摘要
目的探讨转移性宫颈癌组织的旁分泌作用对肿瘤细胞扩散的影响。方法选择22例转移性宫颈鳞癌患者的手术标本(癌组织和癌旁组织)和血清标本,以及22名健康体检者的血清标本,采用逆转录-实时荧光定量PCR(RT-qPCR)检测miR-21-5p在癌组织和癌旁组织中的表达水平;分别转染miR-21-5p模拟物/抑制剂到宫颈癌SiHa细胞,采用RT-qPCR、细胞集落形成实验、Transwell迁移实验、噻唑蓝(MTT)比色法、蛋白免疫印迹法、双荧光素酶报告分析差异;采用高速离心法提取血清外泌体;采用纳米颗粒跟踪分析(NTA)技术、蛋白免疫印迹法、透射电子显微镜(TEM)对外泌体进行表征鉴定;采用RT-qPCR检测宫颈鳞癌患者和健康体检者血清外泌体中miR-21-5p的表达差异;采用受试者操作特征(ROC)曲线分析血清外泌体中miR-21-5p对肿瘤转移的诊断效能。结果miR-21-5p在转移性宫颈鳞癌组织中表达上调;miR-21-5p过表达能促进宫颈鳞癌SiHa细胞集落形成以及细胞增殖与迁移(P均<0.01);双荧光素酶报告显示,miR-21-5p可以靶向快速发育生长因子同源蛋白2抗体(SPRY2),对SPYR2的表达水平进行调控;miR-21-5p模拟物转染的SiHa细胞中,磷酸化丝裂原和应激激活的蛋白激酶、磷酸化核糖体S6蛋白激酶、磷酸化原癌基因C-RAF、磷酸化丝裂原活化蛋白激酶(MAPK)激酶和磷酸化MAPK蛋白的表达均上调(P均<0.05)。miR-21-5p在宫颈鳞癌患者的血清外泌体中上调(P<0.05)。血清外泌体评估宫颈癌转移的ROC曲线下面积为0.9375。结论转移性宫颈鳞癌患者组织中miR-21-5p通过外泌体传递促进肿瘤转移,其机制可能与靶向SPRY2/细胞外调节蛋白激酶/MAPK信号通路有关。
Objective To explore the influence of paracrine function of recurring and metastatic cervical cancer tissues on the spread of cancer cells.Methods Surgical specimens(cancerous and para-cancerous tissues)were obtained from 22 patients with metastatic cervical squamous cancer.Serum specimens were collected from 22 patients with metastatic cervical squamous cancer and 22 healthy volunteers,respectively.The expression levels of miR-21-5 p in the cancerous and para-cancerous tissues were determined by quantitative real-time PCR(RT-qPCR).SiHa cell were transferred with miR-21-5 p mimics/inhibitors,respectively.The differences between two groups were analyzed by RT-qPCR,colony formation test,Transwell migration test,MTT,western blotting and dual luciferase reporter assay.Serum exosomes were extracted by high-speed centrifugation.The exosome characterization was performed by nanoparticle tracking analysis(NTA),western blotting and transmission electron microscopy(TEM).The differences in the expression levels of miR-21-5 p in serum exosomes between cervical squamous cancer patients and healthy volunteers were assessed by RT-qPCR.The diagnostic efficiency of miR-21-5 p in serum exosomes was evaluated by the receiver operating characteristic(ROC)curve.Results The expression level of miR-21-5 p was up-regulated in cervical cancerous tissues compared with para-cancerous tissues.And over-expressed miR-21-5 p could significantly promote SiHa cell colony formation,cell proliferation and migration(all P<0.01).Dual luciferase reporter assay showed that miR-21-5 p could target SPYR2 and regulate the expression level of SPYR2.In SiHa cells transfected with miR-21-5 p mimics,the expression levels of p-MSK,p-90 RSK,p-C-RAF,p-MEK and p-MAPK proteins were significantly up-regulated(all P<0.05).The expression level of miR-21-5 p was significantly up-regulated in serum exosomes of patients with cervical squamous cancer(P<0.05).The area under the ROC curve(AUC)of serum exosomes for the assessment of cervical cancer metastasis was 0.9375.Co
作者
杨翔
陈烨
郑玮
李虎
夏红
Yang Xiang;Chen Ye;Zheng Wei;Li Hu;Xia Hong(Department of Gynecology,Guangzhou Panyu Central Hospital,Guangzhou 511040,China;不详)
出处
《新医学》
CAS
2022年第12期891-898,共8页
Journal of New Medicine
基金
广州市科技计划项目(201904010363)
广州市番禺区科技计划项目(2019-Z04-87)。
