MicroRNA‑34a‑5p serves as a tumor suppressor by regulating the cell motility of bladder cancer cells through matrix metalloproteinase‑2 silencing

Abstract

Bladder cancer (BC), a common urologic cancer, is the fifth most frequently diagnosed tumor worldwide. hsa‑miR‑34a displays antitumor activity in several types of cancer. However, the functional mechanisms underlying hsa‑miR‑34a in BC remains largely unknown. We observed that hsa‑mir‑34a levels were significantly and negatively associated with clinical disease stage as well as regional lymph node metastasis in human BC. In a series of in vitro investigations, overexpression of hsa‑miR‑34a inhibited cell migration and invasion in BC cell lines 5637 and UMUC3 as detected by Transwell assays. We further found that hsa‑miR‑34a inhibited cell migration and invasion by silencing matrix metalloproteinase‑2 (MMP‑2) expression and thus interrupting MMP‑2‑mediated cell motility. Our analysis of BC datasets from The Cancer Genome Atlas database revealed a negative correlation between hsa‑miR‑34a and MMP‑2. Moreover, higher MMP‑2 protein expression was observed in the BC tissues when compared with that noted in the normal tissue. MMP‑2 levels were also significantly associated with clinical disease stage and poor survival rate in human BC. These findings indicate that MMP‑2 plays a critical role in regulating BC progression. Therefore, hsa‑miR‑34a is a promising treatment to target MMP‑2 for the prevention and inhibition of cell migration and invasion in BC.

Keywords: bladder cancer; microRNA-34a-5p; migration; invasion; MMP-2.

Figure 1.

UALCAN portal analysis of BC samples based on the TCGA database. (A) Comparison of hsa-miR-34a expression between male and female individuals in BC samples. (B-F) Expression of hsa-miR-34a for different races, tumor histology, molecular subtypes, clinical stages, and nodal metastasis status of BC samples. (G) Overall survival rate between patients with BC with high and low hsa-miR-34a expression. All data are expressed as the mean ± the SD of triplicate samples. *P<0.05, ***P<0.001 compared with the control group; ns, not significant. BC, bladder cancer; TCGA, The Cancer Genome Atlas.

Figure 2.

hsa-miR-34a affects the functions of cell migration and invasion in BC cells. (A-D) Cells were transfected with control or hsa-miR-34a mimic for 24 h after which cell migration and invasion were performed using Transwell assays. The number of migrated or invaded cells were quantified. All data are expressed as the mean ± SD of triplicate samples. **P<0.01, ***P<0.001, ****P<0.0001 compared with the control group. BC, bladder cancer.

Figure 3.

hsa-miR-34a suppresses MMP-2 expression through direct binding. (A) An miRNA target prediction program (microRNA.org) was used to identify miRNAs that potentially bind to the MMP-2 3′-UTR. (B) Levels of MMP-2 mRNA expression in different BC cell lines were determined by qPCR assay. (C and D) Cells were cotransfected with wild-type or mutant MMP-2 3′-UTR (1 µg/µl) and control mimic or hsa-miR-34a mimic (100 nM) for 24 h, and luciferase activities were measured. (E-G) Cells were transfected with control mimic or hsa-miR-34a mimic for 24 h; the levels of MMP-2 protein and mRNA expression were measured using western blot analysis and qPCR assay, respectively. (H) Correlation between hsa-miR-34a and MMP-2 expression in BC samples. All data are expressed as the mean ± the SD of triplicate samples. *P<0.05, ****P<0.0001 compared with the mimic control group. MMP-2, matrix metalloproteinase-2; BC, bladder cancer.

Figure 4.

Inhibition of MMP-2 expression suppresses cell motility in BC cells stably expressing hsa-miR-34a. (A and B) BC cells stably expressing control or hsa-miR-34a were established. The levels of MMP-2 protein and mRNA expression were determined using western blot analysis and qPCR assay, respectively. (C-F) Cell migration and invasion abilities in stable cells were determined using Transwell assays. The number of migrated or invaded cells were quantified. All data are expressed as the mean ± SD of triplicate samples. **P<0.01, ***P<0.001, ****P<0.0001 compared with the control group. MMP-2, matrix metalloproteinase-2; BC, bladder cancer.

Figure 5.

Levels of MMP-2 expression correlate with clinicopathologic features of BC. (A and B) Representative immunohistochemistry images of MMP-2 protein expression in BC tissues and normal bladder tissues. MMP-2 staining intensity was quantified. (C) Comparison of MMP-2 expression between male and female individuals in BC samples. (D-H) MMP-2 expression for different race, tumor histology, molecular subtypes, clinical stages, and nodal metastasis status of BC samples. (I) Survival probability between patients with BC with high and low MMP-2 expression. All data are expressed as the mean ± SD of triplicate samples. *P<0.05, **P<0.01, ***P<0.001 compared with the control group; ns, not significant. MMP-2, matrix metalloproteinase-2; BC, bladder cancer.

Figure 6.

Schematic image depicting the mechanism by which hsa-miR-34a modulates cell motility through MMP-2 downregulation in BC cells. Modeling revealed that hsa-miR-34a directly targets the 3′-UTR of MMP-2 mRNA and thus suppresses MMP-2 translation. Overexpression of hsa-miR-34a in human BC cells may represent a potential treatment strategy. MMP-2, matrix metalloproteinase-2; BC, bladder cancer.