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HIF-1调控MAN2A1参与胰腺癌转移分子机制及临床意义
中文摘要

背景 胰腺导管腺癌(pancreatic ductal adenocarcinoma,简称PDAC)近些年来,无论是在我国还是西方发达国家,发病率呈现出逐年的上升趋势。众所周知, PDAC的恶性程度很高、侵袭性很强,其患者5年期生存率不到6%。尽管在医学专家及科研人员的不懈努力下,在诸多领域内出现了一些进步,比如外科手术、化疗、靶向药物和放疗的领域,但目前看来PDAC患者的预后仍然非常不容乐观。截止目前外科手术切除仍是当前PDAC治疗中最有效的治疗方法。因此,如何提高对PDAC侵袭、转移机理的认识,如何对PDAC患者的预后精确分层,今后很长一段时间来讲,对于PDAC治疗效果的提高,精准识别,都颇有意义。 众所周知,组织低氧(Hypoxia)是PDAC生长的重要微环境之一。HIF-1 α (Hypoxia-inducible factor-1α)是在组织处于低氧状态时因适应性而产生的一类转录因子,能与具有特异性的低氧反应元件(hypoxia-responsive element, HRE)(5,-RCGTG-3,)特异性结合;而且低氧反应元件HRE区在与HIF1α相互作用中一直处于保守状态结合。国内外有众多课题组的大量研究显示:HIF-1 α在PDAC组织中呈现出高表达状态,而且介导了大量的适应性病理反应与生理反应,其与PDAC的纤维化过程(Fibrosis)、血管生成过程(Angiogenesis)、细胞侵袭过程(Invation)等肿瘤的进展危险因素都密切相关。HIF-1α在参与细胞低氧调控的转录因子中,其作用是非常强的,而且也是比较核心的,因而被国内外科研人员广泛、深入的研究。有诸多的科研机构研究分析发现,HIF-1α在PDAC组织中呈高表达状态,而在正常胰腺实质组织(Parenchymal tissue)中却很难发现HIF-1α的表达,从而研究人员不难推测出HIF-1α很有可能在PDAC恶性表型的进展中发挥了作用。而且也有研究人员发现在PDAC发生侵袭转移(Metastasis)的过程中,HIF-1α可能诱导了多种侵袭转移相关因子和不同酶类的恶性表达,从而使肿瘤细胞的表型发生了巨大转变,最终发生了PDAC的局部侵袭和远处转移。 MAN2A1(mannosidase,alpha,class 2A,member1,简称MAN2A1),属于Ⅱ类α-甘露糖苷酶,有研究指出其在恶性肿瘤细胞的预后不良中发挥了巨大作用。如何进一步认识理解该因子,如何明确其在PDAC中发挥的恶性作用,对于进一步认识MAN2A1及PDAC具有重要意义,或在未来有可能成为肿瘤治疗新靶点。在我们课题组的前期工作中已经揭示,MAN2A1蛋白在PDAC中,相较于正常胰腺组织及细胞系来说,其表达水平更高而且与PDAC的不良预后密切相关。故而课题组设计了本实验,拟从临床、细胞分子等诸多水平探索PDAC中MAN2A1的功能、意义、调控机制,从而加深对MAN2A1及PDAC的认识,提高对PDAC的诊治水平。 方法 1.应用PDAC组织石蜡切片标本,进行HIF-1α、MAN2A1地连续切片免疫组化染色(IHC),分析MAN2A1的不同表达程度和HIF-1α表达的相关性;进一步搜集临床病例资料及预后随访资料,分析MAN2A1表达水平与临床病理学参数之间的关系。 2.应用Western blot等实验技术检验不同处理(乏氧、常氧条件下及不同处理HIF-1α条件下)PDAC细胞系中MAN2A1的表达水平;应用免疫荧光技术(IF)检验MAN2A1在PDAC细胞中的细胞表达及定位。 3.应用瞬转小RNA干扰技术,以及分子克隆的方法,构建HIF-1α及MAN2A1过表达质粒和HIF-1α、MAN2A1 shRNA降表达质粒,以构建不同分子表达的细胞系;用Western blot等检验方法,鉴定HIF-1α、MAN2A1等不同表达的细胞系;应用TRANSWELL迁移、侵袭实验及划痕实验检验所构建细胞表达MAN2A1的不同生物学功能。 4.应用Western blot、CHIP及免疫荧光实验检验HIF-1α与MAN2A1启动子HRE区结合情况,及分析HIF-1α调控MAN2A1基因转录激活的能力,及调控靶标的分子机制。 5.尝试构建裸鼠胰腺原位肿瘤细胞转移模型;体内验证MAN2A1促PDAC转移的作用。 结果 1.在人PDAC切片组织中、新鲜肿瘤组织中及PDAC细胞系中,MAN2A1呈现胞浆高表达,并连续切片组织IHC分析与HIF-1α的表达具有相关性(rs=0.359)。MAN2A1的表达与PDAC的淋巴结(LN)转移相关,并与预后呈负相关。 2.低氧处理人PDAC细胞系,可以诱导MAN2A1在mRNA和蛋白水平的表达上调;通过RNAi干扰技术沉默HIF-1α的表达,可以抑制MAN2A1的表达。CHIP及双荧光素酶实验显示,HIF-1α可以结合于MAN2A1基因启动子HRE区,并可以上调MAN2A1启动子区的活性,激活转录活性。 3.通过RNAi干扰技术沉默MAN2A1的表达可以引起人PDAC迁移侵袭能力下降;而过表达MAN2A1,可以使其能力提升。通过Wetsern blot分析,MAN2A1可以上调Vimentin的表达,减少E-cadherin的表达,引起WNT/SNAIL信号通路的激活,通过上皮间质转化(EMT),引起PDAC迁移侵袭能力的加强。 4.裸鼠胰腺癌原位成瘤转移模型构建失败;未能完成体内动物实验。 结论 1.MAN2A1蛋白在人PDAC切片组织、新鲜组织及细胞系中表达量异常升高;PDAC组织中,MAN2A1表达水平较高的患者预后更差。 2.在PDAC细胞系中,MAN2A1降表达后,抑制了WNT/SNAIL信号通路,波形蛋白(Vimentin)的表达下调,E-钙粘附蛋白(E-cadherin)的表达上调,逆转了上皮间质转化(EMT),从而抑制肿瘤的转移能力。 关键词:胰腺癌;MAN2A1;肿瘤转移;上皮间质转化

英文摘要

Background In recent years, the incidence rate of pancreatic cancer is on the rise, with the high degree of malignancy, and the 5-year survival rate is <5%. Although the current surgery, radiotherapy and chemotherapy and neoadjuvant treatment has made great progress, but the prognosis of pancreatic cancer has not been significantly improved. Surgical resection is the most effective treatment of pancreatic cancer, therefore, pancreatic cancer invasion and metastasis mechanism of in-depth study for improving the clinical treatment of pancreatic cancer has important guiding significance. It is well known that hypoxia is not only an important microenvironment of pancreatic cancer tissue growth. HIF-1α (Hypoxia-inducible factor-1α) is a kind of transcription factor induced by hypoxia. It belongs to bHLH-PAS superfamily and can specifically bind to hypoxia-responsive element (HRE ) (5,-RCGTG-3). HRE is conserved in the promoter of HIF-acting target genes. Studies have shown that HIF-1α is highly expressed in pancreatic cancer tissues and mediates many adaptive pathophysiological responses,which are closely related to tumor progression factors such as fibrosis, angiogenesis and cell invasion of pancreatic cancer. HIF-1α is deeply studied in many of the transcription factors involved in hypoxia regulation of cells because of its strongest effect. A large sample of clinical pathology data found that a higher proportion of pancreatic ductal adenocarcinoma high expression of HIF-1α, and in normal pancreatic tissue is difficult to detect the expression of HIF-1α, so we conclude that HIF-1α may be an important transcription factor for malignant phenotypes. In the process of invasion and metastasis of pancreatic cancer cells, HIF-1α can induce a variety of related factors and enzyme expression, promote tumor cell phenotype transition and then break through the cell matrix of the ultimate transfer of the restraint. MAN2A1 (mannosidase, alpha, class 2A, member 1,referred to as MAN2A1), belongs to class Ⅱ α-mannosidase. Studies have pointed out that N-glycosylation abnormalities of tumor surface in malignant tumor cells play an important role in poor prognosis. It maybe become a new target for the treatment of these diseases. In our previous study, our team revealed that compared with normal tissues and PDAC cell lines, the expression of MAN2A1 protein was highly expressed in pancreatic cancer tissues and cells. We has designed a series of experiments to furtherly clarify the expression level of MAN2A1 in tissues and cell lines of pancreatic cancer. The function, significance and regulation mechanism of MAN2A1 in pancreatic cancer were explored from different levels, including clinical level, cell level and so on. Method 1.Immunohistochemical (IHC) staining of HIF-1α and MAN2A1 was performed. The expression of MAN2A1 and the correlation with HIF-1α were analyzed. The expression of MAN2A1 was correlated with clinical pathology Parameters. 2.The expression level of MAN2A1 in pancreatic cancer cell lines under different treatment (under hypoxia,normoxic and different treatment conditions) was detected by Western blot. The expression of cell localization of MAN2A1 in pancreatic cancer cells was detected. 3.The overexpression plasmids of HIF-1α, MAN2A1 and shRNA plasmids of HIF-1α and MAN2A1 were constructed by molecular cloning method. The stable transfected cells were constructed and the stable transfected cells were established. The expression of HIF-1α and MAN2A1 was detected by Western blot. The biological function of MAN2A1 was detected by scratch test, TRANSWELL migration assay. 4.The combination of HIF-1α and MAN2A1 promoter region was detected by Western blot, CHIP and immunofluorescence assay, and the ability of HIF-1α to regulate the transcriptional activation of MAN2A1 gene and the molecular mechanism of the target. 5.To construct the model of orthotopic tumor cell metastasis in nude mice; to verify the effect of MAN2A1 on PDAC transfer in vivo. The results 1.The expression of MAN2A1 in fresh tumor tissue and PDAC cell line was correlated with the expression of HIF-1α (rs=0.359) in human PDAC slices. The expression of MAN2A1 was associated with PDAC lymph node metastasis and was negatively correlated with prognosis 2.Hypoxia-treated human pancreatic cancer cell lines can induce the up-regulation of mRNA and protein levels in MAN2A1, and silencing the expression of HIF-1α by RNA interference can inhibit the expression of MAN2A1. Further studies have shown that HIF-1α can bind to MAN2A1 gene (HRE region) of the promoter region, and can directly upregulate the activity of the MAN2A1 promoter region, causing transcriptional activation. 3.The expression of MAN2A1 was silenced by RNA interference technique. The expression of MAN2A1 was up-regulated by MAN2A1. The function of migration and invasion of PDAC cancer cells changed significantly. By Wetsern blot analysis, MAN2A1 down-regulates the expression of Vimentin, activates the expression of E-cadherin, and reduces the activation of WNT/SNAIL signaling pathway. It was confirmed by animal model that the pancreatic cancer cell line overexpressing MAN2A1 had a strong ability to metastasize. 4.In situ tumor metastasis model of PDAC in nude mice failed to complete the in vivo animal experiment. Conclusions 1.The MAN2A1 protein was overexpressed in human pancreatic cancer tissues and cell lines. The prognosis of patients with high expression of MAN2A1 in pancreatic cancer tissues was poor. 2.In PDAC cell lines, down-expression of MAN2A1 will inhibit the WNT/SNAIL signaling pathway, and down regulate vimentin expression, increase E-cadherin expression, reversal of EMT, thereby inhibiting tumor metastasis Keywords: Pancreatic Cancer; PDAC; MAN2A1; metastasis; EMT

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