miR-155 overexpression is followed by downregulation of its target gene, NFE2L2, and altered pattern of VEGFA expression in the liver of melanoma B16-bearing mice at the premetastatic stage

Abstract

MicroRNAs are involved in the control of tumour progression and in metastatic cascade dynamics. However, the role of microRNAs in distant organ reorganization at the premetastatic stage is less clear, although the process of premetastatic niche formation is a crucial event according to modern concepts of tumour dissemination. The role of the present study was to investigate the expression levels of miR-155, miR-21, miR-205 and miR-let7b, as well as that of their target genes, in target organs of melanoma metastasis at the premetastatic stage. The expression levels of both the pro-oncogenic miR-155 and the tumour suppressive miR-205 were found to be altered in the premetastatic liver of melanoma B16-bearing mice. Bioinformatics analysis identified the target genes of miR-155 to be nuclear factor, erythroid 2 like 2 (NFE2L2), secretogranin II, miR-205, semaphorin 5A and vascular endothelial growth factor A (VEGFA). Among those, the redox status regulatory factor NFE2L2 was downregulated, which corresponded to increased levels of miR-155. Due to the ability of pro-oxidative events to initiate angiogenesis, VEGFA levels were evaluated in the premetastatic liver by immunohistochemistry, which revealed increased VEGFA expression in the central parts of the organ and diminished expression in the periphery. Taken together, these findings may support the concept of functional organ reorganization due to melanoma progression.

Keywords: erythroid 2 like 2; melanoma; metastasis; miR-155; miR-205; nuclear factor; vascular endothelial growth factor A.

Figure 1

The expression of miR‐155, miR‐21, miR‐205 and miR‐let7b in the premetastatic liver and lung was examined by RT‐qPCR analysis. Expression levels of (A) miR‐155, (B) miR‐let7b, (C) miR‐21 and (D) miR‐205. Data are presented as the mean ± standard error of the mean. ^* P < .05 and ^** P < .01

Figure 2

The expression levels of SEMA5a, VEGFA, NFE2L2 and Scg2 in the premetastatic liver and lung were examined by RT‐qPCR. Expression levels of (A) SEMA5a, (B) VEGFA, (C) NFE2L2 and (D) Scg2. Data are presented as the mean ± standard error of the mean. ^* P < .05 and ^** P < .01. SEMA5a, semaphorin 5A; VEGFA, vascular endothelial growth factor A; NFE2L2, nuclear factor, erythroid 2 like 2; Scg2, secretogranin II

Figure 3

VEGFA immunostaining in the liver of control and melanoma B16‐bearing mice. A, Negative control (immunohistochemical staining without primary antibodies; magnification, ×200). B, Negative control (immunohistochemical staining without primary antibodies; magnification, ×400). C, Weak staining of VEGFA in endothelial cells in the large median hepatic lobe sinusoids (control group; magnification, ×200). D, Intensive VEGFA staining in endothelial cells in the large median hepatic lobe sinusoids of melanoma B16‐bearing mice (experimental group, magnification, ×200). E, VEGFA staining in endothelial cells of liver peripheral sinusoids (control group; magnification, ×400). F, Weak staining of VEGFA in melanoma B16‐mice endothelial cells of peripheral liver sinusoids (experimental group; magnification, ×400). VEGFA, vascular endothelial growth factor A

Figure 4

miR‐155 and miR‐205 induced abnormal redox state and angiogenesis by targeting NFE2L2 and VEGFA respectively. Schematic diagram illustrating the potential role of miR‐155 and miR‐205, and their target genes, NFE2L2 and VEGFA, in target organ remodelling. NFE2L2, nuclear factor, erythroid 2 like 2; VEGFA, vascular endothelial growth factor A