Suppressive role of microRNA-130b-3p in ferroptosis in melanoma cells correlates with DKK1 inhibition and Nrf2-HO-1 pathway activation

Abstract

Cell death pathways related to ferroptosis are implicated in the progression of melanoma. Emerging data reporting the upregulation of microRNA (miR)-130b-3p in melanoma indicate the potential implication of miR-130b-3p in this malignancy. Herein, we aimed to identify whether and how miR-130b-3p regulated ferroptosis in melanoma cells. Melanoma cells (A375, G-361) were treated with erastin or RSL3 to mimic ferroptosis in vitro. Viability, lipid peroxidation level and ferrous ion content in melanoma cells were then assessed in response to manipulation of miR-130b-3p expression. Luciferase assay was conducted to determine the binding of miR-130b-3p to Dickkopf1 (DKK1). Western blot assay was conducted to determine the expression of molecules related to nuclear factor-erythroid 2 p45-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) pathway. The results indicated that miR-130b-3p exerted an inhibitory role in erastin or RSL3-induced ferroptosis, evidenced by reductions in lipid peroxidation and ferrous ion content. By suppressing the expression of target gene DKK1, miR-130b-3p activated the Nrf2/HO-1 pathway, whereby repressing ferroptosis. miR-130b-3p blocked the antitumor activity of erastin. Further, in vitro findings were reproduced in an in vivo murine model. Together, these data suggest the potential of miR-130b-3p to inhibit ferroptosis in melanoma cells and the mechanism was related to DKK1-mediated Nrf2/HO-1 pathway.

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Funding

This study was supported by Hunan Provincial Education Department Project (20C1125); Doctor’s Fund Project of Hunan Provincial People’s Hospital (BSJJ202008); Changsha Municipal Science and Technology Bureau Natural Science Foundation Project (No kq2014202).

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Correspondence to Hao Feng or An Wei.

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The authors declare they have no conflict of interest.

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The current study was performed with the approval of the Animal Ethics Committee of Hunan Provincial People’s Hospital (The First Affiliated Hospital of Hunan Normal University). The animal experiments were conducted strictly obeying the guidelines for the care and use of laboratory animals issued by the National Institutes of Health.

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Supplementary Information

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13577_2021_557_MOESM1_ESM.eps

Supplementary Figure S1 miR-130b-3p is up-regulated in melanoma cells. miR-130b-3p expression in a series of melanoma cell lines (A375, G361, HS1-CLS, MEL-CLS1-4, IGR-1, MEWO, NIS-G, WS1-CLS, and MML1) and a normal skin cell line PIG1. (EPS 446 KB)

13577_2021_557_MOESM2_ESM.eps

Supplementary Figure S2 Representative images of flow cytometry evaluation of the level of lipid ROS using BODIPY-C11 probe after miR-130b-3p overexpression in A375 and G-361 cells treated with erastin for 24 hours or RSL3. The cells were treated with erastin (5 μM in A375 and 10 μM in G-361) or RSL3 (0.1 μM in A375 and 0.5 μM in G-361) for 24 hours. (EPS 1216 KB)

13577_2021_557_MOESM3_ESM.eps

Supplementary Figure S3 Representative images of protein blots and quantitative analysis in Western blot analysis of DKK1 expression in response to miR-130b-3p overexpression alone or in combination with DKK1 overexpression. (EPS 1576 KB)

13577_2021_557_MOESM4_ESM.eps

Supplementary Figure S4 Representative images of flow cytometry evaluation of the level of lipid ROS in A375 cells labeled with C11-BODIPY probe in response to miR-130b-3p overexpression alone or in combination with DKK1 overexpression. (EPS 1232 KB)

13577_2021_557_MOESM5_ESM.eps

Supplementary Figure S5 miR-130b-3p affects tumorigenesis in vivo. Detection of the volume of xenografted tumors in the presence of miR-130b-3p overexpression/inhibition (*p < 0.05). (EPS 477 KB)

Supplementary file6 (DOCX 18 KB)

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Liao, Y., Jia, X., Ren, Y. et al. Suppressive role of microRNA-130b-3p in ferroptosis in melanoma cells correlates with DKK1 inhibition and Nrf2-HO-1 pathway activation. Human Cell (2021). https://doi.org/10.1007/s13577-021-00557-5

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Keywords

  • Melanoma
  • microRNA-130b
  • DKK1
  • Nrf2/HO-1 pathway
  • Ferroptosis
  • Lipid peroxidation
  • Iron accumulation