Background: Microparticles (MPs) are small extracellular plasma membrane particles shed by activated and apoptotic cells, which are involved in the development of atherosclerosis. Our previous study found that microRN...Background: Microparticles (MPs) are small extracellular plasma membrane particles shed by activated and apoptotic cells, which are involved in the development of atherosclerosis. Our previous study found that microRNA (miR)-19b encapsulated within endothelial MPs (EMPs) may contribute to the upregulation of circulating miR-19b in unstable angina patients. Hypoxia is involved in atherosclerosis as a critical pathological stimulus. However, it still remains unclear whether the increase of miR-19b levels in EMPs is related to hypoxia and if the effect of miR-19b - wrapped within EMPs - stimulates hypoxia on vascular endothelial cells. This study aimed to explore the changes of miR-19b in EMPs induced by hypoxia as well as their effects on endothelial cells.Methods: Human umbilical vein endothelial cells (HUVECs) were culturedin vitro and arranged to harvest EMPs in two parts: the first part consisted of EMPcontrol and EMPhypoxia and the second part included EMPvehicle, EMPNC mimic, and EMPmiR-19b mimic. Cell migration was detected by scratch migration and transwell chamber migration. Angiogenesis was assessed by tube formation assays. Furthermore, we predicted the target gene of miR-19b by bioinformatics analysis, and luciferase assay was used to verify the targeted gene of miR-19b. Data were analyzed by one-way analysis of variance. Student’st-test was used when two groups were compared.Results: Compared with EMPcontrol- and EMPhypoxia-inhibited migration of cells by scratch migration assay (80.77 ± 1.10 vs. 28.37 ± 1.40,P < 0. 001) and transwell chamber migration assay (83.00 ± 3.46 vs. 235.00 ± 16.52,P < 0.01), the number of tube formations was markedly reduced by 70% in the EMPhypoxia group (P < 0.001)in vitro analysis of HUVECs. Meanwhile, a strong inhibition of migration and tube formation of HUVECs in the presence of miR-19b-enriched EMPmiR-19b mimic was observed. This effect might be due to the delivery of miR-19b in EMPs. Transforming growth factor-β2 (TGFβ2) was predicted to be one of the target g展开更多
目的:探讨微小RNA-19b(microRNA-19b,miR-19b)在卵巢癌细胞侵袭和迁移中的作用机制。方法:应用qRT-PCR方法检测正常卵巢组织、卵巢癌组织及卵巢癌细胞系(SKOV-3、OVCAR-3)中miR-19b的表达情况。将miR-19b抑制剂或阴性对照寡核苷酸转染到...目的:探讨微小RNA-19b(microRNA-19b,miR-19b)在卵巢癌细胞侵袭和迁移中的作用机制。方法:应用qRT-PCR方法检测正常卵巢组织、卵巢癌组织及卵巢癌细胞系(SKOV-3、OVCAR-3)中miR-19b的表达情况。将miR-19b抑制剂或阴性对照寡核苷酸转染到OVCAR-3细胞中,Transwell实验检测miR-19b的表达对卵巢癌细胞侵袭和迁移能力的影响。免疫印迹分析(Western blot)技术检测miR-19b对第10号染色体缺失的磷酸酶和张力蛋白基因(phosphatase and tensin homolog deleted on chromosome ten,PTEN)的表达及对PTEN/AKT信号通路相关蛋白表达水平的影响。双报告基因实验用于检测miR-19b与PTEN的3'-UTR区之间的相互作用。结果:与正常卵巢组织比较,卵巢癌组织及卵巢癌细胞系SKOV-3和OVCAR-3中miR-19b高表达,其相对表达水平分别为3.56±0.68、3.01±0.02、3.78±0.04(P均<0.05)。Transwell实验表明,抑制miR-19b的表达可降低OVCAR-3细胞的侵袭和迁移能力。上调miR-19b的表达可抑制OVCAR-3细胞中PTEN蛋白的表达水平,并使磷酸化的AKT蛋白的表达水平显著升高。双报告基因检测结果显示,与对照组相比,miR-19b可与PTEN的3'-UTR区结合使其重组载体的荧光素酶活性下降。结论:miR-19b在卵巢癌细胞中存在异常高表达,可促进卵巢癌细胞的侵袭和迁移能力,有望作为卵巢癌患者潜在的生物标志物和治疗靶点。展开更多
基金grants from the National Natural Science Foundation of China (Nos.81770356, 81470473,and 81600340)and the Capital Health Research and Development of Special (No.2016-2-4083).
文摘Background: Microparticles (MPs) are small extracellular plasma membrane particles shed by activated and apoptotic cells, which are involved in the development of atherosclerosis. Our previous study found that microRNA (miR)-19b encapsulated within endothelial MPs (EMPs) may contribute to the upregulation of circulating miR-19b in unstable angina patients. Hypoxia is involved in atherosclerosis as a critical pathological stimulus. However, it still remains unclear whether the increase of miR-19b levels in EMPs is related to hypoxia and if the effect of miR-19b - wrapped within EMPs - stimulates hypoxia on vascular endothelial cells. This study aimed to explore the changes of miR-19b in EMPs induced by hypoxia as well as their effects on endothelial cells.Methods: Human umbilical vein endothelial cells (HUVECs) were culturedin vitro and arranged to harvest EMPs in two parts: the first part consisted of EMPcontrol and EMPhypoxia and the second part included EMPvehicle, EMPNC mimic, and EMPmiR-19b mimic. Cell migration was detected by scratch migration and transwell chamber migration. Angiogenesis was assessed by tube formation assays. Furthermore, we predicted the target gene of miR-19b by bioinformatics analysis, and luciferase assay was used to verify the targeted gene of miR-19b. Data were analyzed by one-way analysis of variance. Student’st-test was used when two groups were compared.Results: Compared with EMPcontrol- and EMPhypoxia-inhibited migration of cells by scratch migration assay (80.77 ± 1.10 vs. 28.37 ± 1.40,P < 0. 001) and transwell chamber migration assay (83.00 ± 3.46 vs. 235.00 ± 16.52,P < 0.01), the number of tube formations was markedly reduced by 70% in the EMPhypoxia group (P < 0.001)in vitro analysis of HUVECs. Meanwhile, a strong inhibition of migration and tube formation of HUVECs in the presence of miR-19b-enriched EMPmiR-19b mimic was observed. This effect might be due to the delivery of miR-19b in EMPs. Transforming growth factor-β2 (TGFβ2) was predicted to be one of the target g
文摘目的:探讨微小RNA-19b(microRNA-19b,miR-19b)在卵巢癌细胞侵袭和迁移中的作用机制。方法:应用qRT-PCR方法检测正常卵巢组织、卵巢癌组织及卵巢癌细胞系(SKOV-3、OVCAR-3)中miR-19b的表达情况。将miR-19b抑制剂或阴性对照寡核苷酸转染到OVCAR-3细胞中,Transwell实验检测miR-19b的表达对卵巢癌细胞侵袭和迁移能力的影响。免疫印迹分析(Western blot)技术检测miR-19b对第10号染色体缺失的磷酸酶和张力蛋白基因(phosphatase and tensin homolog deleted on chromosome ten,PTEN)的表达及对PTEN/AKT信号通路相关蛋白表达水平的影响。双报告基因实验用于检测miR-19b与PTEN的3'-UTR区之间的相互作用。结果:与正常卵巢组织比较,卵巢癌组织及卵巢癌细胞系SKOV-3和OVCAR-3中miR-19b高表达,其相对表达水平分别为3.56±0.68、3.01±0.02、3.78±0.04(P均<0.05)。Transwell实验表明,抑制miR-19b的表达可降低OVCAR-3细胞的侵袭和迁移能力。上调miR-19b的表达可抑制OVCAR-3细胞中PTEN蛋白的表达水平,并使磷酸化的AKT蛋白的表达水平显著升高。双报告基因检测结果显示,与对照组相比,miR-19b可与PTEN的3'-UTR区结合使其重组载体的荧光素酶活性下降。结论:miR-19b在卵巢癌细胞中存在异常高表达,可促进卵巢癌细胞的侵袭和迁移能力,有望作为卵巢癌患者潜在的生物标志物和治疗靶点。
