微小RNA-25通过肿瘤相关成纤维细胞外泌体促进食管癌细胞的远处转移

Exosomal microRNA-25 secreted by cancer-associated fibroblasts promotes the metastasis of esophageal carcinoma cells

目的探讨肿瘤相关成纤维细胞(CAFs)源性外泌体中微小RNA(miR)-25参与促进食管癌细胞转移的作用及机制。方法获取新鲜食管癌及癌旁组织, 通过贴壁法分别获得CAFs和成纤维细胞(NFs), 蛋白质印迹法(Western blot)和免疫荧光法检测α-平滑肌肌动蛋白(α-SMA)、波形蛋白(Vimentin)的表达进行鉴定。用超速离心法提取CAFs和NFs的外泌体, 用Western blot和透射电镜对外泌体进行鉴定, 采用荧光实时定量聚合酶链反应(RT-qPCR)检测食管癌组织和癌旁组织中miR-25的表达, 结果用配对样本t检验进行统计学分析。同时用RT-qPCR检测CAFs、NFs和相应外泌体, 以及外泌体共培养的食管癌TE-1中miR-25的表达, 并将外泌体与TE-1共培养后检测TE-1迁移侵袭能力。Western blot及荧光素酶实验明确miR-25的靶基因, 结果用非配对样本t检验进行统计学分析。结果 RT-qPCR结果提示, 与癌旁组织比较, 食管癌组织中高表达miR-25(n=30, t= 3.46, P<0.05);miR-25在CAFs中的表达高于NFs(t=5.37, P<0.05);免疫荧光结果提示, 相较于NFs, CAFs中α-SMA、Vimentin荧光信号增强;Western blot结果提示外泌体中表达CD9和TSG101蛋白, 且透射电镜鉴定符合外泌体特征;RT-qPCR结果提示, CAFs源性外泌体中miR-25的表达高于NFs外泌体(t=5.45, P<0.05)。Transwell实验表明, NFs外泌体与TE-1共培养组, 每视野TE-1迁移细胞数为(202.700±17.840)个, CAFs外泌体与TE-1共培养组为(558.000±19.430)个(t=13.47, P<0.01, n=3);NFs外泌体与TE-1共培养组, 每视野TE-1侵袭细胞数为(174.000±10.070)个, CAFs外泌体与TE-1共培养组为(428.000±8.888)个(t=18.91, P<0.01, n=3);miR-NC转染TE-1组, 每视野TE-1迁移细胞数为(68.000±2.082)个, miR-25 mimics转染TE-1组为(379.000±6.083)个(t=48.37, P<0.01, n=3);miR-NC转染TE-1组, 每视野TE-1侵袭细胞数为(40.000±2.082)个, miR-25 mimics转染TE-1组为(254.000±2.000)个(t=74.13, P<0.01, n=3), 进一步荧光素酶报告基因结果显示, 过表达miR-25可降低58%的PTEN 3’UTR野生型的荧光素酶活性(野生型:t=7.19, P<0.05), 证实miR-25可靶向促进PTEN的表达。结论 CAFs外泌体miR-25能够通过激活PTEN信号通路, 而促进食管癌细胞的远处转移。

Objective To investigate the role and potential mechanism of exosomal microRNA(miR)-25 from cancer-associated fibroblasts(CAFs)promoting the proliferation and metastasis of esophageal carcinoma cells.Methods CAFs and normal fibroblasts(NFs)were isolated from cancer tissues and adjacent tissues of esophageal carcinoma patients,respectively,and immunofluorescence was used to identifyα-smooth muscle actin(α-SMA)and Vimentin expression in CAFs and NFs.After centrifugation,exosomal RNA was extracted and exosomes was identified by Western blotting and transmission electron microscopy.The quantitative reverse transcriptase polymerase chain reaction(RT-qPCR)was used to detect the expression of miR-25 in esophageal cancer issues and para-canceresophagus tissues.The results were statistically analyzed by paired sample t test,and the miR-25 level in cell lines of esophageal cancer,CAFs,NFs and exosomes was detecd by RT-qPCR.The influence of CAFs-derived exosomes containing miR-25 on the invasion of esophageal cancer cells was evaluated.The confirmation of target gene of miR-25 was performed with luciferase assay and Western blotting.The results were statistically analyzed by unpaired sample t test.Results RT-qPCR indicated that miR-25 was highly expressed in esophageal cancer tissues as compared with para-canceresophagus tissues(n=30,t=3.46,P<0.05).Compared with NFs,α-SMA and Vimentin were highly expressed in CAFs.Western blotting indicated that CD9 and TSG101 proteins were expressed in exosomes.The relative expression of miR-25 in CAFs-derived exosomes was higher than that in NFs-derived exosomes(t=5.45,P<0.05).Transwell assay indicated that the number of TE-1 migrating cells per field was(202.700±17.840)in the NFs-derived exosomes and TE-1 co-culture group,and(558.000±19.430)in the CAFs-derived exosomes and TE-1 co-culture group(t=13.47,P<0.01,n=3).The number of TE-1 invasion cells per field was(174.000±10.070)in the NFs-derived exosomes and TE-1 co-culture group,and(428.000±8.888)in the CAFs-derived exosomes and TE-1 co-culture group(t=18.91,P<0.01,n=3).The number of TE-1 migrating cells per field was(68.000±2.082)in TE-1 cells containing miR-NC,and(379.000±6.083)in the TE-1 cells containing miR-25 mimics(t=48.37,P<0.01,n=3).The number of TE-1 invasive cells per field was(40.000±2.082)in the TE-1 cells containing miR-NC,and(254.000±2.000)in the TE-1 cells containing miR-25 mimics(t=74.13,P<0.01,n=3).Luciferase reporter gene detection showed that overexpression of miR-25 reduced luciferase activity of PTEN 3′UTR wild-type by 58%(wild-type:t=7.19,P<0.05).Conclusion Exosomal miR-25 could regulate the process of CAFs promoting the invasion of esophageal cancer cells.