摘要
目的分析环状RNA 0087432(hsa_circ_0087432)在宫颈癌患者血清外泌体中的表达,探讨其与宫颈癌转移可能存在的关系。方法收集广州市番禺区中心医院2019年6月-2020年6月诊断为宫颈鳞癌且术前未行放化疗的30例患者的宫颈癌组织及血清作为实验组,另将30例未患宫颈癌的女性正常宫颈组织及血清作为对照组。采用实时定量聚合酶链反应(qPCR)检测hsa_circ_0087432在宫颈癌患者与健康人宫颈组织和血清外泌体中的表达水平。通过质粒转染Siha细胞以过表达hsa_circ_0087432,将Siha细胞依不同处理分为3组:control组(未转染质粒),vector组(转染pLC5-ciR)及hsa_circ_0087432组(转染hsa_circ_0087432-pLC5-ciR),并提取各组外泌体;采用透射电镜(TEM)、纳米颗粒跟踪分析技术(NTA)及Western blotting检测外泌体表征。将人脐静脉内皮细胞按不同处理分为3组:control-Exs组(Siha细胞来源外泌体)、vector-Exs组(vector组Siha细胞来源外泌体)、hsa_circ_0087432-Exs组(hsa_circ_0087432组Siha细胞来源外泌体),采用CCK-8试剂盒检测外泌体对人脐静脉内皮细胞增殖的影响,采用划痕实验及Transwell检测外泌体对人脐静脉内皮细胞迁移的影响。结果电镜下观察结果显示,Siha细胞的外泌体大部分呈椭圆形或圆形,粒径分析显示其直径大小分布在30~150 nm;Western blotting检测结果显示,两组囊泡中的CD9、CD81及CD63表达均呈阳性;qPCR结果显示,实验组宫颈组织hsa_circ_0087432表达量明显高于对照组(4.03±1.51 vs.1.00±0.26,P<0.01),且血清外泌体中hsa_circ_0087432表达量也明显高于对照组(1.97±0.04 vs.1.02±0.23,P<0.01)。CCK-8检测结果显示,hsa_circ_0087432-Exs可明显促进人脐静脉内皮细胞增殖(1.57±0.04 vs.1.09±0.11,P<0.05)。划痕实验结果显示,干预12 h后,circ_0087432-Exs组的迁移面积占比高于control-Exs组(24.66%±2.92%vs.15.01%±3.12%,P<0.05),且24 h后hsa_circ_0087432-Exs组的迁移面积占比明显高于control-Ex
Objective To analyze the expression of circRNA0087432(hsa_circ_0087432)in serum exosomes of patients with cervical cancer,and explore its possible relationship with cervical cancer metastasis.Methods The cervical cancer tissue and serum of 30 patients,who were diagnosed as cervical squamous cell carcinoma from June 2019 to June 2020 in Guangzhou Panyu District Central Hospital and untreated with radiotherapy and chemotherapy before surgery,were collected as the experimental group.Normal cervical tissue and serum of 30 women without cervical cancer were used as control group.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression level of hsa_circ_0087432 in cervical tissues and serum exosomes of patients with cervical cancer and healthy people;Siha cells were transfected with plasmid to overexpress hsa_circ_0087432.According to different treatments,Siha cells were divided into three groups:control group(untransfected plasmid),vector group(transfected with pLC5-ciR)and hsa_circ_0087432 group(transfected with hsa_circ_0087432-pLC5-ciR).The exosomes were collected and extracted,and the exosyndrome were characterized by transmission electron microscope(TEM),nanoparticle tracking analysis(NTA)and Western blotting;Endothelial cells were divided into three groups according to different treatments:control-Exs group(extracted exosomes from control group),vector-Exs group(extracted exosomes from vector group)and hsa_circ_0087432-Exs group(extracted exosomes from hsa_circ_0087432 group).CCK-8 kit was used to detect the effect of exosomes on proliferation of endothelial cells.The effect of exosomes on the migration of endothelial cells were detected by scratch test and Transwell.Results The results of TEM showed that most of the exosomes of Siha cells were elliptical or round.The particle size analysis showed that their diameters ranged of 30-150 nm.Western blotting showed that the expressions of CD9,CD81 and CD63 in the two groups of vesicles were positive.The qPCR results showed that the
作者
夏红
杨翔
陈烨
李虎
褚桂芬
Xia Hong;Yang Xiang;Chen Ye;Li Hu;Chu Gui-Fen(Department of Gynecology,Guangzhou Panyu District Central Hospital,Guangzhou 511040,China)
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2021年第4期327-332,共6页
Medical Journal of Chinese People's Liberation Army
基金
广州市科技计划项目(201904010363)。
关键词
环状RNA
外泌体
宫颈癌
增殖
迁移
circRNA
exosome
cervical cancer
proliferation
migration
