circERBB2在卵巢癌中的表达及作用机制研究

Expression of circERBB2 in ovarian cancer and its mechanism

  • 摘要:
    目的 探讨circERBB2在卵巢癌中的表达及作用机制。
    方法 基于GEO数据库筛选卵巢癌差异表达环状RNA(circRNA), 并查找circRNA相关靶基因, 绘制韦恩图得到GSE79572数据集中差异表达基因与Targetscan 7.1预测所得微小RNA-187-3p(miR-187-3p)靶基因的交叉基因。收集20例卵巢癌患者的卵巢癌组织和癌旁正常组织,培养人正常卵巢细胞系HOSEpiC和人卵巢癌细胞系OVCAR-3、SKOV-3、3AO、OV90, 采用实时定量聚合酶链反应(qRT-PCR)检测circERBB2、miR-187-3p、BCL6表达水平。通过双荧光素酶报告基因实验和RNA下拉实验验证circERBB2、miR-187-3p、BCL6三者间的相互作用。通过多项细胞实验分析circERBB2、miR-187-3p、BCL6对卵巢癌细胞侵袭、迁移、增殖及细胞周期的影响。
    结果 数据库分析结果显示,卵巢癌中差异表达circRNA为circERBB2, circERBB2靶向的miRNA为miR-187-3p, 绘制韦恩图取交集后得到BCL6等7个交叉基因, circERBB2与miR-187-3p、miR-187-3p与BCL6存在潜在结合位点。卵巢癌组织中circERBB2、BCL6表达水平高于癌旁组织, miR-187-3p表达水平低于癌旁组织,差异有统计学意义(P < 0.05); 卵巢癌细胞系OVCAR-3、SKOV-3、3AO、OV90中circERBB2表达水平均高于正常卵巢细胞系HOSEpiC, 差异有统计学意义(P < 0.05)。双荧光素酶报告基因实验及RNA下拉实验证实, circERBB2、miR-187-3p、BCL6之间存在相互作用关系。多项细胞实验结果显示,抑制circERBB2表达能够抑制卵巢癌细胞增殖和迁移,这种抑制作用可在过表达miR-187-3p后被逆转; 抑制miR-187-3p表达能够有效促进卵巢癌细胞增殖和迁移,这种促进作用可在抑制BCL6表达后被逆转。
    结论 circERBB2能够通过靶向吸附miR-187-3p促进原癌基因BCL6表达,从而调节卵巢癌发生过程。

     

    Abstract:
    Objective To investigate the expression and mechanism of circERBB2 in ovarian cancer.
    Methods The GEO database was used to screen differentially expressed circular RNAs (circRNAs) in ovarian cancer, and to search for target genes related to circRNAs. A Venn plot was drawn to obtain the cross genes between differentially expressed genes in the GSE79572 dataset and the small RNA-187-3p (miR-187-3p) target genes predicted by Targetscan 7.1. Ovarian cancer tissue and adjacent normal tissue from 20 ovarian cancer patients were collected. Human normal ovarian cell line HOSEpiC and human ovarian cancer cell lines OVCAR-3, SKOV-3, 3AO, OV90 were cultured. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the levels of circERBB2, miR-187-3p, and BCL6. The interactions among circERBB2, miR-187-3p and BCL6 were verified by double Luciferase reporter gene experiment and RNA pull-down experiment. The effects of circERBB2, miR-187-3p, and BCL6 on invasion, migration, proliferation, and cell cycle of ovarian cancer cells were explored.
    Results The database analysis showed that the differentially expressed circRNA in ovarian cancer was circERBB2, and the miRNA targeted by circERBB2 was miR-187-3p. After drawing a Venn plot for intersection, seven cross genes such as BCL6 were obtained. There were potential binding sites between circERBB2 and miR-187-3p, as well as miR-187-3p and BCL6. The levels of circERBB2 and BCL6 in ovarian cancer tissue were higher than those in adjacent tissues, while the level of miR-187-3p was lower than that in adjacent tissues (P < 0.05). The expression levels of circERBB2 in ovarian cancer cell lines OVCAR-3, SKOV-3, 3AO, and OV90 were higher than those in normal ovarian cell lines HOSEpiC (P < 0.05). The double luciferase reporter gene experiment and RNA pull-down experiment confirmed that circERBB2, miR-187-3p and BCL6 had interaction relationships. Multiple cell experiments have shown that silencing circERBB2 expression can inhibit the proliferation and migration of ovarian cancer cells, and this inhibitory effect can be reversed by overexpression of miR-187-3p. Inhibition of miR-187-3p can effectively promote the proliferation and migration of ovarian cancer cells, which can be reversed after inhibiting the expression of BCL6.
    Conclusion CircERBB2 can promote the expression of the oncogene BCL6 through targeted adsorption of miR-187-3p, thereby regulating the occurrence of ovarian cancer.

     

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