摘要
目的探讨血清外泌体miR-454在非小细胞肺癌(NSCLC)中的作用机制。方法纳入我院收治的NSCLC患者40例(NSCLC组),同时选取我院体检中心与之匹配的健康受试者40例(健康对照组),使用实时定量聚合酶链反应(RT-qPCR)和Western blot检测NSCLC组患者和健康对照组血清外泌体中miR-454、PDK1 mRNA和蛋白的表达。RT-qPCR检测人支气管上皮样细胞(HBE)和A549细胞中miR-454、PDK1 mRNA和蛋白的表达。提取NSCLC患者血清外泌体作为外泌体组,将去除外泌体的血清作为血清组,通过透射电镜鉴定外泌体的形态;Western blot检测外泌体表面标志物HSP70、CD63、CD81蛋白的表达。生物信息学网站预测PDK1与miR-454的靶向关系,并通过双荧光素酶报告基因分析进行验证。将NSCLC细胞A549分为血清外泌体组(于含血清来源的外泌体培养基中培养)和对照组(于去除外泌体的血清中培养),探讨血清外泌体对A549细胞增殖、侵袭、迁移及凋亡的影响。将NC mimic、miR-454 mimic、NC inhibitor、miR-454 inhibitor、miR-454 mimic+Vector、miR-454 mimic+pc DNA-PDK1转染到外泌体中,分别设为NC mimic组、miR-454 mimic组、NC inhibitor组、miR-454 inhibitor组、miR-454+Vector组、miR-454+pc DNAPDK1组,探讨外泌体miR-454对A549细胞增殖、侵袭、迁移、凋亡的影响,及外泌体miR-454通过靶向PDK1对NSCLC细胞增殖、侵袭、迁移、凋亡的影响。结果透射电镜结果显示,外泌体形态为典型的脂质双层膜包裹的圆形颗粒。与血清组比较,外泌体组中表面标志物HSP70、CD63和CD81蛋白的表达显著增加(P<0.05)。与健康对照组比较,NSCLC组患者血清外泌体中miR-454的表达显著降低,PDK1 mRNA和蛋白表达水平显著升高,差异均有统计学意义(P<0.05)。在A549细胞中,与对照组比较,NSCLC血清来源的外泌体显著促进细胞增殖、迁移和侵袭,抑制细胞凋亡,差异均有统计学意义(P<0.05)。与HBE细胞相比,A549细胞中miR-454的表达�
Objective To investigate the mechanism of serum exosome miR-454 in non-small cell lung cancer(NSCLC).Methods A total of 40 NSCLC patients admitted to our hospital(the NSCLC group)were included,and 40 matched healthy subjects(the healthy control group)from the physical examination center of our hospital were selected.The expression of miR-454,PDK1 mRNA and protein in serum exosomes of patients in the NSCLC group and the healthy control group were detected by real-time quantitative polymerase chain reaction(RT-qPCR)and Western blot.The expression of miR-454,PDK1 mRNA and protein in human bronchial epithelial cells(HBE)and A549cells were detected by RT-qPCR.The serum exosomes of NSCLC patients were extracted as the exosome group,and the serum removed exosomes was used as the serum group,and the morphology of the exosomes was identified by transmission electron microscopy.The expression of exosome surface markers HSP70,CD63 and CD81 proteins were detected by Western blot.The targeting relationship between PDK1 and miR-454 was predicted by bioinformatics website and verified by dual-luciferase reporter gene analysis.NSCLC cells A549 were divided into the serum exosome group(cultured in serum-derived exosome medium)and the control group(cultured in serum without exosome)to investigate the effects of serum exosomes on the proliferation,invasion,migration and apoptosis of A549 cells.NC mimic,miR-454 mimic,NC inhibitor,miR-454 inhibitor,miR-454 mimic+Vector,and miR-454 mimic+pc DNA-PDK1 were transfected into exosomes and set as the NC mimic group,the miR-454 mimic group,the NC inhibitor group,the miR-454 inhibitor group,the miR-454+Vector group,and the miR-454+pc DNA-PDK1 group,respectively.The effects of exosome miR-454 on the proliferation,invasion,migration,and apoptosis of A549 cells,and the effects of exosome miR-454 on the proliferation,invasion,migration,and apoptosis of NSCLC cells by targeting PDK1 were explored.Results The results of transmission electron microscope showed that the morphology of exosomes was typ
作者
董晓平
刘红岗
张勇
DONG Xiao-ping;LIU Hong-gang;ZHANG Yong(Department of Thoracic Surgery,Second Affiliated Hospital of Air Force Medical University,Xi’an Shaanxi 710038,China)
出处
《局解手术学杂志》
2022年第10期849-856,共8页
Journal of Regional Anatomy and Operative Surgery
基金
陕西省重点研发计划项目(2019SF-273)。
