长链非编码RNA AFAP1-AS1靶向miR-195-5p对肝癌细胞生物学行为的影响

Effect of lncRNA-AFAP1-AS1 targeting miR-195-5p on biological behavior of hepatocellular carcinoma cells

目的探讨长链非编码核糖核酸(lncRNA)AFAP1-AS1及微小核糖核酸-195-5p(miR-195-5p)对肝细胞癌(HCC)细胞生物学行为的影响。方法常规培养人HCC细胞系(HepG2、Hep3B、SMMC-7721、Bel7402、SK-hep1细胞)和正常人肝细胞(HL-7702细胞),用实时荧光定量PCR法检测AFAP1-AS1、miR-195-5p并筛选实验细胞。将对数生长期实验细胞随机分为si-AFAP1-AS1组、si-NC组,miR-195-5pmimics组、mimics-NC组,miR-195-5pinhibitor+si-NC组、miR-195-5p inhibitor+si-AFAP1-AS1组、inhibitor-NC+si-AFAP1-AS1组、inhibitor-NC+si-NC组,用Lipofectamine 2000试剂将相应质粒按照分组转染入细胞。用CCK-8实验、菌落形成实验检测细胞增殖能力,用流式细胞术检测细胞凋亡及细胞周期,用Transwell实验检测细胞迁移及侵袭能力,用双荧光素酶报告基因实验验证AFAP1-AS1与miR-195-5p的靶向关系。结果HCC细胞中AFAP1-AS1表达高于正常人肝细胞(P均<0.05),HCC细胞中miR-195-5p表达低于正常人肝细胞(P均<0.05)。由于SK-hep1细胞中AFAP1-AS1表达最高、miR-195-5p表达最低,因此,后续研究均采用SK-hep1细胞进行实验。与si-NC组比较,si-AFAP1-AS1组中AFAP1-AS1表达低、细胞增殖能力低、迁移和侵袭细胞少、G0/G1期细胞比例高、细胞凋亡率高(P均<0.05)。通过Starbase v3.0在线数据库预测发现,AFAP1-AS1与miR-195-5p有互补的结合位点。双荧光素酶报告基因实验结果显示,miR-NC组、miR-195-5p组共转染WT-AFAP1-AS1后相对荧光素酶活性比较差异有统计学意义(P<0.05),共转染MUT-AFAP1-AS1后相对荧光素酶活性比较差异无统计学意义(P>0.05)。与inhibitor-NC+si-NC组比较,si-NC+miR-195-5p inhibitor组细胞增殖能力高、迁移和侵袭细胞多、G_(0)/G_(1)期细胞比例低、细胞凋亡率低(P均<0.05),而si-AFAP1-AS1+miR-195-5p inhibitor组上述细胞生物学行为则相反。结论lncRNA AFAP1-AS1可能通过竞争性结合miR-195-5p参与调控HCC细胞增殖、迁移、侵袭、凋亡等生物学行为。

Objective To investigate the effects of long non-coding RNA(lncRNA)-AFAP1-AS1 and microRNA(miR)-195-5p on the biological behavior of hepatocellular carcinoma(HCC)cells.Methods Human HCC cell lines(HepG2,Hep3B,SMMC-7721,Bel7402,SK-hep1 cells)and normal human liver cells(HL-7702 cells)were routinely cultured.The expression levels of AFAP1-AS1 and miR-195-5p were detected by real-time fluorescence quantitative PCR to select the experimental cells.The experimental cells in logarithmic growth phase were randomly assigned to different groups:si-AFAP1-AS1 group,si-NC group,miR-195-5p mimics group,mimics-NC group,miR-195-5p inhibitor+si-NC group,miR-195-5p inhibitor+si-AFAP1-AS1 group,inhibitor-NC+si-AFAP1-AS1 group,and inhibitor-NC+si-NC group,respectively.Subsequently,the corresponding plasmids were transfected into the cells using Lipofectamine 2000 reagent based on the grouping.Cell proliferation was assessed by CCK-8 and colony formation experiments,while apoptosis and cell cycle were analyzed using flow cytometry.Cell migration and invasion capabilities were evaluated by Transwell experiment,and the targeted relationship between AFAP1-AS1 and miR-195-5p was confirmed via dual luciferase reporter gene assay.Results In HCC cells,AFAP1-AS1 expression was higher than that in normal liver cells(P<0.05),while miR-195-5p expression was lower than that of the normal liver cells(P<0.05).Given that SK-hep1 cells exhibited the highest AFAP1-AS1 expression and the lowest miR-195-5p expression,subsequent investigations were carried out using SK-hep1 cells for experimental purposes.Compared with the si-NC group,the si-AFAP1-AS1 group displayed reduced AFAP1-AS1 expression,decreased cell proliferation capacity,reduced migration and invasion cells,a higher cell population in the G0/G1 phase,and an elevated apoptosis rate(all P<0.05).The Starbase v3.0 online database predicted that there were complementary binding sites between AFAP1-AS1 and miR-195-5p.Findings from the dual-luciferase reporter gene assay indicated that there was significant difference in the relative luciferase activity between the miR-NC and miR-195-5p after co-transfection of WT-AFAP1-AS1(P<0.05),while no significant difference was found after co-transfection of MUT-AFAP1-AS1(P>0.05).Compared with the inhibitor-NC+si-NC group,the si-NC+miR-195-5p inhibitor group demonstrated enhanced cell proliferation,increased migration and invasion,reduced cell population in the G_(0)/G_(1) phase,and decreased apoptosis rate(all P<0.05).Conversely,the si-AFAP1-AS1+miR-195-5p inhibitor group exhibited the opposite cellular responses.Conclusion The lncRNA AFAP1-AS1 potentially plays a role in regulating various biological processes in HCC cells,including proliferation,migration,invasion,and apoptosis,through its competitive interaction with miR-195-5p.