SAHA-CTSB触发细胞自噬介导乳腺癌分子串说的机制研究

发布时间：2018-05-16 20:46

本文选题：SAHA + CTSB　；参考：《沈阳医学院》2017年硕士论文

【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。
[Abstract]:Breast Cancer (BC) is one of the most common malignant tumors in women. Its incidence is mainly concentrated in perimenopausal women between 40 and 60 years old. In Europe and America, the incidence of breast cancer is the first of the female malignant tumors. In recent years, with the rapid development of modern urban construction in China and the life of the country, The increase of pressure is increasing, the incidence of breast cancer in China's large and middle cities is increasing year by year, and it is becoming younger and threatening the physical and mental health of women. In eukaryotes, the acetylation and deacetylation modification of histone plays an important role in gene expression. The high acetylation state of chromatin can lead to tumor inhibition. Gene or apoptotic genes are highly expressed. Histone deacetylase inhibitor (Histone Deacetylase Inhibitor, HDACi) symplectic two aniline oxime (SuberoylanilideHydroxamicAcid, SAHA) can improve the expression of gene by increasing the level of histone acetylation in a specific chromatin region. It has become a new type of antitumor drug. It is found that SAHA can cause a variety of tumor cell growth stagnation and death, including a variety of breast cancer cell lines MCF-7, MDA-MB-231, MDA-MB-435, SKBr-3 and so on. Cathepsin B (CathepsinB, CTSB) is a cysteine protease in a soluble enzyme. Recent studies have shown that the level of CTSB expression changes with the tumor cells (breast cancer, eggs). The proliferation, invasion, metastasis and angiogenesis are closely related to the proliferation, invasion, metastasis and angiogenesis of nests, bladder cancer, lung cancer, colon cancer, melanoma, gastric cancer and so on. So the overexpression of CTSB in tumor cells is considered to be related to the aggressive and bad prognosis of clinical tumors. However, some studies have also shown that the expression of CTSB also induces the apoptosis and autophagy of some tumor cells. Autophagy (Autophagy) is a mechanism that can remove abnormal proteins and damage organelles in the body, which is beneficial to the homeostasis of all substances in the cells. In the stress state of hypoxia, energy shortage, infection and inflammation, autophagy can inhibit the accumulation of toxic and even carcinogenic proteins and cell organelles, and protect the cells to inhibit cell carcinogenesis. In the late stage of the tumor, autophagy provides the tumor cells with unlimited growth nutrients and promote the proliferation of cancer cells. In recent years, scholars have found that autophagy and its related molecules play an important role in the proliferation and treatment of breast cancer. The effects of SAHA on the proliferation of two breast cancer cell lines, MCF-7 and MDA-MB-231 cells, and the effect on the CTSB expression in two breast cancer cells. Because CTSB is a cysteine protease in the lysosome, however, the key enzymes involved in autophagy are mainly concentrated in the lysosomes. Therefore, we speculate that SAHA-CTSB and some signal pathway are in the lysosome. This study provides a good experimental basis for SAHA-CTSB on the mechanism of breast cancer cell proliferation and autophagy, and provides a theoretical basis for SAHA-CTSB to be a new target for the study of breast cancer gene therapy. Method 1 The effect of MCF-7 and MDA-MB-231.2. on the proliferation of MCF-7 and MDA-MB-231 cells by RTCA and the effect of SAHA on the proliferation of MCF-7 and MDA-MB-231 cells. The morphological changes of the cell proliferation of MCF-7 and MDA-MB-231 cells were observed by morphology. ISA and In Cell Western detection of the changes in the expression of CTSB protein after SAHA and / or Cystatin C acts on MCF-7 and MDA-MB-231 breast cancer cells The expression of LC3 II in cells after the action of SAHA and / or Cystatin C on MCF-7 and MDA-MB-231 cells.8. application RT-QPCR Array, Western Blot detection of SAHA and / or the changes in the expression of autophagy related factors after breast cancer cells The expression of MAPK signaling pathway related factors after MCF-7 and MDA-MB-231 cells. Results 1.RTCA, BioStationIM results showed that SAHA could inhibit the proliferation of MCF-7 and MDA-MB-231 cells of breast cancer,.2.Western Blot, ELISA. The expression, on the contrary, inhibits the anti-tumor effect of SAHA on the.3. cell immunofluorescence, and the Array detection results of the RT-QPCR Array, Western Blot and Human MAPK antibody show that the activation of the mammary cancer cells is induced by the mediation of SAHA, and the activation of the signal pathway plays an important role in this process. A inhibits the proliferation of MCF-7 and MDA-MB-231 cells in breast cancer, and SAHA inhibits the proliferation of breast cancer cells with the high expression of CTSB, which inhibits the anti tumor action of SAHA, and.3.SAHA activates the autophagy of breast cancer cells by activating CTSB, which may be regulated by the MAPK signaling pathway.
【学位授予单位】：沈阳医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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