miR-200b as a prognostic factor in breast cancer targets multiple members of RAB family

Abstract

Background: miR-200b has been reported to be a tumor suppressor and a promising therapeutic target in cancer. miR-200b has been associated with epithelial-mesenchymal transition and chemo-resistance in cancer. The aim of this study is to investigate the expression of miR-200b, its prognostic roles and its potential targets in breast cancer.

Methods: qRT-PCR was used to detect miR-200b expression in breast cancer tissues and cell lines. In situ hybridization of miR-200b on tissue microarray including 134 breast cancer samples was used to evaluate its prognostic role. Novel targets of miR-200b in breast cancer were predicted and confirmed by luciferase reporter assay and western bloting. Immunohistochemical staining was used for protein detection. The biological effects of miR-200b in breast cancer cells were further confirmed by ectopic expression of its mimics followed by MTT assay and invasion test.

Results: miR-200b was downregulated in breast cancer tissues and cell lines and its low-expression correlated with poor outcome in breast cancer patients. Members of RAB family, RAB21, RAB23, RAB18 and RAB3B were predicted to be the targets of miR-200b. The luciferase reporter assay was performed to certificate this prediction. The expressions of RAB21, RAB23, RAB18 and RAB3B were suppressed by transfection of miR-200b in breast cancer cells. Over-expression of miR-200b or knock-down of RAB21, RAB23, RAB18 and RAB3B inhibited breast cancer cell proliferation and invasion in vitro.

Conclusions: Our study provides evidence that miR-200b is a prognostic factor in breast cancer targeting multiple members of RAB family. MiR-200b could be a potential therapeutic target in breast cancer.

Figure 1

miR-200b was down-regulated in breast cancer tissues and cell lines. (A) The expression of miR-200b was detected by qRT-PCR in 40 pairs of normal mammary tissues and breast cancer samples. (B) The expression of miR-200b was detected by qRT-PCR in breast cancer cell lines.

Figure 2

miR-200b as a prognostic factor in breast cancer patients. (A) The ISH staining of miR-200b in breast cancer tissue microarray: ×200. Two miR-200b high samples and two miR-200b low samples were shown as representatives. (B) qRT-PCR results for miR-200b detection in three miR-200b high ISH staining samples and three miR-200b low samples. (C) Survival curves of OS and DFS according to miR-200b expression. Low miR-200b expression correlated with worse outcome.

Figure 3

RAB21, RAB23, RAB18 and RAB3B is a direct target of miR-200b. (A) miR-200b binds to the 3′UTRs of RAB21, RAB23, RAB18, RAB3B, RAB37, RAB8B, RAB7A, RAP1B and RAP2C. Predicted binding between miR-200b and the seeds match in the nine predicted genes’ 3′UTRs. (B) Luciferase reporter assays 48 h after transfection with indicated pMIR-Report plasmids and a renilla transfection control plasmid, co-transfected with miR-200b mimics, or relevant scramble controls. Luciferase activities were significantly inhibited on co-transfection of miR-200b mimics and pMIR-Report plasmids of RAB21, RAB23, RAB18 and RAB3B. Mutation in the corresponding binding sites abolished this inhibition. Data shown were the mean ± s.d. of six replicates and were representative of three independent experiments. *P < 0.05 (C) miR-200b regulated the expression of RAB21, RAB23, RAB18 and RAB3B. Western blot analyzed their expression 48 h after transfection with miR-200b mimics or scramble control in MDA-MB-231 and 4 T1 cells.

Figure 4

IHC staining of RAB21, RAB23, RAB18 and RAB3B in human breast cancer tissues. 10 pairs of tumor tissues and normal tissues from breast cancer patients, who had developed distant metastasis after operation, were detected by ISH or IHC. Compared with normal tissue, miR-200b expression was down-regulated in tumor tissue, while the contrary situations were found for RAB21, RAB23, RAB18 and RAB3B.

Figure 5

Over-expression of miR-200b or knock-down of RAB21, RAB23, RAB18 and RAB3B inhibits breast cancer cell proliferation and invasion. (A) Transfection of miR-200b mimics or knock-down of RAB21, RAB23, RAB18 and RAB3B in MDA-MB-231 and 4 T1 cells markedly attenuated cell proliferation: MTT assay at 24 h, 48 h and 72 h after transfection. (B, C) Transfection of miR-200b mimics or knock-down of RAB21, RAB23, RAB18 and RAB3B in MDA-MB-231 and 4 T1 cells inhibited cell invasion: Data shown in Figure B were the mean ± s.d. of three replicates and were representative of three independent experiments. *P < 0.05. The representing images were shown in Figure C.