Jin Canhui, Wang Aihong, Liu Linbo, Wang Gongping, Li Guangshuai, Han Zhaofeng
Department of Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China.
Department of Gynecologic Oncology, The First Affiliated Hospital, and College of Clinical Medicine of Henan University of Science and Technology, Luoyang, Henan, China.
J Cell Biochem. 2019 Jul;120(7):11115-11126. doi: 10.1002/jcb.28388. Epub 2019 Jan 30.
Compelling evidence shows that deregulated microRNAs (miRNAs) are important regulators in the progression of melanoma. miR-145-5p has been suggested to exhibit antitumorigenic activity in melanoma. However, the molecular mechanism underlying the biological activity of miR-145-5p in melanoma remains to be further understood. Herein, quantitative real-time polymerase chain reaction was used to examine the miR-145-5p expression in malignant melanoma tissues and cells. The interaction between miR-145-5p and toll-like receptor 4 (TLR4) was explored by bioinformatics analyses, luciferase reporter assay, and Western blot. The effects of miR-145-5p or combined with TLR4 on cell proliferation, colony formation, migration, and invasion abilities were investigated by (4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, colony formation, wound healing, and transwell assays, respectively. The melanoma xenograft tumor models were established to determine the biological activity of miR-145-5p in melanoma in vivo. In addition, the changes of the nuclear factor kappa B (NF-κB) pathway were analyzed by detecting the NF-κB activity and the NF-κB p65 protein level. We observed that the miR-145-5p expression was underexpressed in melanoma tissues and cells. miR-145-5p suppressed the TLR4 expression by binding to its 3'untranslated region in melanoma cells. Moreover, TLR4 overexpression abolished the inhibition of cell proliferation, colony formation, migration, and invasion abilities induced by miR-145-5p in melanoma cells. Meanwhile, miR-145-5p was confirmed to restrain melanoma tumor growth in vivo by targeting TLR4. Furthermore, miR-145-5p overexpression inactivated the NF-κB pathway in melanoma in vitro and in vivo, which was reversed by TLR4 overexpression. We concluded that miR-145-5p hindered the occurrence and metastasis of melanoma cells in vitro and in vivo by targeting TLR4 via inactivation of the NF-κB pathway.
有力证据表明，失调的微小RNA（miRNA）是黑色素瘤进展中的重要调节因子。有人提出miR-145-5p在黑色素瘤中具有抗肿瘤活性。然而，miR-145-5p在黑色素瘤中生物学活性的分子机制仍有待进一步了解。在此，采用定量实时聚合酶链反应检测恶性黑色素瘤组织和细胞中miR-145-5p的表达。通过生物信息学分析、荧光素酶报告基因检测和蛋白质免疫印迹法探讨miR-145-5p与Toll样受体4（TLR4）之间的相互作用。分别采用噻唑蓝（MTT）法、集落形成实验、伤口愈合实验和Transwell实验研究miR-145-5p或联合TLR4对细胞增殖、集落形成、迁移和侵袭能力的影响。建立黑色素瘤异种移植瘤模型，以确定miR-145-5p在黑色素瘤体内的生物学活性。此外，通过检测核因子κB（NF-κB）活性和NF-κB p65蛋白水平，分析NF-κB信号通路的变化。我们观察到miR-145-5p在黑色素瘤组织和细胞中表达下调。miR-145-5p通过与黑色素瘤细胞中TLR4的3'非翻译区结合来抑制TLR4的表达。此外，TLR4过表达消除了miR-145-5p对黑色素瘤细胞增殖、集落形成、迁移和侵袭能力的抑制作用。同时，证实miR-145-5p通过靶向TLR4在体内抑制黑色素瘤肿瘤生长。此外，miR-145-5p过表达在体外和体内均使黑色素瘤中的NF-κB信号通路失活，而TLR4过表达可逆转这种失活。我们得出结论，miR-145-5p通过靶向TLR4使NF-κB信号通路失活，从而在体外和体内阻碍黑色素瘤细胞的发生和转移。
