ABSTRACT

Hepatocellular carcinoma (HCC) is a common type of primary liver cancer. Circular RNAs (circRNAs) have been demonstrated to be a crucial player in multiple cancers. However, a large number of circRNAs remain to be explored. Our study focused on investigating hsa_circ_0004018 in HCC. Firstly, we conducted quantitative reverse transcription PCR (RT-qPCR) to find that circ_0004018 was down-regulated in HCC cells. Western blot analysis was performed to detect the protein levels of phosphatase and tensin homologue (PTEN) and related factors of PI3K/AKT signaling pathway. From the results of functional assays, we found that overexpression of circ_0004018 significantly inhibited the proliferative and migratory capacities of HCC cells. The regulatory mechanism of circ_0004018 in HCC was determined by RNA immunoprecipitation (RIP), RNA pull-down, and luciferase reporter assays, thereby we knew that circ_0004018 regulated PTEN by sequestering microRNA-1197 (miR-1197) to modulate PI3K/AKT signaling pathway. Finally, rescue assays verified that circ_0004018 participated in modulation of cell proliferation and migration in HCC via sponging miR-1197 and regulating PTEN. In conclusion, circ_0004018 suppresses the proliferation and migration of HCC cells via sponging miR-1197 to inactivate the PTEN/PI3K/AKT signaling pathway.

Abbreviations: HCC: Hepatocellular carcinoma; circRNAs: Circular RNAs; PTEN: Phosphatase and tensin homologue; miR-1197: microRNA-1197; ceRNA: competitive endogenous RNA; ATCC: American Type Culture Collection; EMEM: Eagle’s Minimum Essential Medium; RT-qPCR: Quantitative real-time PCR; EdU: 5-ethynyl-20-deoxyuridine; FISH: Fluorescent in situ hybridization; RIP: RNA immunoprecipitation; 3ʹ-UTR: 3ʹ-untranslated region; Wt: wild-type; Mut; mutant type; gDNA: genomic DNA; Act D: Actinomycin D; PI3K: phosphatidylinositol-3-kinase; AKT: protein kinase; lncRNAs: long non-coding RNAs

摘要

肝细胞癌（HCC）是一种常见的原发性肝癌。环状 RNA (circRNA) 已被证明是多种癌症的关键参与者。然而，仍有大量的 circRNA 有待探索。我们的研究重点是调查 HCC 中的 hsa_circ_0004018。首先，我们进行定量逆转录PCR（RT-qPCR）发现circ_0004018在HCC细胞中下调。采用蛋白质印迹法检测磷酸酶和张力蛋白同源物（PTEN）蛋白水平及PI3K/AKT信号通路相关因子。从功能测定的结果中，我们发现circ_0004018的过表达显着抑制了HCC细胞的增殖和迁移能力。circ_0004018 在 HCC 中的调控机制通过 RNA 免疫沉淀 (RIP)、RNA pull-down、和荧光素酶报告基因检测，因此我们知道 circ_0004018 通过隔离 microRNA-1197 (miR-1197) 来调节 PTEN，从而调节 PI3K/AKT 信号通路。最后，救援试验证实 circ_0004018 通过海绵化 miR-1197 和调节 PTEN 参与调节 HCC 中的细胞增殖和迁移。总之，circ_0004018 通过海绵化 miR-1197 使 PTEN/PI3K/AKT 信号通路失活，抑制 HCC 细胞的增殖和迁移。

缩写： HCC：肝细胞癌；circRNAs：环状RNA；PTEN：磷酸酶和张力蛋白同源物；miR-1197：微小RNA-1197；ceRNA：竞争性内源性RNA；ATCC：美国典型培养物保藏中心；EMEM：Eagle 的最低基本培养基；RT-qPCR：定量实时 PCR；EdU：5-乙炔基-20-脱氧尿苷；FISH：荧光原位杂交；RIP：RNA免疫沉淀；3ʹ-UTR：3ʹ-非翻译区；Wt：野生型；穆特; 突变型；gDNA：基因组DNA；D法：放线菌素D；PI3K：磷脂酰肌醇-3-激酶；AKT：蛋白激酶；lncRNA：长链非编码 RNA