Department of Dermatology, Shanghai Skin Disease Hospital, No. 1278 Baode Road, Jing'an District, Shanghai, 200443, China.
Mol Cell Biochem. 2020 Dec;475(1-2):227-237. doi: 10.1007/s11010-020-03875-8. Epub 2020 Aug 24.
Many studies confirm that circular RNA (circRNA) plays an important regulatory role in the malignant progression of cancer, including melanoma. However, the role of a novel circRNA, circ_0079593, in melanoma is unclear. The expression levels of circ_0079593 and miR-516b were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was measured by cell counting kit-8 (CCK-8) assay, and cell migration and invasion were evaluated using transwell assay. Meanwhile, western blot (WB) analysis was employed to determine the levels of proliferation and metastasis-related proteins, as well as metabotropic glutamate receptor 3 (GRM3) protein. Furthermore, cell apoptosis was tested by detecting the cell apoptosis rate and Caspase-3 activity. The glucose consumption and lactate production of cells were measured to evaluate cell glucose metabolism. Moreover, dual-luciferase reporter assay and biotin-labeled RNA pull-down assay were used to confirm the interaction between miR-516b and circ_0079593 or GRM3. In addition, mice xenograft models were constructed to explore the effect of circ_0079593 on melanoma tumor growth in vivo. Our results discovered that circ_0079593 was highly expressed in melanoma, and its silencing suppressed melanoma cell proliferation, migration, invasion, glucose metabolism and promoted apoptosis. Moreover, we found that circ_0079593 could serve as a sponge of miR-516b, and miR-516b could target GRM3 in melanoma. The rescue experiments revealed that both miR-516b inhibitor and GRM3 overexpression could reverse the inhibition effect of circ_0079593 knockdown on melanoma progression. Additionally, in vivo experiments also revealed that circ_0079593 interference suppressed melanoma tumor growth. Our study concluded that circ_0079593 accelerated melanoma progression via upregulating GRM3 by sponging miR-516b, which suggested that circ_0079593 had the potential to be a new therapeutic biomarker for melanoma.
许多研究证实,环状 RNA(circRNA)在癌症的恶性进展中发挥着重要的调节作用,包括黑色素瘤。然而,circ_0079593 这种新型 circRNA 在黑色素瘤中的作用尚不清楚。通过实时定量聚合酶链反应(qRT-PCR)检测 circ_0079593 和 miR-516b 的表达水平。通过细胞计数试剂盒-8(CCK-8)测定细胞增殖,通过 Transwell 测定细胞迁移和侵袭。同时,通过 Western blot(WB)分析测定增殖和转移相关蛋白以及代谢型谷氨酸受体 3(GRM3)蛋白的水平。此外,通过检测细胞凋亡率和 Caspase-3 活性来检测细胞凋亡。通过测量细胞葡萄糖代谢来评估细胞葡萄糖消耗和乳酸生成。此外,使用双荧光素酶报告基因检测和生物素标记 RNA 下拉实验来验证 miR-516b 与 circ_0079593 或 GRM3 之间的相互作用。此外,构建了小鼠异种移植模型,以探索 circ_0079593 在体内对黑色素瘤肿瘤生长的影响。我们的研究结果表明,circ_0079593 在黑色素瘤中高表达,其沉默抑制黑色素瘤细胞增殖、迁移、侵袭、葡萄糖代谢并促进凋亡。此外,我们发现 circ_0079593 可以作为 miR-516b 的海绵,miR-516b 可以在黑色素瘤中靶向 GRM3。挽救实验表明,miR-516b 抑制剂和 GRM3 过表达均可逆转 circ_0079593 敲低对黑色素瘤进展的抑制作用。此外,体内实验还表明,circ_0079593 干扰抑制了黑色素瘤肿瘤的生长。我们的研究得出结论,circ_0079593 通过海绵吸附 miR-516b 而上调 GRM3 加速了黑色素瘤的进展,这表明 circ_0079593 有可能成为黑色素瘤的新治疗生物标志物。