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. 2013 Dec 13:12:164.
doi: 10.1186/1476-4598-12-164.

miR-133b, a muscle-specific microRNA, is a novel prognostic marker that participates in the progression of human colorectal cancer via regulation of CXCR4 expression

Fang-Ting Duan, Feng Qian, Ke Fang, Kang-Yu Lin, Wen-Tao Wang, Yue-Qin Chen  1
Affiliations

miR-133b, a muscle-specific microRNA, is a novel prognostic marker that participates in the progression of human colorectal cancer via regulation of CXCR4 expression

Fang-Ting Duan et al. Mol Cancer. .

Abstract

Background: MicroRNA-133b (miR-133b), which is a muscle-specific microRNA, has been reported to be downregulated in human colorectal carcinoma (CRC) when compared to adjacent non-tumor tissue. However, its diagnostic value and role in CRC have yet to be described. CXC chemokine receptor-4 (CXCR4), which participates in multiple cell processes such as cell invasion-related signaling pathways, was predicted to be a potential target of miR-133b. The aim of this study was to investigate the associations and functions of miR-133b and CXCR4 in CRC initiation and invasion.

Methods: Mature miR-133b and CXCR4 expression levels were detected in 31 tumor samples and their adjacent, non-tumor tissues from patients with CRC, as well as in 6 CRC cell lines, using real-time quantitative RT-PCR (qRT-PCR). Luciferase reporter assays and Western blots were used to validate CXCR4 as a putative target gene of miR-133b. Regulation of CXCR4 expression by miR-133b was assessed using qRT-PCR and Western blot analysis, and the effects of exogenous miR-133b and CXCR4 on cell invasion and migration were evaluated in vitro using the SW-480 and SW-620 CRC cell lines.

Results: A significant downregulation of miR-133b was observed in 93.55% of CRC tissues, and the expression of miR-133b was much lower in metastatic tumors (stage C and D, stratified by the Modified Dukes Staging System) than in primary tumors (stage A and B). In contrast, CXCR4 protein expression significantly increased in 52.63% of CRC samples, and increased CXCR4 expression in CRC was associated with advanced tumor stage. CXCR4 was shown to be a direct target of miR-133b by luciferase reporter assays, and transfection of miR-133b mimics inhibited invasion and stimulated apoptosis of SW-480 and SW-620 CRC cells.

Conclusions: Our study demonstrated that downregulated miR-133b contributed to increased cell invasion and migration in CRC by negatively regulating CXCR4. These findings may be significant for the development of therapy target for CRC.

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Figures

Figure 1
Figure 1
Expression of miR-133b was downregulated in CRC. (A-B) miR-133b expression was measured by qRT-PCR in clinical CRC tissues from 31 patients. Data were analyzed in groups, and all data were normalized to U6 snRNA. T, tumor tissue; N, adjacent normal tissues. P, primary tumors; M, metastasis tumors. (C) ROC of miR-133b.
Figure 2
Figure 2
CXCR4 is a direct target of miR-133b. (A) Schematic of the luciferase reporter assay used to validate the interaction between miR-133b and the 3′UTR of CXCR4. Grey font indicates the ‘seed’ regions. The MRE CXCR4 3′UTR of wild, full mutant and full length were separately inserted into a psiCHECK-2 vector downstream from the Renilla luciferase gene. (B-C) Dual luciferase reporter assays performed using the 59 nt flanking the MRE, full mutation in the MRE (B) and the entire 3′UTR of CXCR4 (C). (D) miR-133b expression was measured using qRT-PCR in SW-480 and SW-620 human CRC cell lines and normalized to U6 snRNA expression. (E) miR-133b suppressed CXCR4 expression in CRC cells. CXCR4 protein expression of CRC cells transfected with miR-133b mimic, miR-133 inhibitor, siCXCR4 or a negative control normalized to GAPDH expression. Data are shown as the mean ± SD from three independent assays. *P < 0.05, ** P < 0.01 compared to controls.
Figure 3
Figure 3
The inverse correlation between miR-133b and CXCR4 in CRC cells and clinical samples. (A) CXCR4 protein expression in CRC cells was detected using Western blot analysis normalized to GAPDH expression. Expression of miR-133b and the CXCR4 protein level was inversely correlated. (B) The inverse correlation between miR-133b and CXCR4 in CRC tissues. miR-133b and CXCR4 mRNA were measured using qRT-PCR. Correlation analysis showed a significant relationship between these factors (Spearman r = -0.2840; P = 0.0456).
Figure 4
Figure 4
The effect of the oligonucleotides on proliferation and apoptosis of CRC cells. (A) Colony formation assay performed for SW-480 cells. The number of colonies on the entire plate was counted. (B-D) Induced apoptosis in CRC cells. After 48 h of incubation in the presence of controls or siCXCR4 and following 24-h exposure to cisplatin, all of the cells were stained with Annexin V–FITC and propidium iodide (PI) followed by flow cytometric analysis. Data are expressed as the mean ± SD of three independent experiments. (B) miR-133b mimic strengthened apoptosis in CRC cells. (C) The miR-133b inhibitor suppressed apoptosis in CRC cells. (D) siCXCR4 increased apoptosis in CRC cells.
Figure 5
Figure 5
miR-133b regulates motility of CRC cells. (A) The transwell invasion and a migration assays were used to detect the motility of SW-480 and SW-620 cells transfected with miR-133b mimic, miR-133b inhibitor, siCXCR4 or their corresponding negative controls. (B) The cells that invaded or migrated to the lower upside were counted using a microscope. Original magnification: 200×. (C) Using a wound healing assay, the cell motilities of SW-480 and SW-620 cells transfected with the miR-133b mimic, miR-133b inhibitor, siCXCR4 or their corresponding negative controls were observed at 0, 24 and 48 hours following wounding by a pipette tip. Original magnification: 100×.
Figure 6
Figure 6
qRT-PCR was used to detect the expression of the VEGF and MMP-9 genes in the SW-480 and SW-620 cell lines transfected with the miR-133b mimics, miR-133b inhibitor or CXCR4 siRNA.
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