Dual tumor-suppressors miR-139-5p and miR-139-3p targeting matrix metalloprotease 11 in bladder cancer

Abstract

Our recent study of the microRNA (miRNA) expression signature of bladder cancer (BC) by deep-sequencing revealed that two miRNA, microRNA-139-5p/microRNA-139-3p were significantly downregulated in BC tissues. The aim of this study was to investigate the functional roles of these miRNA and their modulation of cancer networks in BC cells. Functional assays of BC cells were performed using transfection of mature miRNA or small interfering RNA (siRNA). Genome-wide gene expression analysis, in silico analysis and dual-luciferase reporter assays were applied to identify miRNA targets. The associations between the expression of miRNA and its targets and overall survival were estimated by the Kaplan-Meier method. Gain-of-function studies showed that miR-139-5p and miR-139-3p significantly inhibited cell migration and invasion by BC cells. The matrix metalloprotease 11 gene (MMP11) was identified as a direct target of miR-139-5p and miR-139-3p. Kaplan-Meier survival curves showed that higher expression of MMP11 predicted shorter survival of BC patients (P = 0.029). Downregulated miR-139-5p or miR-139-3p enhanced BC cell migration and invasion in BC cells. MMP11 was directly regulated by these miRNA and might be a good prognostic marker for survival of BC patients.

Keywords: Bladder cancer; MMP11; miR-139; miRNA; tumour suppressors.

Figure 1

The expression levels of miR‐139‐5p and miR‐139‐3p and their effects in BC cells. (a) Expression levels of miR‐139‐5p and miR‐139‐3p in BC tissues and BC cell lines and the correlated expression of miR‐139‐5p and miR‐139‐3p were determined by qRT‐PCR. Data were normalized to RNU48 expression. (b) Cell proliferation was determined by XTT assays 72 h after transfection with 10 nM miR‐139‐5p or miR‐139‐3p (*P < 0.0001). (c) Cell migration activity was determined by wound‐healing assays 48 h after transfection with10 nM miR‐139‐5p or miR‐139‐3p (*P < 0.0001). (d) Cell invasion activity was determined by Matrigel invasion assays 48 h after transfection with 10 nM miR‐139‐5p or miR‐139‐3p (*P < 0.0001; **P < 0.005).

Figure 2

Direct regulation of MMP11 by miR‐139‐5p or miR‐139‐3p (a) MMP11 mRNA expression was evaluated by qRT‐PCR 72 h after transfection with 30 nM miR‐139‐5p and miR‐139‐3p. RNU48 was used as an internal control (*P < 0.0001; **P < 0.001). (b) MMP11 protein expression was evaluated by Western blotting 72 h after transfection with 30 nM miR‐139‐5p and miR139‐3p. GAPDH was used as a loading control. (c) miR‐139‐5p or miR‐139‐3p binding site in the 3′‐UTR of MMP11 mRNA. (d) Luciferase reporter assays using vectors encoding a putative miR‐139‐5p and miR‐139‐3p target site of the MMP11 3′‐UTR. Renilla luciferase values were normalized to firefly luciferase values (*P < 0.001).

Figure 3

MMP11 mRNA and protein expression after si‐MMP11 transfection and effects of silencing MMP11 in BC cell lines. (a) MMP11 mRNA expression levels were evaluated by qRT‐PCR 72 h after transfection with 10 nM si‐MMP11. GUSB was used as an internal control (*P < 0.0001). (b) MMP11 protein expression was evaluated by Western blotting 72 h after transfection with si‐MMP11 (10 nM). GAPDH was used as a loading control. (c) Cell proliferation was determined by XTT assays (*P < 0.0001). (d) Cell migration activity was determined by wound healing assays (*P < 0.0001; **P < 0.001). (e) Cell invasion activity was determined by Matrigel invasion assays (*P < 0.0001; ***P < 0.05).

Figure 4

The expression level of MMP11 mRNA in BC clinical specimens. (a) MMP11 mRNA expression in 62 BC specimens and 23 non‐BC specimens. GUSB was used as an internal control. (b) Inverse correlations between MMP11 mRNA and miR‐139‐5p and miR‐139‐3p expression.

Figure 5

The association between the expression level of MMP11 with clinicopathological parameters. The association of MMP11 expression with clinicopathological parameters was determined with the Mann–Whitney U‐test. GUSB was used as an internal control.

Figure 6

The association between the expression level of MMP11 and overall survival. Kaplan–Meier survival curves for overall survival rate based on MMP11 expression in 62 BC patients. P‐values were calculated using the log‐rank test.

Figure 7

Immunohistochemical staining of MMP11 in tissue specimens. (a) There was a significant difference in the expression score of MMP11 of 80 BCs in comparison with 20 NBEs. (b) Immunohistochemical staining of MMP11 in tissue specimens (magnification ×40 and ×200). Immunoreactivity for MMP11 was obtained in the cytoplasm of the BC cells and in the fibrous stroma (Right and middle). Normal bladder epithelia were completely negative or faintly positive (left).