【作者】 李静；

【导师】 周伟强；

【作者基本信息】 沈阳医学院 ， 病原生物学， 2017， 硕士

【摘要】 目的乳腺癌(Breast Cancer,BC)是女性中最常见的恶性肿瘤之一,其发病人群主要集中在40岁～60岁之间的围绝经期女性。在欧美等发达国家中,乳腺癌发病率位居女性恶性肿瘤的首位。近年来,随着我国现代化城市建设的不断加快以及生活压力的不断增加,乳腺癌在我国大中城市的发病率逐年增加,且呈现年轻化趋势,严重威胁着女性的身心健康。在真核生物中,组蛋白的乙酰化与去乙酰化修饰在基因表达中起到重要的调控作用。染色质的高乙酰化状态能导致肿瘤抑制基因或促凋亡基因高表达。组蛋白去乙酰化酶抑制剂(Histone Deacetylase Inhibitor,HDACi)辛二酰苯胺异羟肟酸(SuberoylanilideHydroxamicAcid,SAHA)可通过提高染色质特定区域组蛋白乙酰化水平影响基因的表达,已成为一类新的抗肿瘤药物。大量的研究发现,SAHA能够引发多种肿瘤细胞生长停滞和死亡,其中包括多种乳腺癌细胞系MCF-7、MDA-MB-231、MDA-MB-435、SKBr-3 等。组织蛋白酶B(CathepsinB,CTSB)是一个溶酶体内的半胱氨酸蛋白酶。近年来的研究表明,CTSB表达水平的改变与肿瘤细胞(乳腺癌、卵巢癌、膀胱癌、肺癌、肠癌、黑色素瘤、胃癌等)的增殖、侵袭、转移和血管生成密切相关。因此CTSB在肿瘤细胞中的过表达被认为与临床肿瘤的侵袭性强和不良预后有关。但也有研究表明,CTSB的表达对某些肿瘤细胞的凋亡和自噬也有诱导作用。自噬(Autophagy)是一种能够清除机体内异常蛋白质和损伤细胞器而有利于细胞内各物质保持稳态的机制。在缺氧、能量缺乏、感染炎症等应激状态下,自噬可抑制有毒甚至致癌的受损蛋白质和细胞器的聚集,保护细胞进而抑制细胞癌变;然而在肿瘤后期时,自噬则给肿瘤细胞提供无限生长的养分,促进癌细胞增殖。近年来随着对自噬研究的不断深入,学者们发现自噬及其相关分子在乳腺癌的增殖和治疗中扮演重要的角色,因此越来越受到人们的重视。本研究旨在初步探讨SAHA对两种乳腺癌细胞系MCF-7和MDA-MB-231细胞增殖能力的影响;以及对两种乳腺癌细胞CTSB表达变化的影响。由于CTSB是溶酶体中的半胱氨酸蛋白酶,然而参与自噬发生的关键酶主要集中在溶酶体中,因此我们推测SAHA-CTSB与某种信号通路间存在分子串说,进而促进或抑制自噬来调控乳腺癌的发生和发展。本研究为明确SAHA-CTSB对乳腺癌细胞增殖及自噬产生的作用机制提供了良好的实验基础,也为SAHA-CTSB能够成为乳腺肿瘤基因治疗研究的一个新靶标提供理论依据。方法1.培养乳腺癌细胞MCF-7和MDA-MB-231。2.应用RTCA检测SAHA对MCF-7和MDA-MB-231细胞增殖能力的影响。3.应用BioStationIM活细胞工作站,形态学观察SAHA作用MCF-7和MDA-MB-231细胞后细胞增殖的变化情况。4.利用CTSB抑制剂Cystatin C阻断CTSB的表达。5.Western Blot、ELISA 和 In Cell Western 检测 SAHA 和(或)Cystatin C 作用于MCF-7和MDA-MB-231乳腺癌细胞后,CTSB蛋白表达变化的情况。6.MuseTM流式细胞技术检测SAHA和(或)Cystatin C作用于MCF-7和MDA-MB-231乳腺癌细胞后细胞活力变化的情况。7.应用细胞免疫荧光技术,荧光显微镜下观察SAHA和(或)Cystatin C作用MCF-7和MDA-MB-231细胞后细胞内LC3 Ⅱ的表达情况。8.应用 RT-QPCR Array、Western Blot 检测 SAHA 和(或)Cystatin C 作用乳腺癌细胞后自噬相关因子表达的变化。9.应用 Human MAPK antibody Array 检测 SAHA 和(或)Cystatin C 作用MCF-7和MDA-MB-231细胞后MAPK信号通路相关因子的表达情况。结果1.RTCA、BioStationIM结果显示,SAHA能够抑制乳腺癌MCF-7和MDA-MB-231细胞的增殖。2.Western Blot、ELISA、In Cell Western及MuseTM流式细胞活力检测结果显示,SAHA在抑制乳腺癌细胞增殖时CTSB高表达,相反的是CTSB功能失活时则抑制SAHA的抗肿瘤作用。3.细胞免疫荧光、RT-QPCR Array、Western Blot 及 Human MAPK antibody Array检测结果显示,SAHA通过CTSB的介导诱导乳腺癌细胞自噬的发生,在此过程中MAPK信号通路的激活起着重要的作用。结论1.SAHA能够抑制乳腺癌MCF-7和MDA-MB-231细胞增殖,并且SAHA在抑制乳腺癌细胞增殖时伴随着CTSB的高表达。2.CTSB功能失活抑制SAHA的抗肿瘤作用。3.SAHA通过激活CTSB介导乳腺癌细胞自噬,而这一过程可能主要通过MAPK信号通路调节。更多还原

【Abstract】 ObjectiveBreast cancer(BC)is one of the most common malignant tumors in women,and the age of onset is mainly in 40～60 years-old women in perimenopausal period.In Europe,the United States and other developed countries,the incidence of breast cancer is ranked the first position in women with malignant tumors.In recent years,with the acceleration of city construction and increase of the life pressure,the incidence rate of breast cancers in China is increasing year by year,and the illness age is more and more lowered.It affects women’s health.Acetylation and deacetylation of histone proteins play an important role in the regulation of gene expression in eukaryotes.The acetylation state of chromatin can lead to the expressions of tumor suppressor genes and pro-apoptotic genes.As a Histone deacetylase inhibitors(Histone Deacetylase Inhibitor,HDACi),SAHA(Suberoylanilide Hydroxamic Acid,SAHA)can increase the acetylation status in specific chromatin regions,thereby affecting gene expressions.It has become a new class of anticancer drugs.A large number of studies have found that SAHA can result in growth arrest and death in a variety of tumors,such as MCF-7,MDA-MB-231,MDA-MB-435 and SKBr-3 breast cancer cell lines.Cathepsin B(CTSB)is a lysosomal cysteine protease.Recent studies showed that CTSB expression is involved in the cell proliferation,invasion,metastasis and angiogenesis in tumor cells(breast cancer,ovarian cancer,bladder cancer,lung cancer,colon cancer,melanoma,gastric cancer).Therefore,the overexpression of CTSB in tumor cells is thought to be associated with aggressive and poor prognosis.However,some studies have shown that the expression of CTSB may play an important role in apoptosis and autophagy in some tumor cells.Autophagy is a biological effect that can eliminate the abnormal proteins and aging organelles in the body.It is beneficial to maintain the homeostasis for the cell.In hypoxia,energy deficiency,inflammation and other stress conditions,autophagy can inhibit the functions of toxic or carcinogenic proteins,repress the organelle aggregation,protect cells and depress the carcinogenesis.Furthermore,autophagy can supply unlimited nutrients for tumor growth and promote the proliferation of cancer cells.In recent years,with the deepening research on autophagy,scholars have found that autophagy play an important role in the treatment and prevention of breast cancer,and more and more attention has been paid to it.The purpose of this study is to investigate the role of SAHA in the proliferation of two breast cancer cells MCF-7 and MDA-MB-231,and to assess the expressions of CTSB in breast cancer cells.Belong to the members of lysosomal cysteine protease,CTSB can mediate breast carcinogenesis by autophagy.The study will construct a good experimental basis on breast cancer proliferation regulated by the crosstalk between SAHA-CTSB and other signal pathway,but also provide a theoretical basis on the novel target of gene therapy in breast cancer treatment.Methods1.Culture MCF-7 and MDA-MB-231 breast cancer cells.2.Utilize RTCA to monitor the proliferation changes by SAHA treatment in MCF-7 and MDA-MB-231 cells.3.Employ BioStationIM time-lase live cell imaging acquisition to measure the cell proliferation changes by SAHA treatment in MCF-7 and MDA-MB-231 cells.4.Use CTSB inhibitor Cystatin C to knock down the function of CTSB.5.Employ Western Blot,ELISA and In Cell Western to measure the expression of CTSB and screen the optimal concentrations of Cystatin C.6.Employ MuseTM to analyze the cell viability in MCF-7 and MDA-MB-231 cells with SAHA and Cystatin C treatment.7.Employ immunocytochemistry to measure the expression of LC311 in MCF-7 and MDA-MB-231 cells with SAHA and Cystatin C treatment.8.Analyze the expression of autophagy-related factors in breast cancer with SAHA and Cystatin C treatment by RT-QPCR Array and Western Blot.9.Analyze the expression of MAPK signaling pathway in MCF-7 and MDA-MB-231 cells with SAHA and Cystatin C treatment by MAPK antibody Array.Results1.RTCA and BioStationIM results showed that SAHA could inhibit the proliferation of breast cancer cell line MCF-7 and MDA-MB-231.2.Western Blot,ELISA,In Cell Western and Muse analysis showed that the high expression of CTSB was accompanied by the SAHA inhibitory effect in breast cancer cells.Contrarily,blocking-up the expression of CTSB can reverse the effect of SAHA.3.Immunofluorescence,RT-QPCR Array,Western Blot and MAPK antibody Array showed that SAHA mediated the autophagy by activating CTSB in breast cancer cells,and it was associated with MAPK signaling pathway.Conclusion1.SAHA can inhibit the proliferation of MCF-7 and MDA-MB-231 breast cells with the high expression of CTSB.2.The blocking-up the expression of CTSB can inhibit the anti-tumor effect of SAHA.3.SAHA mediates the autophagy by activating CTSB in breast cancer cells,and it is associated with MAPK signaling pathway.更多还原