miR-145 and miR-133a function as tumour suppressors and directly regulate FSCN1 expression in bladder cancer

Abstract

Background: We have recently identified down-regulated microRNAs including miR-145 and miR-133a in bladder cancer (BC). The aim of this study is to determine the genes targeted by miR-145, which is the most down-regulated microRNA in BC.

Methods: We focused on fascin homologue 1 (FSCN1) from the gene expression profile in miR-145 transfectant. The luciferase assay was used to confirm the actual binding sites of FSCN1 mRNA. Cell viability was evaluated by cell growth, wound-healing, and matrigel invasion assays. BC specimens were subjected to immunohistochemistry of FSCN1 and in situ hybridisation of miR-145.

Results: The miR-133a as well as miR-145 had the target sequence of FSCN1 mRNA by the database search, and both microRNAs repressed the mRNA and protein expression of FSCN1. The luciferase assay revealed that miR-145 and miR-133a were directly bound to FSCN1 mRNA. Cell viability was significantly inhibited in miR-145, miR-133a, and si-FSCN1 transfectants. In situ hybridisation revealed that miR-145 expression was markedly repressed in the tumour lesion in which FSCN1 was strongly stained. The immunohistochemical score of FSCN1 in invasive BC (n=46) was significantly higher than in non-invasive BC (n=20) (P=0.0055).

Conclusion: Tumour suppressive miR-145 and miR-133a directly control oncogenic FSCN1 in BC.

Figure 1

Regulation of FSCN1 expression in the down-regulated microRNA transfectants (T24). (A) FSCN1 mRNA expression after 24 h transfection with 10 nM of microRNAs (miR-145, miR-30a-3p, miR-133a, miR-195, miR-125b, and miR-199a*). FSCN1 mRNA expression was repressed in miR-145 and miR-133a transfectants. (B) FSCN1 protein expression after 72 h transfection of microRNAs. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. The protein expression level of FSCN1 was also repressed in the transfectants.

Figure 2

miR-145- and miR-133a-binding sites in FSCN1 3′-UTR. (A) A luciferase assay using the vector encoding full length of FSCN1 3′-UTR (position 51–1180). BOY cells were transfected with 5 ng vector and 10 nM microRNAs. The Renilla luciferase values were normalised by firefly-luciferase values. (B) Luciferase assays using the vectors encoding putative conserved target sites of FSCN1 3′-UTR identified with the TargetScan database: four conserved sites for miR-145 and one site for miR-133a. (C) Luciferase assays using the mutated vectors in which the specific sites targeted by the microRNAs were deleted.

Figure 3

(A) miR-145 and miR-133a expression in BC cell lines (BOY, T24, KK47) and normal human bladder mucosa (N1 and N2). (B–D) Effect of cell viabilities in miR-145 and miR-133a transfectants: (B) cell growth determined by the XTT assay; (C) cell migration activity determined by the wound-healing assay; and (D) cell invasion activity determined by the matrigel invasion assay in BOY and T24 cell lines transfected with miR-145 and miR-133a. ^*P<0.005, ^**P<0.0001.

Figure 4

FSCN1-knockdown effect on BC cell viability by si-RNA: (A) upper, FSCN1 mRNA expression in three BC cell lines (BOY, T24, KK47) by real-time RT–PCR; (A) lower, western blot revealed that FSCN1 protein was markedly decreased in three si-FSCN1 transfectants compared with the controls; (B) cell growth as revealed by the XTT assay; (C) cell migration activity by the wound-healing assay; and (D) cell invasion activity by the matrigel invasion assay in T24 cell lines transfected with si-FSCN1. si-FSCN1-transfected T24 cell lines exhibited a significant decrease in cell growth, migration, and invasion in comparison with the si-control transfectants. ^**P<0.0001.

Figure 5

In situ hybridisation of miR-145 and immunohistochemistry examination of FSCN1 in clinical BC specimens: (A) H&E staining, tumour, and surrounding smooth muscle; (B) immunohistochemical staining of FSCN1 showing strong expression in tumour lesion; (C) in situ hybridisation of miR-145 showing faint expression in tumour lesion and strong expression in smooth muscle layer; (D) no staining by scramble-control probe; and (E) FSCN1 protein expression in invasive and non-invasive BC specimens. Low-grade bladder cancer without invasion (pTa) (upper panel, original magnification × 400). High-grade bladder cancer with involvement of the muscularis (pT2) (lower panel, original magnification × 400).