摘要
为探讨长链非编码RNA(long non-coding RNA,lncRNA)含5个PH结构域的反义RNA1(containing antisense RNA1 for 5 PH domains,FGD5-AS1)在肝细胞癌(hepatocellular carcinoma,HCC)组织中的表达及对HCC细胞增殖、迁移和侵袭的影响和作用机制,收集53例HCC患者肿瘤组织和对应癌旁组织,并体外培养人肝癌(Hep3B、SK-HEP-1、MHCC97L、MHCC97-H和Huh-7细胞)及健康人肝细胞(LO2),实时荧光定量聚合酶链式反应(real-time fluorescent quantitative polymerase chain reaction,RT-qPCR)检测组织、细胞中FGD5-AS1(lncRNA FGD5-AS1简称)和miR-512-3p表达,Pearson相关性统计分析HCC患者肿瘤组织中FGD5-AS1和miR-512-3p表达水平相关性,Western blotting检测细胞周期蛋白D1(Cyclin D1)表达。体外培养HCC细胞系Huh-7,分别转染lncRNA FGD5-AS1小干扰RNA(small-interfering RNA,siRNA)和miR-512-3p模拟物(mimic),或共转染FGD5-AS1 siRNA和miR-512-3p抑制剂(inhibitor)。CCK8实验、细胞克隆实验和Transwell实验检测各组细胞的增殖、迁移和侵袭情况。双荧光素酶报告基因实验验证FGD5-AS1和miR-512-3p、miR-512-3p和Cyclin D1之间的互作关系。将FGD5-AS1敲低(knockdown,KD)Huh-7细胞接种于裸鼠皮下,观察肿瘤生长情况。与癌旁组织比较,HCC组织中FGD5-AS1和Cyclin D1蛋白表达升高(均P<0.05),miR-512-3p表达降低(P<0.05)。Pearson分析结果表明,FGD5-AS1与miR-512-3p在肝癌组织中的表达呈显著负相关(r=-0.856,P<0.01)。与LO2细胞比较,人肝癌细胞系(Hep3B、SK-HEP-1、MHCC97L、MHCC97-H和Huh-7)中FGD5-AS1表达水平和Cyclin D1蛋白表达量升高,miR-512-3p表达水平降低(均P<0.05)。FGD5-AS1 KD或过表达miR-512-3p降低了Huh-7细胞的增殖活性、克隆形成数、迁移数和侵袭数(均P<0.05)。FGD5-AS1靶向结合miR-512-3p,且KD FGD5-AS1的Huh-7细胞中miR-512-3p表达增加(P<0.05)。Cyclin D1是miR-512-3p的靶基因,过表达miR-512-3p的Huh-7细胞Cyclin D1蛋白表达减少(P<0.05)。下调miR-512-3p逆转了KD FGD5-AS1对Huh-7细胞增殖、迁移、侵袭和Cyclin D1蛋白表达的影响。裸鼠移植瘤实验证实KD FGD5-AS1可抑制Huh-7细胞的在体生长。由此,lncRNA FGD5-AS1在HCC组织中呈高表达,KD FGD5-AS1可通过靶向miR-512-3p/Cyclin D1轴抑制Huh-7细胞增殖、迁移和侵袭。
In order to investigate the expression of long non-coding RNA(lncRNA)containing antisense RNA1 for 5 PH domains(FGD5-AS1)in hepatocellular carcinoma(HCC)tissues,its effects and mechanism on proliferation,migration,and invasion of HCC cells,HCC tissues and corresponding adjacent tissues of 53 HCC patients were collected.Human HCC cell lines(Hep3B,SK-HEP-1,MHCC97L,MHCC97-H,and Huh-7)and normal human hepatocytes(LO2)were cultured in vitro.RT-qPCR was used to detect the expression levels of lncRNA FGD5-AS1 and miR-512-3p in tissues and cell lines and the correlation between the expression levels of FGD5-AS1 and miR-512-3p in HCC patients'cancer tissues were analyzed by Pearson correlation test.Western blotting was used to detect the expression of Cyclin D1 protein.HCC cell line Huh-7 was cultured in vitro,and was transfected with FGD5-AS1 small-interfering RNA(siRNA)or miR-512-3p mimic,respectively,or co-transfected with FGD5-AS1 siRNA and miR-512-3p inhibitor.The proliferation,migration,and invasion of cells in each group were detected by CCK8 assay,cell cloning experiment and Transwell assay,respectively.The dual-luciferase reporter gene experiment was used to verify the binding between FGD5-AS1 and miR-512-3p or miR-512-3p and Cyclin D1.FGD5-AS1 knockdown(KD)Huh-7 cells were subcutaneously inoculated into nude mice to observe tumor growth.The results showed that compared to those of the adjacent tissues,the protein expressions of FGD5-AS1 and Cyclin D1 were increased in HCC tissues(both P<0.05),while the expression of miR-512-3p was decreased(P<0.05).There was a significant negative correlation between FGD5-AS1 and miR-512-3p levels in HCC tissues(r=-0.856,P<0.01).Compared to those of the LO2 cells,the protein expressions of FGD5-AS1 and Cyclin D1 in human HCC cell lines(Hep3B,SK-HEP-1,MHCC97L,MHCC97-H,and Huh-7)were all increased,while the expression level of miR-512-3p was decreased(all with P<0.05).FGD5-AS1 KD or over-expression of miR-512-3p reduced the proliferative activity,number of colony formation,migration,and invasion of Huh-7 cells(all with P<0.05).FGD5-AS1 targeted miR-512-3p,and the expression of miR-512-3p was increased in Huh-7 cells upon FGD5-AS1 KD(P<0.05).Cyclin D1 was the target gene of miR-512-3p,and the expression of Cyclin D1 protein was decreased in Huh-7 cells overexpressing miR-512-3p(P<0.05).Down-regulation of miR-512-3p reversed the inhibition of FGD5-AS1 KD on Huh-7 cells proliferation,migration,invasion,and Cyclin D1 expression.Xenografted nude mice result demonstrated that FGD5-AS1 KD inhibited the growth of Huh-7 cells in vivo.In conclusion,lncRNA FGD5-AS1 is highly expressed in HCC tissues and FGD5-AS1 KD inhibits the proliferation,migration,and invasion of Huh-7 cells by targeting the miR-512-3p/Cyclin D1 axis.
作者
代志强
唐福兴
杨静
DAI Zhiqiang;TANG Fuxing;YANG Jing(Department of Colorectal Surgery,The No.2 Hospital of Baoding,Baoding 071051,China)
出处
《现代免疫学》
2025年第3期259-270,共12页
Current Immunology
基金
2023年度河北省医学科学研究课题(20230251)。
