Yang Chao, Yan Zeqiang, Hu Fen, Wei Wei, Sun Zhihua, Xu Wei
1Department of Oncology, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, No. 136, Jingzhou Street, Xiangcheng District, Xiangyang, 441021 Hubei People's Republic of China.
2Department of Gastroenterology, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, Xiangyang, 441021 People's Republic of China.
Cancer Cell Int. 2020 Jan 29;20:32. doi: 10.1186/s12935-019-1064-y. eCollection 2020.
Melanoma is notoriously resistant to current treatments, and less than 25% of metastatic melanoma cases respond to existing therapies. Growing evidence has shown that microRNAs (miRNAs) play a vital role in the prognosis of melanoma. MiR-517a has been implicated in many types of cancer; however, its expressional features and potential biological functions in melanoma remain unclear. The present study aimed to investigate the possible effects of miR-517a on oxidative stress (OS) in melanoma cells.
miR-517a expression in melanoma was determined using RT-qPCR. After treatment with different concentrations of HO, cell viability was determined in order to identify the most appropriate HO concentration. Through loss and gain of function experiments, the interactions between miR-517a, the cyclin dependent kinase inhibitor 1C (CDKN1C) and the c-Jun NH2-terminal kinase (JNK) signaling pathway, as well as their roles in OS of melanoma cells were identified. Moreover, the expression of Cleaved Caspase-3, extent of ERK1/2 phosphorylation, Bax/Bcl-2 ratio, levels of T-AOC, ROS and MDA, and SOD activity were also tested. Finally, melanoma cell viability and apoptosis were detected.
MiR-517a was upregulated, while CDKN1C was downregulated in melanoma tissues and cells. MiR-517a targets CDKN1C and consequently reduced its expression. Inhibition of miR-517a was shown to increase Cleaved Caspase-3 expression, Bax/Bcl-2 ratio, levels of ROS and MDA, as well as cell apoptosis but decrease extent of ERK1/2 phosphorylation, T-AOC levels, SOD activity, along with cell proliferation and mitochondrial membrane potential.
Overall, silencing miR-517a results in upregulated CDKN1C expression, and inhibited JNK signaling pathway activation, consequently promoting OS in melanoma cells.
黑色素瘤对当前治疗具有显著抗性，转移性黑色素瘤病例中对现有疗法有反应的不到25%。越来越多的证据表明，微小RNA（miRNA）在黑色素瘤的预后中起着至关重要的作用。MiR-517a已被证明与多种类型的癌症有关；然而，其在黑色素瘤中的表达特征和潜在生物学功能仍不清楚。本研究旨在探讨miR-517a对黑色素瘤细胞氧化应激（OS）的可能影响。
使用RT-qPCR测定黑色素瘤中miR-517a的表达。在用不同浓度的HO处理后，测定细胞活力以确定最合适的HO浓度。通过功能丧失和功能获得实验，确定了miR-517a、细胞周期蛋白依赖性激酶抑制剂1C（CDKN1C）和c-Jun氨基末端激酶（JNK）信号通路之间的相互作用，以及它们在黑色素瘤细胞OS中的作用。此外，还检测了Cleaved Caspase-3的表达、ERK1/2磷酸化程度、Bax/Bcl-2比值、总抗氧化能力（T-AOC）、活性氧（ROS）和丙二醛（MDA）水平以及超氧化物歧化酶（SOD）活性。最后，检测了黑色素瘤细胞的活力和凋亡情况。
在黑色素瘤组织和细胞中，MiR-517a上调，而CDKN1C下调。MiR-517a靶向CDKN1C并因此降低其表达。抑制miR-517a可增加Cleaved Caspase-3表达、Bax/Bcl-2比值、ROS和MDA水平以及细胞凋亡，但降低ERK1/2磷酸化程度、T-AOC水平、SOD活性，以及细胞增殖和线粒体膜电位。
总体而言，沉默miR-517a导致CDKN1C表达上调，并抑制JNK信号通路激活，从而促进黑色素瘤细胞中的OS。
