摘要
目的探讨神经纤毛蛋白1(NRP-1)和血管内皮生长因子(VEGF)在多形性胶质母细胞瘤(GBM)复发和耐药性中的机制。方法体外培养人源性GBM1A和GBM22细胞株,通过感染慢病毒shRNA的方式沉默VEGF或NRP-1。根据感染慢病毒shRNA的种类,将两种细胞株分别分为shCont组(感染无义序列shRNA)、shNRP-1组(感染NRP-1慢病毒shRNA)及shVEGF组(感染VEGF慢病毒shRNA)。采用实时荧光定量聚合酶联链式反应(qRT-PCR)和蛋白质免疫印迹法检测肿瘤细胞株在沉默NRP-1或VEGF基因后干细胞标志物[包括:性别决定区Y盒2(Sox2)、表皮生长因子受体(EGFR)、N-钙黏附素(N-cadherin)、CD133、细胞间质上皮转换因子(c-Met)]的基因和蛋白表达情况。采用细胞划痕实验、Transwell小室测定法、神经球形成实验及MTT法检测肿瘤细胞的迁移能力、侵袭能力、神经球形成能力及细胞代谢活性。采用细胞生长抑制实验检测肿瘤细胞株在沉默VEGF或NRP-1后对替莫唑胺(TMZ)、紫杉醇(PTX)和卡博替尼(XL-184)的敏感性。向GBM1A和GBM22细胞株中的shCont组和shNRP-1组分别加入10μg重组VEGF,分为shCont组、shCont+VEGF组、shNRP-1组、shNRP-1+VEGF组,判断NRP-1与VEGF之间的相互作用。结果GBM1A细胞株中,shVEGF组和shNRP-1组Sox2、EGFR、N-cadherin,CD133及c-Met的mRNA含量均较shCont组下降(均P<0.05)。GBM1A细胞株中,shNRP-1组和shVEGF组EGFR、CD133、N-Cadherin的蛋白质相对表达量、24 h和48 h时的细胞划痕愈合率及发生细胞迁移的比例均较shCont组低(均P<0.05),但shNRP-1组和shVEGF组间的差异均无统计学意义(均P>0.05);GBM22细胞株得到相似的结果。在GBM1A细胞株和GBM22细胞株中,3组细胞活力的差异均无统计学意义(均P>0.05)。根据绘制的剂量-生长曲线,GBM1A细胞株中,shNRP-1组和shVEGF组TMZ、PTX、XL-184的半抑制浓度(IC50)均低于shCont组(均P<0.05);在GBM22细胞株得到相似的结果(均P<0.05)。GBM1A细胞株中,shCont组和shCont+VEGF组形成神经球的数量均高于shNRP-1组和shNRP-1+VEGF组(均P<0.05),其中shCont+VEGF组高于shCont组(P<0.05),而shNRP-1组和shNRP-1+VEGF组间的差异无统计学意义(P>0.05);GBM22细胞株得到相似的结果。结论沉默VEGF或NRP-1基因在不影响GBM本身活力的情况下可有效抑制其干细胞性,降低其迁移能力及侵袭力,并恢复其对化疗药物的敏感性;VEGF介导的细胞迁移可能依赖NRP-1发挥作用。
Objective To investigate the mechanism of neuropilin-1(NRP-1)and vascular endothelial growth factor(VEGF)in the recurrence and drug resistance of glioblastoma multiforme(GBM).Methods GBM1A and GBM22 cell lines were cultured in vitro,and VEGF or NRP-1 was silenced by infecting lentivirus shRNA.According to the type of lentivirus shRNA infected,the two cell lines were divided into shCont group(infected with nonsense sequence shRNA),shNRP-1 group(infected with NRP-1 lentivirus shRNA)and shVEGF group(infected with VEGF lentivirus shRNA).The gene and protein expression of stem cell markers[sex determining region Y-box 2(Sox2),epidermal growth factor receptor(EGFR),N-cadherin,leukocyte mediated antigen(CD133)and cellular-mesenchymalepithelial transition factor(c-Met)]were detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)and Western blotting.Cell scratch test,Transwell chamber assay,neurosphere formation test and MTT method were used to detect the migration ability,invasion ability,neurosphere formation ability and metabolic activity of tumor cells.The sensitivity of tumor cells to temozolomide(TMZ),paclitaxel(PTX)and cabotinib(XL-184)was detected by cell growth inhibition assay.Tenμg of recombinant VEGF were added to the shCont group and shNRP-1 group in GBM1A and GBM22 cell lines,which were divided into shCont group,shCont+VEGF group,shNRP-1 group,shNRP-1+VEGF group,and the interaction between NRP-1 and VEGF was explored.Results In GBM1A cell line,the mRNA contents of Sox2,EGFR,N-cadherin,CD133 and c-Met in shVEGF group and shNRP-1 group were lower than those in shCont group(all P<0.05).In GBM1A cell line,the relative protein expressions of EGFR,CD133 and N-cadherin in shNRP-1 group and shVEGF group were lower than those in shCont group(all P<0.05),the cell scratch healing rate at 24 and 48 hours was lower than that in shCont group(both P<0.05),and the proportion of cell migration was lower than that in shCont group(P<0.05),while there was no significant difference between shNRP-1 group and shVEGF group(all P>0.05).Similar results were obtained in GBM22 cell line.There was no significant difference in cell viability between GBM1A cell line and GBM22 cell line(both P>0.05).According to the dose growth curve,the IC50 values of TMZ,PTX and XL-184 in shNRP-1 group and shVEGF group were lower than those in shCont group(all P<0.05).Similar results were obtained in GBM22 cell line(all P<0.05).In GBM1A cell line,the number of neurospheres formed in shCont+VEGF group was higher than that in shCont group(P<0.05),while there was no significant difference between shNRP-1 group and shNRP-1+VEGF group(P>0.05).Similar results were obtained in GBM22 cell line.Conclusions Silencing VEGF or NRP-1 gene can effectively inhibit the stem cell viability,reduce its migration ability and invasion,and restore its drug sensitivity without affecting the activity of GBM itself.VEGF-mediated cell migration may depend on NRP-1 to play a role.
作者
任佳星
白慧茹
窦长武
张龙骥
包洪恩
岳鹏
王飞
Ren Jiaxing;Bai Huiru;Dou Changwu;Zhang Longji;Bao Hongen;Yue Peng;Wang Fei(Department of Neurosurgery,Affiliated Hospital of Inner Mongolia Medical University,Huhhot 010050,China;Forensic Identification Center,Basic Medical College of Inner Mongolia Medical University,Huhhot 010050,China)
出处
《中华神经外科杂志》
CSCD
北大核心
2021年第12期1277-1285,共9页
Chinese Journal of Neurosurgery
基金
内蒙古自治区自然科学基金(2017MS0833)
内蒙古自治区科技计划项目(2019GG9044)。
作者简介
通信作者:王飞,Email:doctorwangfei@163.com。
