胃癌细胞MKN-45外泌体携带miR-552对HUVEC细胞增殖、迁移和血管生成能力的影响

Effects of gastric cancer cell exosomes carrying miR-552 on the proliferation,migration,and angiogenesis of HUVEC cells

目的探讨MKN-45细胞外泌体(exosome)携带的微小RNA-552(miR-552)对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)增殖、迁移和血管生成能力的影响。方法(1)将MKN-45细胞分为MKN-45空白对照组(不转染任何质粒)、MKN-45 miR-552抑制组[转染抑制miR-552表达的质粒(miR-552抑制质粒)]、MKN-45阴性对照组[转染阴性对照质粒(空质粒)]。提取、纯化和鉴定MKN-45细胞外泌体,采用免疫印迹法检测外泌体标志物分化抗原63(CD63)、分化抗原9(CD9)和肿瘤易感基因101(tumor susceptibility gene 101,TSG101)蛋白的表达。(2)将HUVEC细胞分为HUVEC对照组(加入PBS溶液)、HUVEC-外泌体组(加入MKN-45细胞来源外泌体)、HUVEC-阴性对照外泌体组(加入转染阴性对照质粒的MKN-45细胞来源外泌体)和HUVEC-miR-552抑制外泌体组(加入转染miR-552抑制质粒的MKN-45细胞来源外泌体),采用外泌体示踪实验检测外泌体是否进入HUVEC细胞;采用实时荧光定量PCR法检测miR-552的表达;采用噻唑蓝法检测HUVEC细胞的增殖情况;采用Transwell小室法检测HUVEC细胞的迁移情况;采用血管生成实验检测血管生成能力。结果本研究成功从MKN-45胃癌细胞中提取了外泌体,透射电镜下观察外泌体均呈圆形或椭圆形,直径为100~150 nm,可见外泌体囊泡结构;免疫印迹法法检测到外泌体表面表达标志物CD63、CD9和TSG101蛋白。示踪实验结果显示,MKN-45细胞来源的外泌体成功被HUVEC细胞内化。与MKN-45空白对照组和MKN-45阴性对照组相比较,MKN-45 miR-552抑制组外泌体中的miR-552相对表达水平降低(P<0.05);与HUVEC对照组相比,HUVEC-外泌体组24、48和72 h的细胞增殖率较高,且迁移细胞数、小管生成节点数以及miR-552相对表达水平均升高(P<0.05);与HUVEC-阴性对照外泌体组相比较,HUVEC-miR-552抑制外泌体组24、48和72 h的细胞增殖率降低,且迁移细胞数、小管生成节点数以及miR-552相对表达水平均降低(P<0.05)。结论MKN-45细胞外泌体携带的miR-552可以显著促进HUVEC细胞的增殖、迁移以及血管生成。

Objective To investigate the effects of the MKN-45 gastric cancer cell exosomes carrying microRNA-552(miR-552)on the proliferation,migration,and angiogenesis of human umbilical vein endothelial cells(HUVEC).Methods(1)The MKN-45 cells were divided into MKN-45 blank control group(no transfection),MKN-45 miR-552inhibitor group[transfection of plasmid inhibiting mir-552 expression(mir-552 inhibitor plasmid)],and MKN-45negative control group[transfection of negative control plasmid(empty plasmid)],the exosomes were extracted,purified,and identified.Western blotting was used to detect the protein expression of exosomal markers[CD63,CD9,and tumor susceptibility gene 101(TSG101)].(2)The HUVEC cells were divided into HUVEC control group(added PBS),HUVECexosome group(co-cultured with exosomes of MKN-45 cell),HUVEC-negative control exosome group(co-cultured with exosomes of MKN-45 cell transfected with negative control plasmid),and HUVEC-miR-552 inhibitor exosome group(cocultured with exosomes of MKN-45 cell transfected with miR-552 inhibitor plasmid),exosomes tracing experiment was used to detect whether exosomes entered HUVEC cells.Real-time fluorescent quantitative PCR method was used to detect the expression of miR-552,the MTT method was used to detect the proliferation of HUVEC cells,the Transwell chamber method was used to detect the migration of HUVEC cells,the angiogenesis test was used to detect the angiogenesis ability.Results This study successfully extracted exosomes from MKN-45 gastric cancer cells.Observed by transmission electron microscope,the exosomes were all round or elliptical,with a diameter of 100–150 nm,and the exosomal vesicle structure could be seen.Western blotting detection showed that the surface markers of exosomes(CD63,CD9,and TSG101 protein)were expressed in exosomes.The results of the tracing experiment showed that exosomes derived from MKN-45 cells were successfully internalized by HUVEC cells.After MKN-45 cells were transfected with miR-552inhibitor plasmid,compared with the MKN-45 blank control group and MKN-45 negative control group,the relative expression level of miR-552 in the exosomes decreased(P<0.05).Compared with the HUVEC control group,the cell proliferation rate at 24,48 and 74 h increased,as well as number of migration,tubule formation nodes,and relative expression level of miR-552 in the HUVEC-exosomes group increased(P<0.05).Compared with the HUVEC-negative control exosome group,the cell proliferation rate at 24,48 and 74 h decreased,as well as the number of migration,tubule formation nodes,and relative expression level of miR-552 in the HUVEC-miR-552 inhibitor exosome group decreased(P<0.05).Conclusion The exosomes of gastric cancer cells carrying miR-552 can significantly promote the proliferation,migration,and angiogenesis of HUVEC cells.