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环状RNA hsa_circ_0091579对肝癌细胞增殖、迁移和侵袭的影响
DOI: 10.3969/j.issn.1001-5256.2021.05.029
利益冲突声明本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突。
作者贡献声明：于维凯、冯婷婷、冯万文负责课题设计，资料分析，撰写论文；陈晓兵、骆继业参与收集数据，修改论文；于维凯、王言理负责拟定写作思路，指导撰写文章并最后定稿。
Effect of circRNA hsa_circ_0091579 on the proliferation, migration, and invasion of hepatoma cells
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摘要:
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。 Abstract: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. -
Key words:
- Carcinoma, Hepatocellular /
- DNA, Circular /
- Cell Physiological Phenomena
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图 2 hsa_circ_0091579 siRNA设计及沉默效率
注：a，hsa_circ_0091579 siRNA靶序列；b，qRT-PCR验证在HepG2和HuH-7细胞中转染降低hsa_circ_0091579。与NC组比较，*P < 0.05。
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