鼻咽癌外泌体来源的长链非编码RNA结肠癌相关转录本2促进血管生成的实验研究  

Long non-coding RNA colon cancer associated transcript-2 from nasopharyngeal carcinoma-derived exosomes promotes angiogenesis

作　　者：周盛恺 高峰 钟志生 姚浩 Zhou Shengkai;Gao Feng;Zhong Zhisheng;Yao Hao(Department of Otorhinolaryngology Head and Neck surgery,Affiliated Hospital of Nantong University,Institute of Otorhinolaryngology Head and Neck Surgery,Affiliated Hospital of Nantong University,Nantong 226000,Jiangsu Province,China;Department of Otorhinolaryngology,Hai′an People′s Hospital,Hai′an 226600,Jiangsu Province,China;Department of Otorhinolaryngology,Changshu Hospital Affiliated to Soochow University,Changshu No.1 People′s Hospital,Changshu 215500,Jiangsu Province,China)

机构地区：[1]南通大学附属医院耳鼻咽喉头颈外科南通大学附属医院耳鼻咽喉头颈外科研究所,226000 [2]江苏省海安市人民医院耳鼻咽喉科,226600 [3]苏州大学附属常熟医院常熟市第一人民医院耳鼻咽喉科,江苏省常熟市215500

出　　处：《中华耳鼻咽喉头颈外科杂志》2020年第10期944-951,共8页Chinese Journal of Otorhinolaryngology Head and Neck Surgery

摘　　要：目的探究鼻咽癌来源外泌体中含有的长链非编码RNA(lncRNA)结肠癌相关转录本(CCAT2)对鼻咽癌血管生成的影响。方法将人脐静脉内皮细胞(HUVEC)分为鼻咽癌细胞(CNE2)上清共培组与正常鼻咽上皮细胞(NP69)上清共培组,CCK8法检测两组HUVEC增殖能力。实时荧光定量PCR(qRT-PCR)检测CNE2上清与HUVEC共培后lncRNA CCAT2的表达。将HUVEC分为鼻咽癌患者血清共培组、健康人血清共培组,CCK8法检测两组HUVEC增殖能力,qRT-PCR检测两组HUVEC中lncRNA CCAT2的表达。超速离心法提取CNE2、NP69细胞上清外泌体。将HUVEC分为CNE2上清外泌体共培组与NP69细胞上清外泌体共培组,CCK8等血管生成实验比较两组HUVEC功能差异。在HUVEC中转染短发夹RNA(shRNA)抑制lncRNA CCAT2表达,CCK8等血管生成实验比较不同表达lncRNA CCAT2的HUVEC功能差异。通过shRNA转染CNE2,提取上清外泌体RNA,qRT-PCR检测不同表达lncRNA CCAT2的CNE2上清外泌体中lncRNA CCAT2的差异,将上述两组外泌体分别与HUVEC共培,qRT-PCR检测共培后HUVEC中lncRNA CCAT2的表达差异,同时迁移实验检测HUVEC的功能差异。用GraphPad Prism 8作图及统计分析。结果与NP69上清共培组相比,CNE2上清共培组HUVEC增殖能力较高。CNE2上清与HUVEC共培48 h与0 h相比,lncRNA CCAT2表达较高(1比1.40±0.01,t=42.43,P=0.000)。与健康人血清共培组相比,鼻咽癌患者血清共培组HUVEC增殖能力较高,48 h lncRNA CCAT2表达较高(1比1.25±0.03,t=14.43,P=0.001)。与NP69细胞上清外泌体共培组相比,CNE2上清外泌体共培组HUVEC增殖能力较高,迁移实验中穿过小室的细胞数较多(53.12±2.13比154.74±4.17,t=37.73,P=0.000),小管生成实验中结点数较多(10.72±1.02比53.65±3.21,t=22.63,P=0.000)。与sh-NC组相比,sh-CCAT2组HUVEC增殖能力较低,迁移实验中穿过小室的细胞数较少(401.34±22.15比138.25±6.85,t=23.19,P=0.000),裸鼠皮下基质胶栓塞实验中微血管计数较少(41.00±0.32比27.15±0.23,t=61.Objective To evaluate the role of exosomal long non-coding RNA(lncRNA)colon cancer associated transcript-2(CCAT2)in angiogenesis in nasopharyngeal carcinoma(NPC).Methods Human umbilical vein endothelial cells(HUVECs)were divided into CNE2 supernatant coculture group and NP69 supernatant coculture group.The proliferation ability of HUVEC in each group was examined by CCK8.The lncRNA CCAT2 level in HUVEC was detected using real-time fluorescent quantitative PCR(qRT-PCR).HUVECs were divided into coculture group of serum in patients with NPC and coculture group of serum in healthy donors.The proliferation ability of HUVEC in each group was examined by CCK8.The lncRNA CCAT2 level in each group was detected using qRT-PCR.Exosomes from CNE2,NP69 supernatant were obtained by ultracentrifugation.HUVECs were divided into coculture group of CNE2′s supernatant exosomes and coculture group of NP69′s supernatant exosomes.Biological experiments like CCK8 were conducted to compare the angiogenesis ability of HUVEC in each group.shRNA transfection was used in HUVEC to suppress the expression of lncRNA CCAT2.Biological experiments like CCK8were conducted to compare the HUVEC′s function with different expression of lncRNA CCAT2.CNE2 was transfected with shRNA,the total supernatant exosomal RNA was extracted,and the difference of exosomal lncRNA CCAT2 in CNE2 supernatant with low expression and normal expression of lncRNA CCAT2 was detected by qRT-PCR.The above mentioned two groups of exosomes were cocultured with HUVEC respectively.The difference of lncRNA CCAT2 expression in each group′s HUVEC was detected by qRT-PCR and the function of HUVEC was examined with migration assay.GraphPad Prism 8 software was used for graphing and statistical analysis.Results Compared with those in the NP69 supernatant coculture group,HUVECs in the CNE2 supernatant coculture group showed a higher proliferation ability and higher expression of lncRNA CCAT2(1 vs.1.40±0.01,t=42.23,P=0.000).Compared with those in serum of healthy donors,HUVECs in

关 键 词：鼻咽肿瘤 外泌体 血管生成 长链非编码RNACCAT2 

分 类 号：R739.63[医药卫生—肿瘤]