Oxymatrine induces anti-tumor response in cervical cancer by modulating circ_0008460/miR-197-3p/ribonucleotide reductase subunit M2 (RRM2)

Oxymatrine (OMT) has exhibited an anti-cancer role in human cancers, including cervical cancer (CC). The dysregulated circular RNAs (circRNAs) are key regulators in cancer biology, and circ_0008460 was upregulated in CC. This study was performed to investigate the circRNA-based molecular mechanism for OMT in CC. RNA detection for circ_0008460, microRNA-197-3p (miR-197-3p), or ribonucleotide reductase subunit M2 (RRM2) was completed using reverse transcription-quantitative polymerase chain reaction assay. Cell behaviors were assessed by Cell Counting Kit-8 assay for cell viability, colony formation assay or Edu assay for cell proliferation, flow cytometry for cell apoptosis, and wound healing assay/transwell assay for migration/invasion. Protein expression examination was conducted using western blot. Dual-luciferase reporter assay and RNA pull-down assay were applied to confirm target binding. Tumor xenograft assay was performed for OMT research <i>in vivo</i>. OMT induced circ_0008460 downregulation in CC cells. OMT-induced inhibitory effects on cell growth, migration, and invasion but promoting effect on cell apoptosis were attenuated by circ_0008460. Circ_0008460 directly interacted with miR-197-3p, and OMT inhibited malignant behaviors of CC cells via mediating circ_0008460/miR-197-3p axis. RRM2 acted as a target for miR-197-3p and circ_0008460 affected the RRM2 level through absorbing miR-197-3p. OMT upregulated miR-197-3p to inhibit RRM2 expression to impede CC cell development. CC tumorigenesis was suppressed by OMT via targeting circ_0008460/miR-197-3p/RRM2 axis <i>in vivo</i>. These results suggested that OMT restrained CC cell progression <i>in vitro</i> and tumor growth <i>in vivo</i> by downregulating circ_0008460 to mediate miR-197-3p/RRM2 axis.

氧化苦参碱（Oxymatrine, OMT）在包括宫颈癌（cervical cancer, CC）在内的人类恶性肿瘤中已展现出抗癌活性。失调的环状RNA（circular RNAs, circRNAs）是肿瘤生物学的关键调控因子，而circ_0008460在宫颈癌中呈上调表达状态。本研究旨在探究氧化苦参碱调控宫颈癌发生发展的环状RNA分子机制。采用逆转录-定量聚合酶链反应（reverse transcription-quantitative polymerase chain reaction）实验检测circ_0008460、微小RNA-197-3p（microRNA-197-3p, miR-197-3p）及核糖核苷酸还原酶亚基M2（ribonucleotide reductase subunit M2, RRM2）的转录水平。通过细胞计数试剂盒-8（Cell Counting Kit-8）实验检测细胞活力，集落形成实验或EdU实验（Edu assay）评估细胞增殖能力，流式细胞术（flow cytometry）检测细胞凋亡水平，划痕愈合实验（wound healing assay）/Transwell小室实验（transwell assay）检测细胞迁移与侵袭能力。采用蛋白质印迹法（western blot）检测目标蛋白的表达水平。通过双荧光素酶报告基因实验（dual-luciferase reporter assay）与RNA下拉实验（RNA pull-down assay）验证靶基因间的直接结合关系。采用裸鼠移植瘤实验（tumor xenograft assay）开展体内（in vivo）功能研究。实验结果表明，氧化苦参碱可显著下调宫颈癌细胞中circ_0008460的表达水平。氧化苦参碱对宫颈癌细胞增殖、迁移及侵袭的抑制作用，以及对细胞凋亡的促进作用，均可被circ_0008460所削弱。circ_0008460可直接结合miR-197-3p，氧化苦参碱通过调控circ_0008460/miR-197-3p信号轴抑制宫颈癌细胞的恶性生物学行为。RRM2是miR-197-3p的下游靶基因，circ_0008460可通过吸附miR-197-3p调控RRM2的表达水平。氧化苦参碱通过上调miR-197-3p的表达抑制RRM2的转录与翻译，从而阻滞宫颈癌细胞的恶性进展。体内实验证实，氧化苦参碱可通过靶向circ_0008460/miR-197-3p/RRM2信号轴抑制宫颈癌的体内成瘤能力。综上，氧化苦参碱可通过下调circ_0008460介导miR-197-3p/RRM2信号轴，从而在体外（in vitro）抑制宫颈癌细胞的恶性进展，并在体内（in vivo）阻滞肿瘤生长。