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shRNA干扰KAP-1基因表达对胰腺癌肿瘤干细胞自我更新的影响

ISSN号：1007-8118
期刊名称：《中华肝胆外科杂志》
时间：0
分类：R735.9[医药卫生—肿瘤;医药卫生—临床医学]
作者机构：[1]贵阳医学院附属医院肝胆外科,550004
相关基金：国家自然科学基金资助项目（81160311）;贵州省科教青年英才培养工程基金资助项目[黔省专合字（2012）177号];贵州省科技厅2011年度社会攻关计划基金资助项目[黔科合SY字（2011）3007];贵州省肝胰疾病研究科技创新人才团队基金资助项目[黔科合人才团队（2010）4010]

关键词：胰腺肿瘤, 干细胞, 基因表达, Pancreatic neoplasm, Stem cells, The mechanism is probably related to the up- Gene expression

中文摘要：

目的研究KAP-1对胰腺癌肿瘤干细胞自我更新能力的影响。方法采用免疫组织化学方法检测14例胰腺癌组织标本和配对癌旁组织标本KAP1蛋白的表达情况。PCR扩增KAP1shRNA干扰序列，克隆至pGC—LV慢病毒表达载体；双酶切及测序鉴定正确后进行慢病毒包装与滴度检测。构建成功后感染人胰腺癌细胞株Panc-1细胞48h后，通过Westernblot实验和RT—qPCR实验检测KAP-1的表达量以及该细胞系相关EMT标志蛋白波形蛋白的表达。采用悬浮法培养人胰腺癌Panc-1细胞生成肿瘤干细胞球，通过测定初代以及次代肿瘤干细胞球的成球直径和数量判断KAP1基因敲减对肿瘤干细胞球成球能力及自我更新能力的影响。结果在14例胰腺癌组织标本和癌旁组织标本的免疫组织化学检测中，胰腺癌组织标本KAP1阳性数为13例（P=0．002）。构建的慢病毒载体感染细胞48h后，在倒置荧光显微镜下观察可见绿色荧光。RTqPCR实验显示感染KAP-1RNA干扰慢病毒载体的Panc-1细胞KAP-1及EMT标志蛋白波形蛋白的表达量较未感染病毒细胞以及感染空载体细胞显著增高。KAP1基因敲低表达的人胰腺癌Panc-1肿瘤干细胞球的初代以及次代的成球直径相较于转染空载体的Panc1肿瘤干细胞球发生明显减小，在成球数量上也明显减少。结论KAP-1在胰腺癌组织中存在表达，且与正常组织表达量具有明显差异。构建了高效稳定表达KAP-1的RNA干扰慢病毒转染系统。KAP1转录因子表达量降低可以减弱人类胰腺癌细胞Panc-1细胞系生成干细胞球的自我更新能力。其机制可能与敲低KAP-1表达影响波形蛋白下调有关。

英文摘要：

Objective To investigate whether KAP-1 could induce CSC-self renewal in pancreat- ic cancer cell lines. Methods KAP-1 expression was examined in 14 cases of pancreatic cancer with immunohistochemistry. KAP-1 shRNA amplified by PCR was inserted into pGC-LV in vector, and then identified by restriction endonuclease digestion and nucleotide sequencing. The lentiviral vector pGC-shRNA-KAP 1 was co transfected with pHelper 1.0 and pHelper 2.0 packaging plasmids into HEK 293T cells, and the lentivirus was collected and virus titer was measured. The expressions of KAP-1 and vimentin were detected by Western blot and RT-qPCR when human pancreatic cancer cell line Panc I was infected .by the lentivirus. Sphere forming assay was conducted to assess the capacity of CSC or CSC-like cell selPrenewal in this study. Results The KAP-1 expression level in cancerous tissues on immunohistochemistry was significantly higher than in the corresponding normal tissues （P=0. 002）. After infected by lentivirus, the expressions of KAP-1 and vimentin were knocked down, which could be detected by Wesren blot and RT-qPCR. Compared to Pane-1 GFP （NC） ceils,the outcomes suggested that knocking down the expression of KAP-1 could decrease the formation of pancreatospheres, which further suggested the capacity of CSC-self-renewal in primary and secondary pancreatospheres of Panml shRNA KAP-1 and NC cells. Conclusions KAP-1 expression in pancreat- ic cancer tissues has been identified. The lentiviral vector for shRNAs targeting KAP 1 was construc- ted successfully. The formation of pancreatospheres decreased by knocking down the KAP-1 gene. KAP-1 is involved in the regulation of CSC phenotype regulated expression of the EMT marker vimentin.