软木肟酸激活Notch-1信号通路通过P53诱导甲状腺未分化癌细胞凋亡
发布时间：2018-09-19 08:06
【摘要】：目的:甲状腺未分化癌(anaplastic thyroid carcinoma,ATC)是甲状腺癌中发病率较低但恶性度很高预后极差的恶性肿瘤,目前治疗的方法主要有传统手术和放化疗结合手术的方式,但效果都不理想。随着ATC遗传和分子发病机制研究的不断深入,基因靶向治疗也逐渐兴起,并有望成ATC治疗的发展方向之一。Notch信号通路在进化上高度保守,通过相邻细胞之间的相互作用调节细胞、组织、器官的分化和发育。软木肟酸(Suberoyl bis-hydroxamic acid,SBHA)属于第二代异羟肟酸类组蛋白乙酰化酶抑制剂,它可以诱导激活Notch1信号通路,在神经内分泌肿瘤中起到抑癌作用。本研究探讨在甲状腺未分化癌细胞中应用软木肟酸激活Notch1信号通路,是否可以抑制肿瘤细胞增殖诱导细胞凋亡,在此基础上探讨Notch1信号通路是否通过P53通路发挥作用。方法:1.收集天津医科大学肿瘤医院甲状腺未分化癌组织标本16例,应用免疫组织化学染色检测Notch1和P53蛋白表达情况。应用Western blot技术检测正常甲状腺细胞系、甲状腺乳头状癌系和甲状腺未分化癌细胞系中Notch1蛋白的表达情况。2.应用不同浓度SBHA作用于甲状腺未分化癌细胞系,应用MTT实验判断SBHA对甲状腺未分化癌细胞增殖情况的影响,应用流式细胞术判断SBHA对甲状腺未分化癌细胞促凋亡效应,应用Western blot技术检测Notch1、cleaved caspase3、cleaved PRAP和Bax蛋白的表达情况,来验证细胞凋亡情况。应用Real-time PCR技术检测Notch1和Hes1的m RNA水平,来判断Notch1信号通路的激活情况。应用siRNA干扰细胞系Notch1的表达,再应用SBHA激活该信号通路,判断SBHA是否作用于Notch1通路。3.应用不同浓度SBHA作用于甲状腺未分化癌细胞系,应用Western blot技术检测P53和P21蛋白的表达情况,来判断细胞凋亡是否通过该通路完成,并分析和Notch1通路是否有关。应用Real-time PCR技术检测P53和P21的mRNA情况验证P53和P21激活情况。应用免疫共沉淀实验检测Notch1蛋白和P53蛋白是否存在联系。应用shRNA干扰细胞系P53的表达,再应用SBHA激活Notch1通路,验证Notch1和P53两者的关系。结果:1.免疫组织化学结果显示,16例组织切片中仅有1例切片呈现Notch1和P53阳性结果,且为同一患者组织。Western blot检测结果显示正常甲状腺细胞系中有Notch1蛋白表达,甲状腺乳头状癌系中Notch1蛋白低表达而甲状腺未分化癌细胞系中Notch1蛋白不表达。2.应用不同浓度SBHA作用于甲状腺未分化癌细胞系,可以诱导Notch1mRNA和蛋白的表达,活化Notch1信号通路,有效的抑制细胞的增殖,诱导促凋亡蛋白的表达,促进细胞凋亡,并且呈时间和剂量双重依赖性。3.在甲状腺未分化癌细胞系中应用不同浓度SBHA可以诱导P53和P21蛋白和mRNA时间和剂量依赖性的表达,这与Notch1一致。免疫共沉淀实验提示两者存在关联,shRNA干扰实验提示SBHA激活Notch1信号通路是通过P53通路诱导细胞凋亡的。结论:1.Notch1和P53蛋白在甲状腺未分化癌组织中处于低表达状态,Notch1蛋白在甲状腺未分化癌细胞系中低表达或不表达。2.SBHA在甲状腺未分化癌细胞系中可以激活Notch1信号通路,抑制细胞的增殖,并且呈时间和剂量双重依赖诱导细胞凋亡。3.在甲状腺未分化癌细胞系中,Notch1和P53两者存在联系,SBHA激活Notch1信号通路是通过P53通路诱导细胞凋亡的。
[Abstract]:Objective: Undifferentiated thyroid carcinoma (ATC) is a malignant tumor with a low incidence but a high degree of malignancy and a poor prognosis. Currently, the main treatment methods are traditional surgery combined with radiotherapy and chemotherapy, but the effect is not satisfactory. Notch signaling pathway is highly conserved in evolution and regulates the differentiation and development of cells, tissues and organs through the interaction between adjacent cells. Suberoyl bis-hydroxamic acid (SBHA) belongs to the second generation of isohydroxamic acid histone acetylation. Enzyme inhibitors, which can induce and activate Notch1 signaling pathway, play an inhibitory role in neuroendocrine tumors. This study was to investigate whether pyramidal acid activates Notch1 signaling pathway in undifferentiated thyroid cancer cells and inhibits proliferation and induces apoptosis of tumor cells. On this basis, we investigated whether Notch1 signaling pathway is mediated by P53. Methods: 1. The expression of Notch1 and P53 proteins in 16 undifferentiated thyroid carcinoma tissues collected from the Tumor Hospital of Tianjin Medical University was detected by immunohistochemical staining. The expression of Notch1 protein in normal thyroid cell line, thyroid papillary carcinoma line and thyroid undifferentiated carcinoma cell line was detected by Western blot. Expression of SBHA in undifferentiated thyroid carcinoma cell lines was determined by MTT assay. The effect of SBHA on the proliferation of undifferentiated thyroid carcinoma cells was evaluated by flow cytometry. Notch 1, cleaved caspase 3, cleaved PRA were detected by Western blot. Real-time PCR was used to detect the level of Notch1 and Hes1 m RNA and to determine the activation of Notch1 signaling pathway. Western blot was used to detect the expression of P53 and P21 proteins in undifferentiated thyroid carcinoma cell lines to determine whether apoptosis was completed through this pathway and whether there was any relationship between the pathway and Notch1. Real-time PCR was used to detect the mRNA of P53 and P21 to verify the activation of P53 and P21. Immunocoprecipitation assay was used to detect the expression of P53 and P21 proteins. The relationship between Notch1 and P53 was examined by interfering with the expression of P53 by shRNA and activating Notch1 pathway by SBHA. Results: 1. Immunohistochemistry showed that only one of the 16 tissue sections showed Notch1 and P53 positive results, and were the same patient tissue. The results showed that Notch1 protein was expressed in normal thyroid cell lines, but not in undifferentiated thyroid carcinoma cell lines. 2. The expression of Notch1 mRNA and protein was induced and Notch1 signaling pathway was activated by different concentrations of SBHA in undifferentiated thyroid carcinoma cell lines. 3. Different concentrations of SBHA could induce time and dose dependent expression of P53 and P21 proteins and mRNA in undifferentiated thyroid carcinoma cell lines, which was consistent with Notch1. Conclusion: 1. Notch1 and P53 proteins are low expressed in undifferentiated thyroid carcinoma tissues, and Notch1 protein is low or not expressed in undifferentiated thyroid carcinoma cell lines. 2. SBHA is not expressed in undifferentiated thyroid carcinoma cell lines. In undifferentiated thyroid carcinoma cell lines, Notch1 and P53 are related, and SBHA activates Notch1 signaling pathway to induce apoptosis through P53 pathway.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R736.1
本文编号：2249534
[Abstract]:Objective: Undifferentiated thyroid carcinoma (ATC) is a malignant tumor with a low incidence but a high degree of malignancy and a poor prognosis. Currently, the main treatment methods are traditional surgery combined with radiotherapy and chemotherapy, but the effect is not satisfactory. Notch signaling pathway is highly conserved in evolution and regulates the differentiation and development of cells, tissues and organs through the interaction between adjacent cells. Suberoyl bis-hydroxamic acid (SBHA) belongs to the second generation of isohydroxamic acid histone acetylation. Enzyme inhibitors, which can induce and activate Notch1 signaling pathway, play an inhibitory role in neuroendocrine tumors. This study was to investigate whether pyramidal acid activates Notch1 signaling pathway in undifferentiated thyroid cancer cells and inhibits proliferation and induces apoptosis of tumor cells. On this basis, we investigated whether Notch1 signaling pathway is mediated by P53. Methods: 1. The expression of Notch1 and P53 proteins in 16 undifferentiated thyroid carcinoma tissues collected from the Tumor Hospital of Tianjin Medical University was detected by immunohistochemical staining. The expression of Notch1 protein in normal thyroid cell line, thyroid papillary carcinoma line and thyroid undifferentiated carcinoma cell line was detected by Western blot. Expression of SBHA in undifferentiated thyroid carcinoma cell lines was determined by MTT assay. The effect of SBHA on the proliferation of undifferentiated thyroid carcinoma cells was evaluated by flow cytometry. Notch 1, cleaved caspase 3, cleaved PRA were detected by Western blot. Real-time PCR was used to detect the level of Notch1 and Hes1 m RNA and to determine the activation of Notch1 signaling pathway. Western blot was used to detect the expression of P53 and P21 proteins in undifferentiated thyroid carcinoma cell lines to determine whether apoptosis was completed through this pathway and whether there was any relationship between the pathway and Notch1. Real-time PCR was used to detect the mRNA of P53 and P21 to verify the activation of P53 and P21. Immunocoprecipitation assay was used to detect the expression of P53 and P21 proteins. The relationship between Notch1 and P53 was examined by interfering with the expression of P53 by shRNA and activating Notch1 pathway by SBHA. Results: 1. Immunohistochemistry showed that only one of the 16 tissue sections showed Notch1 and P53 positive results, and were the same patient tissue. The results showed that Notch1 protein was expressed in normal thyroid cell lines, but not in undifferentiated thyroid carcinoma cell lines. 2. The expression of Notch1 mRNA and protein was induced and Notch1 signaling pathway was activated by different concentrations of SBHA in undifferentiated thyroid carcinoma cell lines. 3. Different concentrations of SBHA could induce time and dose dependent expression of P53 and P21 proteins and mRNA in undifferentiated thyroid carcinoma cell lines, which was consistent with Notch1. Conclusion: 1. Notch1 and P53 proteins are low expressed in undifferentiated thyroid carcinoma tissues, and Notch1 protein is low or not expressed in undifferentiated thyroid carcinoma cell lines. 2. SBHA is not expressed in undifferentiated thyroid carcinoma cell lines. In undifferentiated thyroid carcinoma cell lines, Notch1 and P53 are related, and SBHA activates Notch1 signaling pathway to induce apoptosis through P53 pathway.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R736.1
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