环状RNA hsa_circ_0091579对肝癌细胞增殖、迁移和侵袭的影响

Effect of circRNA hsa_circ_0091579 on the proliferation,migration,and invasion of hepatoma cells

目的探究环状RNA(circRNA)hsa_circ_0091579在肝细胞癌(HCC)细胞系中的表达及其对HCC细胞增殖、迁移和侵袭的影响。方法体外培养人肝癌细胞系SMMC-7721、HuH-7、MHCC-97H、HepG2和人正常肝细胞系HL-7702。从细胞中提取RNA,用实时荧光定量PCR(qRT-PCR)检测hsa_circ_0091579在HCC细胞系及人正常肝细胞系中的表达,并进行对比。针对hsa_circ_0091579的环化拼接位点设计siRNA,在体外HepG2和HuH-7细胞中转染hsa_circ_0091579 siRNA,并用qRT-PCR验证其有效性。实验分为siRNA组(hsa_circ_0091579 siRNA)和NC组(negative control siRNA),并在HepG2和HuH-7细胞中用CCK8实验、流式细胞术、划痕实验以及Transwell实验分别研究hsa_circ_0091579对细胞增殖、凋亡、迁移和侵袭的影响。使用双荧光素酶报告基因实验对预测的靶点进行验证。计量资料两组间比较采用t检验。结果与hsa_circ_0091579在人正常肝细胞系HL-7702中的表达水平相比,其在HCC细胞系SMMC-7721、HuH-7、MHCC-97H、HepG2中的表达水平均明显升高(t值分别为14.27、36.34、26.70、36.16,P值均<0.001)。与NC组相比,hsa_circ_0091579 siRNA可在HepG2和HuH-7细胞中有效沉默hsa_circ_0091579(t值分别为14.22、27.20,P值分别为0.005、0.001)。CCK8实验和流式细胞术结果显示,与NC组相比,siRNA组HepG2和HuH-7细胞的增殖活性明显降低,凋亡率明显升高,差异均有统计学意义(P值均<0.05);划痕实验和Transwell实验显示,与NC组相比,siRNA组HepG2和HuH-7细胞的迁移和侵袭能力明显减弱(t值分别为19.63、13.61、20.75、18.45,P值分别为0.003、0.005、0.002、0.003)。荧光素酶报告基因实验结果显示,与NC组相比,miR-149、miR-490-5p和miR-502-5p均能明显降低野生型荧光素酶质粒的活性(t值分别为10.01、9.13、61.49,P值分别为0.010、0.012、<0.001)。结论hsa_circ_0091579在HCC细胞系中高表达,可能通过抑制miR-149、miR-490-5p和miR-502-5p发挥其癌基因的作用。

Objective To investigate the expression of circular RNA(cirRNA)hsa_circ_0091579 in human hepatocellular carcinoma(HCC)cell lines and its effect on the proliferation,migration,and invasion of HCC cells.Methods Human HCC cell lines SMMC-7721,Huh-7,MHCC-97H,and HepG2 and normal human liver cell line HL-7702 were cultured in vitro.RNA was extracted from cells and quantitative real-time PCR(qRT-PCR)was used to measure the expression of hsa_circ_0091579 in HCC cell lines and normal human liver cell line.Small interfering RNA(siRNA)was designed for the cyclic splicing site of hsa_circ_0091579,and HepG2 and Huh-7 cells were transfected with hsa_circ_0091579 siRNA in vitro;qRT-PCR was used to verify transfection efficiency.The experiment was divided into siRNA group(transfected with hsa_circ_0091579 siRNA)and NC group(transfected with negative control siRNA),and CCK-8 assay,flow cytometry,wound healing assay,and Transwell assay were used to investigate the effect of hsa_circ_0091579 on cell proliferation,apoptosis,migration,and invasion in HepG2 and Huh-7 cells.Dual luciferase reporter gene assay was used to verify the predicted target.The t-test was used for comparison of continuous data between the two groups.Results Compared with the normal human liver cell line HL-7702,the HCC cell lines SMMC-7721,Huh-7,MHCC-97H,and HepG2 had a significant increase in the expression level of hsa_circ_0091579(t=14.27,36.34,26.70,and 36.16,all P<0.001).Compared with the NC group,hsa_circ_0091579 siRNA effectively silenced hsa_circ_0091579 in HepG2 and Huh-7 cells(t=14.22 and 27.20,P=0.005 and 0.001).CCK-8 assay and flow cytometry showed that compared with the NC group,the siRNA group had a significant reduction in the proliferative activity of HepG2 and Huh-7 cells and a significant increase in apoptosis rate(all P<0.05);wound healing assay and Transwell assay showed that compared with the NC group,the siRNA group had significant reductions in the migration and invasion abilities of HepG2 and Huh-7 cells(t=19.63,13.61,20.75,and 18.45,P=0.003,0.005,0.002,and 0.003).Luciferase reporter assay showed that compared with the NC group,miR-149,miR-490-5p,and miR-502-5p significantly reduced the activity of wild-type luciferase plasmids(t=10.01,9.13,and 61.49,P=0.010,P=0.012,and P<0.001).Conclusion Hsa_circ_0091579 is highly expressed in HCC cell lines and may play the role of oncogene by inhibiting miR-149,miR-490-5p,and miR-502-5p.