The circular RNA RNF13 (circ-RNF13; ID: hsa_circ_0067717) is a novel identified abnormally upregulated circRNA in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) patients. However, its role and mechanism remain to be further annotated. The expression of circ-RNF13, microRNA (miR)-424-5p, and TGFβ-induced factor homeobox 2 (TGIF2) were detected by real-time quantitative PCR and western blotting, and their interaction was confirmed by dual-luciferase reporter assay. Functional assays were performed using MTS assay, colony formation assay, flow cytometry, enzyme-linked immunosorbent assay, transwell assay, and xenograft tumor model, along with real-time quantitative PCR. Circ-RNF13 was upregulated in HBV-infected human HCC tissues and HBV-expressing cells (Huh7-HBV and Hep3B-HBV), accompanied with TGIF2 upregulation and miR-424-5p downregulation. Blocking circ-RNF13 enhanced the apoptosis rate of Huh7-HBV and Hep3B-HBV cells but inhibited cell viability, colony formation, migration, and invasion, along with suppressed tumor growth in vivo. Besides, HBV DNA copies and levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) were diminished by circ-RNF13 knockdown in Huh7-HBV and Hep3B-HBV cells. Mechanistically, circ-RNF13 and TGIF2 served as competing endogenous RNAs (ceRNAs) for miR-424-5p. Overexpressing miR-424-5p mimicked and silencing miR-424-5p counteracted the effects of circ-RNF13 depletion in HBV-expressing HCC cells in vitro. Consistently, TGIF2 restoration partially abrogated the role of miR-424-5p upregulation in Huh7-HBV and Hep3B-HBV cells. The circ-RNF13 sponged miR-424-5p to suppress HBV-associated HCC cells malignant progression and HBV infection by regulating TGIF2, providing a novel insight into the occurrence and treatment of HBV-associated HCC.

中文翻译：

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Circ-RNF13作为一种癌基因，通过circ-RNF13/miR-424-5p/TGIF2 ceRNA通路调控HBV相关肝细胞癌细胞的恶性进展以及HBV的表达和复制。

环状 RNA RNF13 (circ-RNF13; ID: hsa_circ_0067717) 是一种新发现的在乙型肝炎病毒 (HBV) 相关的肝细胞癌 (HCC) 患者中异常上调的环状 RNA。但其作用和机制仍有待进一步阐明。通过实时定量PCR和蛋白质印迹检测circ-RNF13、microRNA (miR)-424-5p和TGFβ诱导因子同源框2 (TGIF2)的表达，并通过双荧光素酶报告基因分析证实它们的相互作用。使用 MTS 测定、集落形成测定、流式细胞术、酶联免疫吸附测定、transwell 测定和异种移植肿瘤模型以及实时定量 PCR 进行功能测定。Circ-RNF13 在 HBV 感染的人 HCC 组织和 HBV 表达细胞（Huh7-HBV 和 Hep3B-HBV）中上调，伴随着 TGIF2 上调和 miR-424-5p 下调。阻断 circ-RNF13 可提高 Huh7-HBV 和 Hep3B-HBV 细胞的凋亡率，但抑制细胞活力、集落形成、迁移和侵袭，同时抑制体内肿瘤生长。此外，Huh7-HBV 和 Hep3B-HBV 细胞中的 circ-RNF13 敲低降低了 HBV DNA 拷贝和乙型肝炎表面抗原 (HBsAg) 和乙型肝炎 e 抗原 (HBeAg) 水平。机制上，circ-RNF13 和 TGIF2 充当 miR-424-5p 的竞争内源性 RNA (ceRNA)。过表达 miR-424-5p 模拟和沉默 miR-424-5p 抵消了体外表达 HBV 的 HCC 细胞中 circ-RNF13 耗竭的影响。一致地，TGIF2 恢复部分消除了 miR-424-5p 在 Huh7-HBV 和 Hep3B-HBV 细胞中上调的作用。