下调ZEB1表达抑制黑色素瘤干细胞的致瘤性和肺转移研究

发布时间：2018-01-12 09:20

本文关键词：下调ZEB1表达抑制黑色素瘤干细胞的致瘤性和肺转移研究　出处：《东南大学》2015年硕士论文　论文类型：学位论文

【摘要】：恶性黑色素瘤(malignant melanoma, MM)是来源于黑色素细胞的一种恶性肿瘤。在过去的20多年中,世界范围内恶性黑色素瘤的发病率呈逐步增高的趋势。皮肤是恶性黑素色瘤的主要发病部位,占皮肤恶性肿瘤的死亡率约70%,也可起源于眼、鼻腔等处,其高度侵袭与转移的能力是恶性黑色素瘤患者死亡的主要原因,早期可转移至肺、脑等部位,晚期随体循环扩散至全身。而上皮-间充质转换(epithelialmesenchymal transition, EMT)在其中扮演着重要角色,EMT被认为是启动肿瘤侵袭转移的重要事件。研究表明,历经EMT的恶性黑色素瘤细胞能从肿瘤的原发灶逃离,侵袭到周围其他组织,经过血液或淋巴途径到达更远处的器官形成转移灶。新近研究还发现,有EMT表型的恶性黑色素瘤细胞更具耐药性、免疫抑制及类干细胞等特性。在EMT的发生发展过程中最主要的调控对象是E-钙黏着蛋白(E-cadherin)。该蛋白是维持上皮细胞特性的重要分子,E-cadherin的丢失是EMT的一个重要特征,若E-cadherin表达下降,细胞则会呈现许出多非上皮细胞的特征,如细胞极性丢失,细胞间的粘附性降低,转变成更易发生转移的间质细胞,而表达波形蛋白(Vimentin)上调,这些蛋白表达改变是EMT的一个重要标志。因此,探究EMT的发生和调控机制,对于寻找治疗肿瘤细胞转移的靶点有重要意义。随着分子生物学的发展,研究者发现肿瘤组织中有极少数细胞具有类干细胞的特性：能无限增殖、不断分化,对化疗、放疗不敏感,而这些细胞成为肿瘤发生、侵袭转移和复发的关键细胞,即肿瘤干细胞(tumor stem cells, TSCs)或癌干细胞(cancer stem cells, CSCs)。近来对肿瘤发生发展过程的研究表明,锌指结合蛋白(1 zinc finger E-box-binding protein 1, ZEB1)在肿瘤转移过程中发挥着重要作用。ZEB1是锌指结构转录因子中的E盒结合锌指蛋白,它不仅能调节EMT的发生和发展,还能通过调节细胞周期、细胞凋亡、衰老、侵袭转移和间质血管生成等,进而调节肿瘤的进展。本课题组前期实验结果显示：降低B16F10细胞中ZEB1的表达,可在一定程度上降低B16F10细胞在体外的克隆形成能力、增殖能力、耐药性、侵袭转移能力及体内致瘤能力,但对B16F10细胞中CSCs的影响如何,期待进一步探讨。正因如此,本研究选择小鼠黑色素瘤细胞系B16F10细胞中CD133+ CD44+CSCs作为研究对象,将其从B16F10细胞中分离,探讨下调ZEB1的表达对CD44+CD133+CSCs的致瘤性和肺转移的影响,从而为恶性黑色素瘤的治疗提供新的思路。目的：探讨下调黑色素瘤CSCs中ZEB1的表达对其致瘤性和肺转移的影响,为寻找黑色素瘤新的治疗靶点提供实验依据。方法：1.将构建好的靶向鼠ZEB1基因的pSUPER-EGFP1-ZEB1-shRNA干扰质粒通过脂质体转染B16F10细胞,并通过G418筛选稳定转染细胞株。2.用免疫磁选技术从稳定转染pSUPER-EGFPl-ZEB1-shRNA的B16F10克隆中分离获取ZEB1-shRNA2CD 133+CD44+CSCs以及scramble-shRNA CD133+ CD44+CSCs;从B16F10细胞中分选出CD133+CD44+CSCs。3.通过划痕实验、侵袭性实验,体内致瘤性、荷瘤鼠生存实验及肺转移实验,综合分析下调ZEB1表达后对B16F10 CD44+CD133+ CSCs的致瘤性和肺转移的影响。4.用2×104B16F10 CD44+CD133+CSCs、ZEB1-shRNA CD44+CD133+CSCs、 scramble-pSUPER-EGFP CD44+CD133+CSCs分别攻击C57BL/6小鼠,通Western Blotting实验和免疫组化实验分别检测瘤组织中EMT相关蛋白ZEB1, E-cadherin N-cadherin及Vimentin的表达。结果：1.分离出经G418筛选出稳定转染pSUPER-EGFP1-ZEB1-shRNA的B16F10细胞克隆；2.用免疫磁选技术成功分离出ZEB1-shRNA CD133+CD44+CSCs、scramble-pSUPER-EGFP CD133+CD44+CSCs以及B16F10 CD133+CD44+CSCs;划痕实验、侵袭性实验、小鼠体内致瘤性和肺转移实验结果显示：B16F10 CD44+CD133+ CSCs及scramble-pSUPER-EGFP CD44+CD133+CSCs的侵袭性和转移性以及致瘤性均高于ZEB1-shRNA CD44+CD133+CSCs,差异有显著性意义(P0.01)3. qRT-PCR法、Western Blotting及免疫组化检测瘤组织中EMT相关蛋白显示：ZEB1-shRNA CD44+CD133+CSCs的靶基因ZEB1表达减低,间质型蛋白Vimentin和N-cadherin表达下降,而上皮型蛋白E-cadherin的表达升高；肺组织黑色素瘤转移结节显著减少,与B16F10 CD44+CD133+CSCs和scramble-pSUPER-EGFP CD44+CD133+CSCs组相比,差异有显著性意义(P0.05或P0.01)。结论：下调黑色素瘤B16F10 CD44+CD133+CSCs中ZEB1的表达,可在一定程度上降低其在体外的增殖能力、克隆形成能力,侵袭力及小鼠体内致瘤性和抑制肺转移能力,提示ZEB1可作为黑素瘤CSCs治疗的新靶点。
[Abstract]:Malignant melanoma (malignant melanoma MM) is a malignant tumor derived from melanoma cells. In the past 20 years, the worldwide incidence of malignant melanoma rate is gradually increased. The skin is the main pathogenesis of malignant tumor plain black, skin tumor mortality accounted for about 70% also, originated in the eyes, nasal cavity, the high ability of invasion and metastasis is the main cause of death in patients with malignant melanoma, early metastasis to the lung, brain and other parts, with advanced systemic spread to the whole body. The epithelial mesenchymal transition (epithelialmesenchymal transition EMT) plays an important role among them, EMT is considered to be an important event in tumor invasion and metastasis promoter. The results show that malignant melanoma after EMT from primary tumor escape, invasion to the surrounding tissues, blood or lymphatic pathway after arrival The more distant organ forming metastases. Recent studies also found that malignant melanoma EMT cells more resistant phenotype, characteristics of immune suppression and stem. In the development of EMT control is the most important E- cadherin (E-cadherin). The protein is an important molecule to maintain epithelial cells the characteristics of E-cadherin, the loss is an important feature of EMT, if E-cadherin was decreased and the cells are non epithelial cells Xu characteristics, such as loss of cell polarity, cell to cell adhesion decreased into more prone to metastasis mesenchymal cells, and up-regulated expression of vimentin (Vimentin), the the expression change is an important sign of EMT. Therefore, exploring the occurrence and regulation mechanism of EMT, is of great significance to target the tumor cell metastasis. With the development of molecular biology, the researchers found that tumor In a few cells have characteristics of stem cells: proliferation, differentiation, not sensitive to chemotherapy and radiotherapy, and these cells become cancer cells, key to metastasis and recurrence, tumor stem cells (tumor stem cells, TSCs) or cancer stem cells (cancer stem cells, CSCs). Recently the research of cancer occurrence and development process showed that the zinc finger binding protein (1 zinc finger E-box-binding protein 1, ZEB1) plays an important role in the process of tumor metastasis is.ZEB1 zinc finger transcription factor E box structure in combination with zinc finger protein, it can not only regulate the occurrence and development of EMT, but also through the adjustment cell cycle, apoptosis, senescence, invasion and angiogenesis, and regulate the progress of tumors. Our previous experimental results show that the decreased expression of ZEB1 in B16F10 cells, B16F10 can be reduced to a certain extent Cell formation ability in vitro clonal proliferation, drug resistance, invasion and tumorigenicity in vivo, but the effect of CSCs in B16F10 cells to look forward to further discussion. Because of this, this study selected CD133+ B16F10 cells in CD44+CSCs mouse melanoma cell line as the research object, separating it from B16F10 cells in the study of CD44+CD133+CSCs induced down-regulation of ZEB1 expression in vitro and lung metastasis, thus provide a new way of thinking for the treatment of malignant melanoma. Objective: To investigate the effect of down-regulation of ZEB1 melanoma in CSCs on the expression of the tumorigenic effect and lung metastasis, and provide experimental basis for the search for new treatment targets melanoma. Methods: 1. constructed pSUPER-EGFP1-ZEB1-shRNA interference targeting ZEB1 gene plasmid were transfected into B16F10 cells with liposome and screened by G418 stable transfected cell line.2. with immune Magnetic separation technology to obtain ZEB1-shRNA2CD CD44+CSCs isolated from CD133+ scramble-shRNA 133+CD44+CSCs and B16F10 pSUPER-EGFPl-ZEB1-shRNA in the stably transfected clones were selected by CD133+CD44+CSCs.3.; the scratch test from the B16F10 cells, the invasive experiments in vivo tumorigenicity of tumor bearing mice survival experiment and lung metastasis experiment, comprehensive analysis of expression of ZEB1 on B16F10 CD44+CD133+ CSCs induced.4. tumor and the lung metastasis with 2 * 104B16F10 CD44+CD133+CSCs, ZEB1-shRNA CD44+CD133+CSCs, scramble-pSUPER-EGFP CD44+CD133+CSCs were challenged C57BL/6 mice, Western Blotting assay and immunohistochemical experiments were detected in tumor tissues of EMT related protein ZEB1, expression of E-cadherin N-cadherin and Vimentin. Results: 1. isolated screened with G418 B16F10 cell clones stably transfected with pSUPER-EGFP1-ZEB1-shRNA; 2. by magnetic separation technology of immune The successful isolation of ZEB1-shRNA CD133+CD44+CSCs, scramble-pSUPER-EGFP CD133+CD44+CSCs and B16F10 CD133+CD44+CSCs; scratch test, invasive experiment, mice tumorigenicity and metastasis experimental results show that B16F10 CD44+CD133+ CSCs and scramble-pSUPER-EGFP CD44+CD133 +CSCs invasion and metastasis and tumorigenicity were higher than that of ZEB1-shRNA CD44+CD133+CSCs, there were significant differences (P0.01) 3. qRT-PCR Western, Blotting and immunohistochemistry in tumor tissues of EMT associated protein showed that the target gene ZEB1 ZEB1-shRNA expression of CD44+CD133+CSCs decreased and decreased interstitial protein Vimentin and N-cadherin expression, and the expression of E-cadherin protein E-cadherin increased; lung metastasis of melanoma nodules was significantly reduced, compared with B16F10 CD44+CD133+CSCs and scramble-pSUPER-EGFP CD44+CD133+CSCs group, a significant differences (P0.05 or P 0.01). Conclusion: the expression of ZEB1 in melanoma B16F10 CD44+CD133+CSCs, to a certain extent, reduce the ability of in vitro, colony forming ability, and inhibit lung metastasis of tumor invasion and in vivo in mice, suggesting that ZEB1 can be used as a new target for treatment of CSCs melanoma.

【学位授予单位】：东南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R739.5

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