Melanoma is a type of aggressive skin cancer with a poor survival rate. The resistance to conventional therapy of this disease is, at least in part, attributed to its cancer stem cell population. However, the mechanism of survival and stemness maintenance of cancer stem cells remains to be investigated. Tumorsphere formation assay was used to study the stem-like property of melanoma stem-like cells (MSLC). Chromatin immunoprecipitation (ChIP), promoter luciferase reporter assay were included for exploring the role of MCL-1 in MSLC and electrophoretic mobility shift assay were used to evaluate the interaction between shrimp miR-965 and human Ago2 protein. Melanoma xenograft nude mice were used to study the inhibition of tumor development. In the present study, our results showed that myeloid cell leukemia sequence 1 (MCL-1) knocking down induced ER stress and apoptosis, and the expression reduction of stemness associated genes in MSLC, which implied a significant role of MCL-1 in MSLC. Further study indicated that ER stress agonist (tunicamycin) treatment in MSLC results in the translocation of XBP1, an ER stress sensor, into the nucleus to induce MCL-1 expression through direct binding to the − 313- to − 308-bp region of MCL-1 promoter. In addition, we found that a shrimp-derived miRNA (shrimp miR-965) could interact with the human Ago2 protein and suppressed the human MCL-1 expression by binding to the 3′ UTR of MCL-1 mRNA, thereby inhibiting the MSLC proliferation and stemness in vitro and in vivo in a cross-species manner. In conclusion, we identified an important role of MCL-1-ER stress-XBP1 feedback loop in the stemness and survival maintenance of MSLC, and shrimp miR-965, a natural food derived miRNA, could regulate MSLC stemness and survival by targeting MCL-1 and disrupting the balance of MCL-1-ER stress-XBP1 feedback loop. In conclusion, this study indicated an important mechanism of the regulation of MSLC stemness and survival, otherwise it also demonstrated the significance of cross-species-derived miRNA as promising natural drugs in melanoma therapy.

中文翻译：

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虾miR-965通过跨物种方式破坏MCL-1-ER应激-XBP1反馈环路，诱导人黑色素瘤干细胞样细胞凋亡并抑制其干细胞性。

黑色素瘤是一种侵袭性皮肤癌，存活率很低。这种疾病对传统疗法的抵抗至少部分归因于其癌症干细胞群。然而，癌症干细胞的存活和干性维持机制仍有待研究。肿瘤球形成实验用于研究黑色素瘤干细胞样细胞（MSLC）的干细胞特性。染色质免疫沉淀 (ChIP)、启动子荧光素酶报告基因测定用于探索 MCL-1 在 MSLC 中的作用，电泳迁移率变动测定用于评估虾 miR-965 与人 Ago2 蛋白之间的相互作用。使用黑色素瘤异种移植裸鼠来研究对肿瘤发展的抑制作用。在本研究中，我们的结果表明，骨髓细胞白血病序列1（MCL-1）敲低可诱导内质网应激和细胞凋亡，以及MSLC中干性相关基因的表达减少，这表明MCL-1在MSLC中发挥着重要作用。进一步的研究表明，MSLC 中的 ER 应激激动剂（衣霉素）治疗会导致 ER 应激传感器 XBP1 易位到细胞核中，通过直接结合到 MCL 的 − 313- 至 − 308-bp 区域来诱导 MCL-1 表达-1个启动子。此外，我们发现虾源性miRNA（虾miR-965）可以与人Ago2蛋白相互作用，并通过与MCL-1 mRNA的3'UTR结合来抑制人MCL-1表达，从而抑制MSLC增殖以及跨物种方式的体外和体内干性。总之，我们确定了 MCL-1-ER 应激 XBP1 反馈环路在 MSLC 干性和生存维持中的重要作用，虾 miR-965（一种天然食品来源的 miRNA）可以通过靶向 MCL-1-ER 应激 XBP1 反馈环来调节 MSLC 干性和生存。 1 并破坏 MCL-1-ER 应激-XBP1 反馈环路的平衡。总之，这项研究表明了调节 MSLC 干性和存活的重要机制，同时也证明了跨物种来源的 miRNA 作为有前景的天然药物在黑色素瘤治疗中的重要性。