LncRNA CBR3-AS1调节miR-409-3p/hnRNPA2B1轴对肝细胞癌细胞恶性生物学行为的影响  

LncRNA CBR3-AS1 promotes the malignant biological behaviors of hepatocellular carcinoma cells by targeting the miR-409-3p/hnRNPA2B1 axis

作　　者：陈仕[1] 黎慧娟 许若思 张彩英 陈露 CHEN Shi;LI Huijuan;XU Ruosi(Department of Gastroenterology,the Second People’s Hospital of Hunan Province,Hunan,Changsha 410007,China)

机构地区：[1]湖南省第二人民医院消化内科,长沙市410007

出　　处：《河北医药》2023年第24期3685-3690,共6页Hebei Medical Journal

基　　金：湖南省卫生健康委员会资助项目(编号:D202303107884)。

摘　　要：目的探讨长链RNA CBR3-AS1(LncRNA CBR3-AS1)靶向微小RNA-409-3p(miR-409-3p)/核不均一核糖核蛋白A2B1(hnRNPA2B1)轴对肝细胞癌(HCC)细胞恶性生物学行为的影响。方法qRT-PCR法分析肝癌组织中LncRNA CBR3-AS1和miR-409-3p表达水平。双荧光素酶检测LncRNA CBR3-AS1和miR-409-3p及hnRNPA2B1和miR-409-3p的靶向关系。将Hep G2细胞分为si-NC组、si-CBR3-AS1组、si-CBR3-AS1+anti-miR-NC组、si-CBR3-AS1+anti-miR-409-3p组、miR-NC组、miR-409-3p mimics组、miR-409-3p mimics+pcDNA组、miR-409-3p mimics+hnRNPA2B1组。平板克隆检测细胞增殖;Annexin V-FITC/PI法检测细胞凋亡;划痕实验检测细胞迁移;Transwell实验检测细胞侵袭;Western blot分析法检测细胞核增殖抗原标记物(Ki67)、细胞周期负调控因子(P21)、B淋巴细胞瘤(Bcl)-2、Bcl-2相关X蛋白(Bax)、基质金属蛋白酶2(MMP-2)、MMP-9蛋白的表达变化。小鼠移植瘤实验检测LncRNA CBR3-AS1对肝癌肿瘤生长及miR-409-3p/hnRNPA2B1的影响。结果与癌旁组织比较,癌组织中LncRNA CBR3-AS1表达显著升高,miR-409-3p表达显著降低(P<0.05)。StarBase预测显示LncRNA CBR3-AS1与miR-409-3p有靶向结合位点。双荧光素酶实验显示,miR-409-3p mimics与LncRNA CBR3-AS1-WT共转染Hep G2细胞相对荧光素酶活性显著降低(P<0.05)。与si-NC组比较,si-CBR3-AS1组LncRNA CBR3-AS1表达明显下降,miR-409-3p表达明显上升(P<0.05);与si-CBR3-AS1+anti-miR-NC组比较,si-CBR3-AS1+anti-miR-409-3p组miR-409-3p表达明显下降(P<0.05)。与si-NC组比较,si-CBR3-AS1组Hep G2细胞集落形成数、划痕愈合率、侵袭数量及Ki67、Bcl-2、MMP-2、MMP-9表达水平显著降低,细胞凋亡率及P21、Bax蛋白表达水平显著升高(P<0.05);与si-CBR3-AS1+anti-miR-NC组比较,si-CBR3-AS1+anti-miR-409-3p组Hep G2细胞集落形成数、划痕愈合率、侵袭数量及Ki67、Bcl-2、MMP-2、MMP-9表达水平显著升高,细胞凋亡率及P21、Bax蛋白表达水平显著降低(P<0.05)。生物信息学分析显示miR-409-3p与hnRNObjective To investigate the regulatory effects of long non-coding RNA CBR3-AS1(lncRNA CBR3-AS1)on the malignant biological behaviors of hepatocellular carcinoma(HCC)cells by targeting the microRNA-409-3p(miR-409-3p)/heteronuclear ribonucleoprotein A2B1(hnRNPA2B1)axis.Methods Expression levels of lncRNA CBR3-AS1 and miR-409-3p in HCC tissues were detected by quantitative real-time polymerase chain reaction(qRT-PCR).The binding relationship between lncRNA CBR3-AS1 and miR-409-3p,and that between miR-409-3p and hnRNPA2B1 were assessed by dual-luciferase reporter assay.HepG2 cells were transfected with si-NC,si-CBR3-AS1,si-CBR3-AS1+anti-miR-NC,si-CBR3-AS1+anti-miR-409-3p,miR-NC,miR-409-3p mimics,miR-409-3p mimics+pcDNA,miR-409-3p mimics+pcDNA-hnRNPA2B1.Cell proliferation,apoptosis,migration,and invasion were detected by colony formation assay,Annexin V-fluorescein isothiocyanate/propidium iodide(Annexin V-FITC/PI)staining,wound healing assay and Transwell assay,respectively.Protein expressions of nuclear proliferation antigen marker(Ki67),cell cycle negative regulator(P21),B-cell lymphoblastoma(Bcl-2),Bcl-2 associated X protein(Bax),matrix metalloproteinase-2(MMP-2),and MMP-9 were detected by Western blot.A xenograft model in nude mice was created to validate the in vivo influence of lncRNA CBR3-AS1 on HCC growth and the regulatory effect on the miR-409-3p/hnRNPA2B1 axis.Results Significantly upregulated lncRNA CBR3-AS1 and downregulated miR-409-3p were detected in HCC tissues than those of paracancerous tissues(P<0.05).The binding site in the promoter regions of lncRNA CBR3-AS1 and miR-409-3p was predicted in StarBase.Dual-luciferase reporter assay showed significantly decreased relative luciferase activity in HepG2 cells co-transfected with lncRNA CBR3-AS1 wild-type plasmid and miR-409-3p mimics than that of other groups(P<0.05).LncRNA CBR3-AS1 was significantly downregulated in HepG2 cells transfected with si-CBR3-AS1 than that in cells transfected with si-NC,while miR-409-3p was significantly upregulated(P<0.05)

关 键 词：长链RNA CBR3-AS1 微小RNA-409-3p 核不均一核糖核蛋白A2B1 肝癌 增殖 凋亡 迁移 侵袭 

分 类 号：R735.7[医药卫生—肿瘤]