Department of Burns & Skin Wounds Repair Center, The Third Hospital of Wuhan, Wuhan, 430070, Hubei, People's Republic of China.
Department of Plastic & Cosmetic Surgery, Tongji Medical College Hospital, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, People's Republic of China.
Mol Biotechnol. 2023 Oct;65(10):1619-1631. doi: 10.1007/s12033-023-00672-8. Epub 2023 Feb 3.
In this study, we aimed to assess the biological functions of HAGLR and its underlying mechanisms in melanoma. HAGLR and ASB11 were knocked down by transfection with the corresponding siRNAs. Meanwhile, miR-4644 was downregulated using the miR-4644 inhibitor treatment. The target interactions among the three molecules were demonstrated using dual-luciferase reporter and RNA immunoprecipitation assays. The levels of HAGLR, miR-4644, and ASB11 in melanoma cells and tissues were assessed using quantitative real‑time PCR and western blotting. The functions and mechanisms underlying HAGLR action in melanoma progression were examined using Cell Counting Kit-8, Transwell, Caspase-3 activity, and xenograft tumor formation assays. HAGLR and ASB11 expression were elevated, whereas that of miR-4644 was downregulated in melanoma cells and tissues. The viability and migration of melanoma cells (A875 and A375) were markedly suppressed by the knockdown of HAGLR and ASB11 but promoted following miR-4644 inhibitor transfection. In contrast, apoptosis showed the opposite trend. In vivo, tumor weight declined considerably with downregulation of HAGLR. Mechanistically, HAGLR sponges miR-4644, increasing the levels of ASB11 and further aggravating melanoma. It latter negatively targets ASB11 in melanoma cells. Hence, the HAGLR-miR-4644-ASB11 axis may be a promising target for melanoma treatment.
在这项研究中，我们旨在评估 HAGLR 的生物学功能及其在黑色素瘤中的潜在机制。通过转染相应的 siRNA 来敲低 HAGLR 和 ASB11。同时，使用 miR-4644 抑制剂处理来下调 miR-4644。使用双荧光素酶报告和 RNA 免疫沉淀测定来证明这三个分子之间的靶相互作用。使用定量实时 PCR 和 Western blot 来评估黑色素瘤细胞和组织中的 HAGLR、miR-4644 和 ASB11 水平。使用细胞计数试剂盒-8、Transwell、Caspase-3 活性和异种移植肿瘤形成测定来检查 HAGLR 在黑色素瘤进展中的作用及其机制。HAGLR 和 ASB11 的表达在黑色素瘤细胞和组织中上调，而 miR-4644 的表达下调。HAGLR 和 ASB11 的敲低显著抑制黑色素瘤细胞（A875 和 A375）的活力和迁移，但 miR-4644 抑制剂转染后促进其活力和迁移。相反，细胞凋亡呈现相反的趋势。在体内，下调 HAGLR 后肿瘤重量明显下降。机制上，HAGLR 海绵吸附 miR-4644，增加 ASB11 的水平，进一步加重黑色素瘤。随后它在黑色素瘤细胞中负向靶向 ASB11。因此，HAGLR-miR-4644-ASB11 轴可能是黑色素瘤治疗的一个有前途的靶点。
