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. 2013 Nov 2;11:276.
doi: 10.1186/1479-5876-11-276.

MicroRNA-124-3p Inhibits Cell Migration and Invasion in Bladder Cancer Cells by Targeting ROCK1

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Free PMC article

MicroRNA-124-3p Inhibits Cell Migration and Invasion in Bladder Cancer Cells by Targeting ROCK1

Xianglai Xu et al. J Transl Med. .
Free PMC article

Abstract

Background: Increasing evidence has suggested that dysregulation of certain microRNAs (miRNAs) may contribute to human disease including carcinogenesis and tumor metastasis in human. miR-124-3p is down-regulated in various cancers, and modulates proliferation and aggressiveness of cancer cells. However, the roles of miR-124-3p in human bladder cancer are elusive. Thus, this study was conducted to investigate the biological functions and its molecular mechanisms of miR-124-3p in human bladder cancer cell lines, discussing whether it has a potential to be a therapeutic biomarker of bladder cancer.

Methods: Three human bladder cancer cell lines and samples from ten patients with bladder cancer were analyzed for the expression of miR-124-3p by quantitative RT--PCR. Exogenetic overexpression of miR-124-3p was established by transfecting mimics into T24, UM-UC-3 and J82 cells, after that cell proliferation and cell cycle were assessed by MTT assay, flow cytometry and Colony-forming assay. Cell motility and invasion ability were evaluated by wound healing assay and transwell assay. Tissue microarray, and immunohistochemistry with antibodies against ROCK1, MMP2 and MMP9 was performed using the peroxidase and DAB methods. The target gene of miR-124-3p was determined by luciferase assays, quantitative RT--PCR and western blot. The regulation of epithelial-to-mesenchymal transition by miR-124-3p was analyzed by western blot.

Results: miR-124-3p is frequently down-regulated in bladder cancer both in three bladder cancer cell lines, T24, UM-UC-3, J82 and clinical samples. Overexpression of miR-124-3p induced G1-phase arrest in T24, UM-UC-3 and J82 cell lines and suppressed cell growth in colony-forming assay. miR-124-3p significantly repressed the capability of migration and invasion of bladder cancer cells. In addition, ROCK1 was identified as a new target of miR-124-3p. ROCK1, MMP2, MMP9 were up-regulated in bladder cancer tissues. Furthermore, we demonstrated miR-124-3p could inhibit bladder cancer cell epithelial mesenchymal transfer, and regulated the expression of c-Met, MMP2, MMP9.

Conclusions: miR-124-3p can repress the migration and invasion of bladder cancer cells via regulating ROCK1. Our data indicate that miR-124-3p could be a tumor suppressor and may have a potential to be a diagnostics or predictive biomarker in bladder cancer.

Figures

Figure 1
Figure 1
Expression patterns of miR-124-3p in urinary bladder cancer tissues and BCa cell lines. (A) qPCR detection of miR-124-3p in T24, UM-UC-3 and J82 cell lines versus SV-HUC-1. It was normalized with U6 snRNA in quantitative real-time RT-PCR analysis. (B) The miR-124-3p levels were determined in 10 surgical specimens of human bladder cancer tissues and were normalized to that in the adjacent normal bladder tissues, which is defined as 1. *P <0.05, **P < 0.01, ****P < 0.0001.
Figure 2
Figure 2
miR-124-3p induces G1-phase arrest and inhibits clonogenicity in bladder cancer cell lines. (A) The T24, UM-UC-3 and J82 cells, transfected with NC RNA or miR-124-3p, were subjected to flow cytometry for cell cycle analysis. Three independent experiments were performed in each group. (B) Decreased clonogenicity in bladder cancer cells treated with miR-124-3p (Representative wells were presented). (C) The colony count was significantly lower for miR-124-3p treated group compared with NC treated group (P < 0.05).
Figure 3
Figure 3
Forced expression of miR-124-3p suppresses cell motility in wound healing assay. T24, UM-UC-3 and J82 cells were transfected with NC, miR-124-3p mimics or siROCK1 and were performed wound healing assays with a 24-h recovery period; Scale bars = 500 μm.
Figure 4
Figure 4
Forced expression of miR-124-3p and silenced expression of ROCK1 suppresses cell migration and invasion in transwell assay. (A) T24, UM-UC-3 and J82 cells were transfected with NC or miR-124-3p mimics. Cell migration and invasion were assessed after 24 hours incubation by transwell assay. (B) T24, UM-UC-3 and J82 cells were transfected with NC or siROCK1. Cell migration and invasion were evaluated after 24 hours incubation by transwell assay; Scale bars = 100 μm.
Figure 5
Figure 5
IHC analysis of ROCK1, MMP2 and MMP9 expression pattern in bladder cancer tissues. (A) Representative images of IHC staining for ROCK1, MMP2 and MMP9 in human bladder cancer tissues and NT tissues were captured at 400× magnification. Scale bars = 100 μm. (B) Positive strength of ROCK1, MMP2 and MMP9 were significantly higher in bladder cancer tissues compared with paired non-tumor tissues; P < 0.05.
Figure 6
Figure 6
miR-124-3p directly targets ROCK1. (A) Oligonucleotides containing the predicted miR-124-3p binding sites in the 3′UTR of ROCK1 mRNA was synthetized, while mutations on the “seed” sequences are designed as below. Alignment between the predicted miR-34a target sites and miR-124-3p is marked with black color. (B) HEK293T cells were co-transfected with 50 nM of either miR-124-3p mimics or NC and 100 ng pmirGLO Dual-Luciferase miRNA Target Expression Vector with Wt or Mut 3′-UTR of ROCK1. The relative luciferase activity was measured 48 h after transfection. *P < 0.05. (C) The down-expression of ROCK1 was confirmed by western blotting, after miR-124-3p or siROCK1 transfected, GAPDH was used as control. (D) Quantitative real-time PCR analysis indicated that the relative mRNA level of ROCK1 was significantly decreased after miR-124-3p or RNAi treatment. GAPDH was used as control. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 7
Figure 7
Forced expression of ROCK1 rescues miR-124-3p-associated decrease in cell migration and invasion. miR-124-3p inhibits T24 EMT. (A) T24 cells were transfected as in (B) and then used in a transwell migration assay (top) or an invasion assay (bottom). Scale bars = 100 μm (B) Either miR-124-3p mimics or NC oligos were co-transfected with the pT-ROCK1 or the empty pTarget vector, pT-NULL, into T24 cells. Western blot was then performed to detect the expression of ROCK1. GAPDH was used as control. (C) Expression of epithelial markers and mesenchymal markers were compared by western blot between NC and miR-124-3p or siROCK1 groups. GAPDH was used as a loading control. (D) Expression of p-p38MAPK and p38MAPK were determined by Western blot analysis.

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