KAP-1对胰腺癌细胞Capan-2上皮细胞间质转化的促进作用  

Effects of KAP-1 in promoting the epithelial-mesenchymal transition of pancreatic cancer cell line Capan-2

作　　者：孙诚谊[1] 江建新[1] 潘耀振[1] 詹磊[1] 

机构地区：[1]贵阳医学院附属医院肝胆外科,550001

出　　处：《中华消化外科杂志》2013年第8期586-591,共6页Chinese Journal of Digestive Surgery

基　　金：国家自然科学基金（81160311）;贵州省科教青年英才培养工程基金（黔省专合字[2012]177）;贵州省科技厅2011年度社会攻关计划基金（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（黔科合人才团队[2010]4010）

摘　　要：目的研究KAP-1对胰腺癌细胞Capan-2上皮细胞间质转化（EMT）的调节作用。方法构建LV-plenti-GFP。KAP-1慢病毒载体。将Capan-2细胞分为实验组（转染LV-plenti-GFP-KAP-1慢病毒载体）、阴性对照组（转染空载体）以及空白对照组1（含10％小牛血清的1640培养基）；而后再将实验组Capan-2细胞分为抑制剂组（转染化学合成的miR-100-5p抑制剂）、空载体对照组（转染无关序列micro-RNAs抑制剂）、空白对照组2（含10％小牛血清的1640培养基）。用双酶切及测序鉴定慢病毒并计算病毒滴度。LV-plenti-GFP-KAP-1慢病毒载体稳定转染至Capan-2细胞后，观察细胞的形态学改变。应用实时定量PER检测各组细胞中KAP-1、EMT标志蛋白以及miR-100-5pmRNA的表达情况。应用Westernblot法检测各组细胞中KAP-1、EMT标志蛋白表达水平。计量资料采用x-±s表示，组间比较均采用方差分析。结果成功构建LV-plenti-GFP-KAP-1慢病毒载体，病毒滴度为2×10^8 TU／ml。慢病毒载体转染Capan-2细胞48h后，实验组细胞与对照组比较其形态有明显间质样改变。实验组、阴性对照组、空白对照组1细胞中各目标蛋白mRNA的相对表达量：KAP-1分别为1．77±0．83、5．03±0．29、5．13±1．14，N-钙黏蛋白分别为2．62±0．71、5．07±1．53、5．81±1．49，波形蛋白分别为2．50±0．21、3．83±0．57、4．92±0．90，E-钙黏蛋白分别为7．20±1．17、7．83±0．78、3．07±0．36，miR-100-5p分别为1．81±0．40、7．01±0．96、6．87±0．35，3组比较，差异有统计学意义（F=5．99，7．62，7．88，6．62，4．64，P〈0．05）。抑制剂组、空载体对照组、空白对照组2细胞中KAP-1mRNA的相对表达量分别为1．56±0．42、4．89±0．61、5．20±0．38，3组比较，差异有统计学意义（F=5．14，P〈0．05）；波形蛋白mRNA的相对表达量分别为3．10±1．37、3．44±0．94、3．08±1．16，3组比较，差异无Objective To investigate the effects of KAP-1 in promoting the epithelial-mesenchymal transi- tion （EMT） of pancreatic cancer cell line Capan-2. Methods The lentiviral vector of LV-plenti-GFP-KAP-1 was constructed. Capan-2 cells were divided into the experimental group （cells transfected by lentiviral vector of LV- plenti-GFP-KAP-1 ） , negative control group （ cells transfected by empty vector） and blank control group 1 （ cells cultured in 1640 medium plus 10% fetal calf serum）. Capan-2 cells in the experimental group were subdividedinto the miR-100-5p inhibitor transfection group （cells transfected with miR-100-5p inhibitor）, empty vector control group （ ceils transfected with microRNAs inhibitor）, blank control group 2 （ cells cultured in 1640 medium plus 10% fetal calf serum）. The lcntivirus was identified by double endonuclease restriction and sequencing ,and the virus titer was detected. The morphological changes of the cells were observed after transfecting lentiviral vector of LV-plenti-GFP-KAP-1 to the Capan-2 cells. The expressions of KAP-1, genes of EMT proteins and mRNA of miR-100-5p were detected by real-time quantitative polymerase chain reaction. The protein expressions of KAP-1, EMT proteins in all the groups were detected by Western blot. The measurement data were presented by mean ± standard deviation, and were analyzed using the analysis of variance. Results The lentiviral vector of LV-plenti- GFP-KAP-1 was successfully constructed, and the virus titer was 2 ×10^8 TU/ml. Compared with the control group, the mesenchymal transition of the Capan-2 cells was detected in the experimental group after transfecting the Capan-2 cells with lentiviral vector of LV-plenti-GFP-KAP-1 for 48 hours. The relative mRNA expressions of KAP-1, N-cadherin, vimentin, E-cadherin, miR-100-5p were 1.77 ±0.83, 2.62 ±0.71, 2.50 ±0.21, 7.20 ± 1.17 and 1.81 ±0.40 in the experimental group, 5.03 ±0.29, 5.07 ± 1.53, 3.83 ±0.57, 7.83 ±0.78, 7.01 ± 0.96 in the negative control group, 5.13

关 键 词：胰腺肿瘤 KAP-1 上皮细胞间质转化 微小RNA 

分 类 号：R735.9[医药卫生—肿瘤]