Department of Hematology and Oncology (Key Discipline of Jiangsu Medicine), Medical School, Zhongda Hospital, Southeast University, Dingjiaqiao No.87, Gulou District, 210009 Nanjing, Jiangsu, Nanjing, People's Republic of China.
Interventional Vascular Department, Jianhu People's Hospital, Yancheng, Jiangsu, China.
BMC Cancer. 2022 Dec 7;22(1):1284. doi: 10.1186/s12885-022-10367-z.
Circular RNAs (circRNAs) are enriched in exosomes and are extremely stable. Exosome-mediated intercellular transfer of specific biologically active circRNA molecules can drive the transformation of the tumor microenvironment and accelerate or inhibit the local spread and multifocal growth of hepatocellular carcinoma (HCC). In this study, we explored in depth about the biological roles of HCC cell-derived exosomes and exosome-transported circRNAs on HCC in vivo and in vitro.
Exosomes extracted from HCC cells (Huh7 and HA22T) were characterized using transmission electron microscopy, nanoparticle size tracer analysis, and western blotting. Exosomes were observed for endocytosis using fluorescent labeling. The effects of HCC cell-derived exosomes and the circ_002136 they carried on cell growth, metastasis and apoptosis were determined by CCK-8 assay, transwell assay, flow cytometry analysis and TUNEL staining, respectively. The expressions of circ_002136, miR-19a-3p and RAB1A were detected by quantitative RT-PCR (qRT-PCR). Targeted binding between miR-19a-3p and circ_002136 or RAB1A was predicted and verified by bioinformatics analysis, dual-luciferase reporter and RNA pull-down experiments. The in vivo effect of circ_002136 was determined by constructing a xenograft tumor model.
The findings revealed that Huh7 and HA22T exosomes conferred enhanced viability as well as invasive ability to recipient HCC cells. Circ_002136 was shown for the first time to be differentially upregulated in HCC tissues and cells and transferred by HCC cell-derived exosomes. More importantly, selective silencing of circ_002136 depleted the malignant biological behaviors of HCC exosome-activated Huh7 and HA22T cells. Depletion of circ_002136 in vivo effectively retarded the growth of HCC xenograft tumors. Furthermore, a well-established circ_002136 ceRNA regulatory network was constructed, namely circ_002136 blocked miR-19a-3p expression, elevated RAB1A expression activity and stimulated HCC development. Finally, high levels of circ_002136 or RAB1A, as well as low levels of miR-19a-3p, negatively affected HCC patient survival.
The study on circ_002136 provides good data to support our insight into the mechanism of to-be-silenced circRNA as a therapeutic agent in the progression of HCC.
环状 RNA（circRNAs）在细胞外囊泡（exosomes）中丰富存在，且极其稳定。外泌体介导的特定具有生物活性的 circRNA 分子的细胞间转移可以驱动肿瘤微环境的转化，并加速或抑制肝细胞癌（HCC）的局部扩散和多灶性生长。在这项研究中，我们深入探讨了 HCC 细胞来源的 exosomes 和外泌体转运的 circRNAs 对 HCC 的体内和体外生物学作用。
通过透射电子显微镜、纳米颗粒大小示踪分析和 Western blot 对来自 HCC 细胞（Huh7 和 HA22T）的 exosomes 进行了表征。通过荧光标记观察 exosomes 的内吞作用。通过 CCK-8 测定、Transwell 测定、流式细胞术分析和 TUNEL 染色分别确定 HCC 细胞来源的 exosomes 及其携带的 circ_002136 对细胞生长、转移和凋亡的影响。通过定量 RT-PCR（qRT-PCR）检测 circ_002136、miR-19a-3p 和 RAB1A 的表达。通过生物信息学分析、双荧光素酶报告基因和 RNA 下拉实验预测和验证 miR-19a-3p 与 circ_002136 或 RAB1A 之间的靶向结合。通过构建异种移植肿瘤模型确定 circ_002136 的体内作用。
研究结果表明，Huh7 和 HA22T exosomes 赋予受体 HCC 细胞更强的活力和侵袭能力。circ_002136 首次在 HCC 组织和细胞中差异上调，并由 HCC 细胞来源的 exosomes 转移。更重要的是，选择性沉默 circ_002136 可耗尽 HCC 外泌体激活的 Huh7 和 HA22T 细胞的恶性生物学行为。体内沉默 circ_002136 可有效抑制 HCC 异种移植瘤的生长。此外，构建了一个成熟的 circ_002136 ceRNA 调控网络，即 circ_002136 阻断 miR-19a-3p 的表达，提高 RAB1A 表达活性，并刺激 HCC 的发展。最后，高水平的 circ_002136 或 RAB1A 以及低水平的 miR-19a-3p 均对 HCC 患者的生存产生负面影响。
对 circ_002136 的研究为我们深入了解作为 HCC 进展治疗剂的沉默 circRNA 的机制提供了良好的数据支持。
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