutant plasmids into prostate cancer cell line, it was found that the self-renewal of YAP regulatory cells was dependent on the tea interaction region of YAP domain. In vivo model of TRAMP transgenic mice, we found that inhibiting YAP signaling pathway can increase the therapeutic effect of ADT. Conclusion it is necessary to maintain the self-renewal and dedifferentiation of prostate cancer cells in vitro and in vivo by the interaction between TEAD interaction region of YAP domain and tumor stem cell markers such as OCT4Nanog and Sox2. It is expected to be a new key molecular mechanism for the development and metastasis of prostate cancer. Blocking YAP signaling pathway can increase the therapeutic effect of ADT. In addition, YAP may be a new tumor marker, which can lay a foundation for clinical diagnosis and treatment of prostate cancer, play an important role in judging prognosis, and put forward a new idea for the treatment of advanced metastatic prostate cancer.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.25

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