Department of Infectious Diseases, People's Hospital of Hanchuan, Hanchuan, Hubei, China.
Bosn J Basic Med Sci. 2021 Oct 1;21(5):555-568. doi: 10.17305/bjbms.2020.5266.
Circular RNA RNF13 (circ-RNF13; ID: hsa_circ_0067717) is newly identified to be abnormally upregulated in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) patients. However, its role and mechanism remain to be further annotated. First of all, real-time quantitative PCR (RT-qPCR) was utilized to examine RNA expression, and circ-RNF13 was upregulated in HBV-infected human HCC tissues and HBV-expressing cells (Huh7-HBV and Hep3B-HBV), accompanied with TGFβ-induced factor homeobox 2 (TGIF2) upregulation and microRNA (miR)-424-5p downregulation. Loss-of-functional experiments were performed using MTS assay, colony formation assay, flow cytometry, enzyme-linked immunosorbent assay, transwell assay, and xenograft tumor model. As a result, blocking circ-RNF13 enhanced the apoptosis rate of Huh7-HBV and Hep3B-HBV cells, but inhibited cell proliferation, colony formation, migration, and invasion in vitro, along with suppressed tumor growth in vivo. Besides, RT-qPCR data showed that 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 could directly interacting with miR-424-5p according to dual-luciferase reporter assay, suggesting that circ-RNF13 and TGIF2 served as competing endogenous RNAs (ceRNAs) for miR-424-5p. Functionally, overexpressing miR-424-5p mimicked and silencing miR-424-5p counteracted the effects of circ-RNF13 depletion in HBV-expressing HCC cells in vitro; TGIF2 restoration partially abrogated the role of miR-424-5p upregulation. In conclusion, circ-RNF13 might sponge 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.
环状 RNA RNF13（circ-RNF13；ID：hsa_circ_0067717）在乙型肝炎病毒（HBV）相关肝细胞癌（HCC）患者中被新鉴定为异常上调。然而，其作用和机制仍有待进一步注释。首先，利用实时定量 PCR（RT-qPCR）检测 RNA 表达，发现 HBV 感染的人 HCC 组织和 HBV 表达细胞（Huh7-HBV 和 Hep3B-HBV）中 circ-RNF13 上调，同时 TGFβ诱导因子同源盒 2（TGIF2）上调和 microRNA（miR）-424-5p 下调。利用 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进行功能丧失实验。结果表明，阻断 circ-RNF13 可增强 Huh7-HBV 和 Hep3B-HBV 细胞的凋亡率，但可抑制细胞增殖、集落形成、迁移和侵袭体外，同时抑制体内肿瘤生长。此外，RT-qPCR 数据显示，circ-RNF13 敲低可减少 Huh7-HBV 和 Hep3B-HBV 细胞中的 HBV DNA 拷贝数以及乙型肝炎表面抗原（HBsAg）和乙型肝炎 e 抗原（HBeAg）水平。机制上，根据双荧光素酶报告基因测定，circ-RNF13 和 TGIF2 可与 miR-424-5p 直接相互作用，表明 circ-RNF13 和 TGIF2 作为 miR-424-5p 的竞争性内源 RNA（ceRNA）。功能上，过表达 miR-424-5p 模拟和沉默 miR-424-5p 可逆转 circ-RNF13 缺失在 HBV 表达 HCC 细胞中的作用体外；TGIF2 恢复部分消除了 miR-424-5p 上调的作用。总之，circ-RNF13 可能通过调节 TGIF2 来吸附 miR-424-5p 以抑制 HBV 相关 HCC 细胞的恶性进展和 HBV 感染，为 HBV 相关 HCC 的发生和治疗提供了新的见解。
