Overexpression of long non-coding RNA NORAD promotes invasion and migration in malignant melanoma via regulating the MIR-205-EGLN2 pathway
Overexpression of long non-coding RNA NORAD promotes invasion and migration in malignant melanoma via regulating the MIR-205-EGLN2 pathway
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长链非编码RNA NORAD的过度表达通过调节MIR-205-EGLN2通路促进恶性黑色素瘤的侵袭和迁移
DOI：
10.1002/cam4.2046
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发表时间：
2019-04-01
期刊：
影响因子：
4
通讯作者：
Liu, Yan
中科院分区：
文献类型：
--
作者：
Chen, Yong;Cao, Ke;Liu, Yan
越来越多的证据表明，长链非编码RNA NORAD和miR-205在调节癌症进展和转移中起着重要作用。本研究首次在黑色素瘤组织和人恶性黑色素瘤细胞系中观察到NORAD的高表达，旨在研究NORAD在恶性黑色素瘤细胞侵袭和迁移过程中的相互作用。通过qRT-PCR检测MM细胞中NORAD、miR-205和EGLN 2的mRNA水平。采用原位杂交（ISH）技术检测NORAD在MM组织标本中的表达。Western blot检测NORAD和miR-205对MM细胞中脯氨酰羟化酶2（EGLN 2）表达的影响。采用双荧光素酶报告基因检测NORAD与miR-205以及miR-205与EGLN 2的相互作用关系。采用Transwell法检测NORAD和miR-205对体外培养的人胃癌细胞的影响。裸鼠移植瘤实验证实NORAD和miR-205在体内的作用。体外实验中，NORAD基因敲低可显著抑制恶性黑色素瘤细胞的迁移和侵袭，并上调miR-205的表达，在RNA诱导沉默复合物中，miR-205与NORAD之间存在相互作用。与乱序对照相比，miR-205的上调诱导了迁移和侵袭能力的显著抑制。然而，下调NORAD在很大程度上逆转了这种影响。此外，miR-205对EGLN 2水平的调节作用和内质网应激的诱导被NORAD逆转。在体内，miR-205的缺失在裸鼠中诱导肿瘤生长。NORAD可能通过调控miR-205-EGLN 2通路在恶性黑色素瘤的发生、发展中发挥重要作用，有望成为新的治疗靶点。
Growing evidence suggests that long non-coding RNAs NORAD and miR-205 play a significant role in regulating cancer progression and metastasis. In this study, high expression of NORAD was firstly observed in melanoma tissues and human malignant melanoma cell lines, our aim was to study the interaction of them in the process of invasion and migration of malignant melanoma cells. NORAD, miR-205, and EGLN2 mRNA level in MM cells was detected by qRT-PCR. In situ hybridization (ISH) was performed to detect NORAD expression in MM tissues specimens. Effects of NORAD and miR-205 on Prolyl hydroxylase 2 (EGLN2) expression was explored by western blot in MM cells line. Dual-luciferase reporter assay was performed to verify the interaction relationship between NORAD and miR-205, as well as, miR-205 and EGLN2. Transwell assay was conducted to explore the effects of NORAD and miR-205 in vitro. Xenografts in nude mice experiment were used to confirm the role of NORAD and miR-205 in vivo. In vitro, NORAD knockdown significantly inhibited migration and invasion of malignant melanoma cells and elevated the expression of miR-205, there was an interaction between miR-205 and NORAD in the RNA-induced silencing complex. Upregulation of miR-205 induced significant inhibition of migratory and invasive ability compared with the scrambled control. However, downregulating NORAD largely reversed this effect. Furthermore, the regulatory effects of miR-205 on EGLN2 levels and the induction of endoplasmic reticulum stress were reversed by NORAD. In vivo, deletion of miR-205 induced tumor growth in nude mice. NORAD may play critical roles in tumorigenesis and progression of malignant melanoma by regulating of the miR-205-EGLN2 pathway, and may serve as a new therapeutic target.