Department of Clinical Laboratory, Affiliated Hospital of Chifeng University, Chifeng, China.
Thorac Cancer. 2022 Aug;13(15):2223-2234. doi: 10.1111/1759-7714.14550. Epub 2022 Jul 5.
The competing endogenous RNA (ceRNA) activity of circular RNAs (circRNAs) has been implicated in the pathogenesis of cancers, including esophageal squamous cell carcinoma (ESCC). Here, we identified the ceRNA mechanism of circ_0000654 regulation in ESCC.
The levels of circ_0000654, E2F transcription factor 3 (E2F3), and microRNA (miR)-375 were gauged by quantitative real-time PCR (qRT-PCR) and western blot. Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and 5-ethynyl-2'-deoxyuridine (EdU) assays. Cell apoptosis was detected by flow cytometry. Cell colony formation was tested by colony formation assay. Dual-luciferase reporter, RNA pull-down and RNA immunoprecipitation (RIP) assays were performed to confirm the direct relationship between miR-375 and circ_0000654 or E2F3. Xenograft model assays were used to evaluate the effect of circ_0000654 in vivo.
Circ_0000654 and E2F3 were upregulated in ESCC. Circ_0000654 depletion enhanced cell apoptosis and hindered cell proliferation and glycolysis in vitro, as well as weakened tumor growth in vivo. Increased expression of E2F3 counteracted the effects of circ_0000654 depletion. Mechanistically, E2F3 was a target of miR-375, and circ_0000654 modulated E2F3 expression through sequestering miR-375. Furthermore, miR-375 upregulation phenocopied circ_0000654 knockdown in inhibiting ESCC progression.
Our findings identify a new circ_0000654/miR-375/E2F3 ceRNA crosstalk for the oncogenic role of circ_0000654 in ESCC and establish a notion that targeting circ_0000654 and its pathways may have the potential to improve ESCC outcome.
环状 RNA（circRNA）的竞争内源性 RNA（ceRNA）活性已被认为与癌症的发病机制有关，包括食管鳞状细胞癌（ESCC）。在这里，我们确定了 circ_0000654 在 ESCC 中的调控 ceRNA 机制。
通过定量实时 PCR（qRT-PCR）和 Western blot 检测 circ_0000654、E2F 转录因子 3（E2F3）和 microRNA（miR）-375 的水平。通过 3-(4,5-二甲基噻唑-2-基）-5-(3-羧基甲氧基苯基）-2-(4-磺苯基）-2H-四唑（MTS）和 5-乙炔基-2'-脱氧尿苷（EdU）测定评估细胞增殖。通过流式细胞术检测细胞凋亡。通过集落形成实验检测细胞集落形成。进行双荧光素酶报告、RNA 下拉和 RNA 免疫沉淀（RIP）实验以证实 miR-375 与 circ_0000654 或 E2F3 之间的直接关系。进行异种移植模型实验以评估 circ_0000654 在体内的作用。
circ_0000654 和 E2F3 在 ESCC 中上调。circ_0000654 耗竭增强了细胞凋亡，抑制了细胞增殖和糖酵解，减弱了体内肿瘤生长。E2F3 的表达增加抵消了 circ_0000654 耗竭的作用。从机制上讲，E2F3 是 miR-375 的靶标，circ_0000654 通过结合 miR-375 调节 E2F3 的表达。此外，miR-375 的上调模拟了 circ_0000654 敲低在抑制 ESCC 进展中的作用。
我们的研究结果确定了一种新的 circ_0000654/miR-375/E2F3 ceRNA 串扰，用于环状 RNA 在 ESCC 中的致癌作用，并建立了一种靶向 circ_0000654 及其途径可能改善 ESCC 结局的概念。
