摘要
目的探讨外泌体长链非编码RNA H19(lncRNA H19)调控人肺腺癌细胞迁移和侵袭作用及机制。方法收集确诊肺腺癌并经影像学诊断已发生远处转移患者(转移组)或无远处转移患者(非转移组)以及正常人群(正常对照组)各30例外周血标本,分离单核细胞获得血浆上清液分离外泌体,同时取培养人肺腺癌细胞系A549细胞时收集的培养上清液分离外泌体,实时荧光定量PCR(qRT-PCR)法检测并比较组间外泌体lncRNA H19 mRNA表达差异。并采用慢病毒介导的lncRNA H19特异性小干扰RNA(shRNA)构建稳定敲低lncRNA H19的人肺腺癌A549细胞模型,荧光素酶验证lncRNA H19通过吸附结合微RNA-7(miR-7),划痕实验、Transwell实验检测稳定敲低lncRNA H19对A549细胞迁移、侵袭能力的影响,蛋白免疫印迹法检测稳定敲低lncRNA H19对miR-7及其下游的信号通路蛋白Kruppel样因子4(KLF4)和血管内皮生长因子(VEGF)蛋白表达水平的影响。结果外泌体被成功分离。与非转移组肺腺癌患者相比,转移组肺腺癌患者外周血外泌体lncRNA H19 mRNA表达增加(P <0.01)。A549细胞中,与对照细胞相比,稳定敲低lncRNA H19细胞的外泌体lncRNA H19 mRNA表达减少(P <0.01)。细胞划痕实验和Transwell实验显示,敲低lncRNA H19的A549细胞迁移和侵袭的能力下降。野生型lncRNA H19与miRNA-7共转染后A549细胞荧光度值下降(P <0.01),突变型lncRNA H19与miRNA共转染后A549细胞荧光度值未见明显变化(P> 0.05)。A549细胞中,稳定敲低lncRNA H19后KLF4和VEGF蛋白表达水平均有所下调(P均<0.05)。结论外泌体lncRNA H19可能在肺腺癌细胞迁移和侵袭过程中发挥作用,且这种作用可能与竞争性结合miR-7及KLF4/VEGF信号通路有关。
Objective To investigate the effect and mechanism of exosomes long non-coding RNA H19(lncRNA H19) on the migration and invasion of human lung adenocarcinoma cells. Methods Peripheral blood samples were collected from 30 lung adenocarcinoma patients with distant metastasis(metastasis group), 30 patients without distant metastasis(non-metastasis group) and 30 healthy controls(normal control group).The exosomes were isolated from plasma supernatant obtained from separating monocytes, and the exosomes were separated from culture supernatant collected from human lung adenocarcinoma cell line A549. The expression levels of exosomes lncRNA H19 mRNA were detected and compared among three groups by real-time fluorescence quantitative PCR(qRT-PCR). A549 cell models with stable H19 knockdown were constructed by lentivirus-mediated H19 shRNA. The binding between lncRNA H19 and microRNA-7(miR-7) was verified by luciferase assay. The effect of stable lncRNA H19 knockdown upon the migration and invasion of A549 cells was evaluated by wound healing test and Transwell chamber test. The effect of stable lncRNA H19 knockdown upon the expression levels of KLF4 and VEGF proteins was assessed by Western Blot. Results The exosomes were successfully isolated. Compared with the non-metastasis group, the expression level of lncRNA H19 mRNA in the peripheral blood exosomes in the metastasis group was significantly up-regulated(P < 0.01).Compared with the control cells, the expression level of lncRNA H19 mRNA in the exosomes of A549 cells with stable lncRNA H19 knockdown was significantly down-regulated(P < 0.01). Wound healing test and Transwell chamber test demonstrated that the migration and invasion of A549 cells were significantly decreased after lncRNA H19 knockdown. The fluorescent intensity of A549 cells was significantly decreased after wild-type lncRNA H19 was co-transfected with miR-7(P < 0.01), whereas the fluorescent intensity of A549 cells did not significantly change after the co-transfection of mutant lncRNA H19 and miR-7(P
作者
王林
易基群
江高峰
Wang Lin;Yi Jiqun;Jiang Gaofeng(Department of Oncology,Guangzhou Red Cross Hospital,Medical College of Jinan University,Guangzhou 510220,China)
出处
《新医学》
CAS
2021年第10期745-751,共7页
Journal of New Medicine
基金
国家自然科学基金(81773264)
广东省医学科学技术研究基金(A2021173)
广州市医药卫生科技项目(20201A011019)。
