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. 2020 Dec 12;8(12):599.
doi: 10.3390/biomedicines8120599.

Regulation of Oncogenic Targets by the Tumor-Suppressive miR-139 Duplex (miR-139-5p and miR-139-3p) in Renal Cell Carcinoma

Reona Okada  1 , Yusuke Goto  1   2 , Yasutaka Yamada  1   2 , Mayuko Kato  1   2 , Shunichi Asai  1 , Shogo Moriya  3 , Tomohiko Ichikawa  2 , Naohiko Seki  1
Affiliations

Regulation of Oncogenic Targets by the Tumor-Suppressive miR-139 Duplex (miR-139-5p and miR-139-3p) in Renal Cell Carcinoma

Reona Okada et al. Biomedicines. .

Abstract

We previously found that both the guide and passenger strands of the miR-139 duplex (miR-139-5p and miR-139-3p, respectively) were downregulated in cancer tissues. Analysis of TCGA datasets revealed that low expression of miR-139-5p (p < 0.0001) and miR-139-3p (p < 0.0001) was closely associated with 5-year survival rates of patients with renal cell carcinoma (RCC). Ectopic expression assays showed that miR-139-5p and miR-139-3p acted as tumor-suppressive miRNAs in RCC cells. Here, 19 and 22 genes were identified as putative targets of miR-139-5p and miR-139-3p in RCC cells, respectively. Among these genes, high expression of PLXDC1, TET3, PXN, ARHGEF19, ELK1, DCBLD1, IKBKB, and CSF1 significantly predicted shorter survival in RCC patients according to TCGA analyses (p < 0.05). Importantly, the expression levels of four of these genes, PXN, ARHGEF19, ELK1, and IKBKB, were independent prognostic factors for patient survival (p < 0.05). We focused on PXN (paxillin) and investigated its potential oncogenic role in RCC cells. PXN knockdown significantly inhibited cancer cell migration and invasion, possibly by regulating epithelial-mesenchymal transition. Involvement of the miR-139-3p passenger strand in RCC molecular pathogenesis is a new concept. Analyses of tumor-suppressive-miRNA-mediated molecular networks provide important insights into the molecular pathogenesis of RCC.

Keywords: miR-139-3p; miR-139-5p; microRNA; paxillin (PXN); renal cell carcinoma (RCC); tumor suppressor.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The expression and clinical significance of miR-139-5p and miR-139-3p in renal cell carcinoma (RCC) clinical specimens. (A) Expression of miR-139-5p and miR-139-3p were significantly reduced in The Cancer Genome Atlas—Kidney Renal Clear Cell Carcinoma (TCGA-KIRC) cancer specimens compared with adjacent normal specimens (p < 0.001). (B) Spearman’s rank test showed positive correlations between the expression levels of miR-139-5p and miR-139-3p in TCGA-KIRC clinical specimens (R = 0.8278, p < 0.001). (C) Kaplan–Meier survival curves of patients from TCGA-KIRC cohort. Patients were divided into two groups according to the median expression levels of miR-139-5p or miR-139-3p: high- and low-expression groups. Both miR-139-5p and miR-139-3p expression levels were significantly associated with the 5-year survival rate of RCC patients (p < 0.0001).
Figure 2
Figure 2
Functional assays of cell proliferation, migration, and invasion following ectopic expression of miR-139-5p and miR-139-3p in RCC cell lines A498 and 786-O. N.S.: not significant. (A) Cell proliferation was assessed using XTT assays. Data were collected 72 h after miRNA transfection. (B) Cell migration was assessed using wound healing assays. (C) Cell invasions were determined 48 h after seeding miRNA-transfected cells into chambers using Matrigel invasion assays.
Figure 3
Figure 3
Clinical significance of miR-139-5p or miR-139-3p target genes in TCGA-KIRC database. High expression of two miR-139-5p target genes (PLXDC1 and TET3) and six miR-139-3p target genes (PXN, ARHGEF19, ELK1, CSF1, IKBKB, and DCBLD1) were significantly associated with poor prognosis in patients with RCC. Kaplan–Meier curves for 5-year overall survival according to the expression of each miR-139-3p target gene are shown.
Figure 4
Figure 4
Forest plot showing the results of multivariate analyses of eight genes (PLXDC1, TET3, PXN, ARHGEF19, ELK1, CSF1, IKBKB, and DCBLD1). In addition to the gene expression level, the tumor stage, pathological grade, and age group were evaluated as potential independent factors associated with survival. Numbers of cases per each group are shown in Table S2.
Figure 5
Figure 5
Expression levels of miR-139-5p or miR-139-3p target genes (PLXDC1 and TET3 for miR-139-5p, PXN, ARHGEF19, ELK1, CSF1, IKBKB, and DCBLD1 for miR-139-3p) in TCGA-KIRC cohort. Seven genes (PLXDC1, TET3, PXN, ELK1, CSF1, IKBKB, and DCBLD1) were confirmed to be significantly upregulated in clinical specimens. N.S.: not significant.
Figure 6
Figure 6
Expression of paxillin (PXN) was regulated directly by miR-139-3p in RCC cells. (A) Expression of PXN mRNA was significantly suppressed in miR-139-3p-transfected A498 and 786-O cells (48 h after transfection). Expression of GAPDH was used as an internal control. (B) Expression of PXN protein was reduced in miR-139-3p-transfected RCC cells (48 h after transfection). GAPDH was used as a loading control. (C) The TargetScanHuman database predicted one putative miR-139-3p-binding site in the 3’-UTR of PXN. (D) Dual-luciferase reporter assays showed decreased luminescence activity in RCC cells co-transfected with miR-139-3p together with a vector harboring the “wild-type sequence”. Normalized data were calculated as Renilla/firefly luciferase activity ratios. N.S.: not significant.
Figure 7
Figure 7
Effects of PXN knockdown on cell proliferation, migration, and invasion in RCC cells. (A,B) Expression of PXN was successfully reduced after siRNA transfection in RCC cells. (C) Cell proliferation was determined by XTT assays. N.S.: not significant. (D) Cell migration was determined by wound healing assays. (E) Cell invasion was determined by Matrigel invasion assays.
Figure 8
Figure 8
Pathways enriched among the differentially expressed genes in the high PXN expression group according to gene set enrichment analysis. The six significantly enriched pathways (FDR q-value < 0.05) are shown.
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