Regulation of VEGFA, KRAS, and NFE2L2 Oncogenes by MicroRNAs in Head and Neck Cancer

Abstract

Mutations and alterations in the expression of VEGFA, KRAS, and NFE2L2 oncogenes play a key role in cancer initiation and progression. These genes are enrolled not only in cell proliferation control, but also in angiogenesis, drug resistance, metastasis, and survival of tumor cells. MicroRNAs (miRNAs) are small, non-coding regulatory RNA molecules that can regulate post-transcriptional expression of multiple target genes. We aimed to investigate if miRNAs hsa-miR-17-5p, hsa-miR-140-5p, and hsa-miR-874-3p could interfere in VEGFA, KRAS, and NFE2L2 expression in cell lines derived from head and neck cancer (HNC). FADU (pharyngeal cancer) and HN13 (oral cavity cancer) cell lines were transfected with miR-17-5p, miR-140-5p, and miR-874-3p microRNA mimics. RNA and protein expression analyses revealed that miR-17-5p, miR-140-5p and miR-874-3p overexpression led to a downregulation of VEGFA, KRAS, and NFE2L2 gene expression in both cell lines analyzed. Taken together, our results provide evidence for the establishment of new biomarkers in the diagnosis and treatment of HNC.

Keywords: angiogenesis; miR-140-5p; miR-17-5p; miR-874-3p.

Figure 1

TCGA expression data of (a) VEGFA (p = <0.001), (b) KRAS (p = 0.80) and (c) NFE2L2 (p = 0.70) in HNC, comparing normal tissue and primary tumor.

Figure 2

(a). VEGFA gene expression after miR-17-5p, miR-140-5p and miR-874-3p transfection compared to the negative control (RQ = 1). (b) Mean relative quantification (RQ) of VEGFA gene expression in HNC cell lines transfected with miR-17-5p, miR-140-5p, and miR-874-3p. * Significant values.

Figure 3

(a) KRAS gene expression after miR-17-5p, miR-140-5p and miR-874-3p transfection compared to the negative control (RQ = 1). (b) Mean relative quantification (RQ) of KRAS gene expression in HNC cell lines transfected with miR-17-5p, miR-140-5p, and miR-874-3p. * Significant values.

Figure 4

(a) NFE2L2 gene expression after miR-17-5p, miR-140-5p, and miR-874-3p transfection compared to the negative control (RQ = 1). (b) Relative quantification mean (RQ) of NFE2L2 gene expression in HNC cell lines transfected with miR-17-5p, miR-140-5p, and miR-874-3p. * Significant values.

Figure 5

(a) VEGFA protein expression in HNC cancer cell lines expressing miRNAs. (b) KRAS protein expression in HNC cancer cell lines expressing miRNAs. (c) NFE2L2 protein expression in HNC cancer cell lines expressing miRNAs. Abbreviation: MNC, mimic negative control. For the evaluation of protein expression, the images were analyzed and quantified using ImageJ 4.0 software.

Figure 6

Pathways of action of the VEGFA, KRAS, and NFE2L2 genes. VEGFA binds to its membrane receptor VEGFR2 leading to activation of the signaling cascade RAS/RAF. NFE2L2 is regulated by the KEAP1 protein which, when inhibited, increases the levels of NEF2L2 that is released into the cell nucleus. The MAPK pathway is also related to the VEGF, KRAS and NFE2L2 pathway. Adapted: Cuzziol et al., 2020 [35].