Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Feb 1:12:51.
doi: 10.1186/1471-2407-12-51.

Loss of miR-133a expression associated with poor survival of breast cancer and restoration of miR-133a expression inhibited breast cancer cell growth and invasion

Zheng-sheng Wu  1 , Chao-qun Wang, Ru Xiang, Xue Liu, Shan Ye, Xue-qing Yang, Gui-hong Zhang, Xiao-chun Xu, Tao Zhu, Qiang Wu
Affiliations

Loss of miR-133a expression associated with poor survival of breast cancer and restoration of miR-133a expression inhibited breast cancer cell growth and invasion

Zheng-sheng Wu et al. BMC Cancer. .

Abstract

Background: miRNAs, endogenous oligonucleotide RNAs, play an important role in mammary gland carcinogenesis and tumor progression. Detection of their expression and investigation of their functions could lead to discovery of novel biomarkers for breast cancer.

Methods: In situ hybridization was used to detect miR-133a expression in formalin-fixed paraffin-embedded breast surgical specimens from 26 benign, 34 pericancerously normal and 90 cancerous tissues. qRT-PCR was performed to assess miR-133a levels in 6 breast cell lines and 10 benign and 18 cancerous fresh breast tissue specimens. Cell viability, migration, and invasion assays were used to determine the role of miR-133a in regulation of breast cancer cell growth, migration, and invasion, respectively. Luciferase assay was performed to assess miR-133a binding to FSCN1 gene.

Results: Expression of miR-133a was reduced from normal through benign to cancerous breast tissues. Expression of miR-133a was also low in breast cancer cell lines. The reduced miR-133a expression was associated with lymph nodes metastasis, high clinical stages, and shorter relapse-free survivals of patients with breast cancer. Furthermore, transfection of miR-133a oligonucleotides slightly inhibited growth but significantly decreased migration and invasion capacity of breast cancer cells, compared with negative controls, whereas knockdown of miR-133a expression induced breast cancer cell migration and invasion. In addition, we identified a putative miR-133a binding site in the 3'-untranslated region (UTR) of Fascin1 (FSCN1) gene using an online bioinformatical tool. We found that miR-133a transfection significantly reduced expression of FSCN1 mRNA and protein. The luciferase reporter assay confirmed that FSCN1 was the direct _target gene of miR-133a.

Conclusions: miR-133a expression was lost in breast cancer tissues, loss of which was associated with lymph nodes metastasis, high clinical stages and shorter relapse-free survivals of patients with breast cancer. Functionally, miR-133a can suppress tumor cell invasion and migration and _targeted the expression of FSCN1. Future study will verify whether detection of miR-133a expression can served as a novel biomarker for breast cancer progression and patient prognosis.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Expression of miR-133a in breast tissue specimens and cell lines. A. Strongly positive expression of miR-133a in tissues of breast benign disease. Magnification: ×200. B. Strongly positive expression of miR-133a in an adjacent normal breast tissue. Magnification: ×200. C. Weakly positive expression of miR-133a in an invasive ductal carcinoma. Magnification: ×200. D. Fresh tissue samples from patients with breast cancer or benign breast diseases were obtained from the surgery room. Total miRNA was extracted from the tissues and subjected to qRT-PCR analysis. E. Expression of miR-133a and FSCN1 in breast cancer cell lines: Breast cell lines were grown in monolayer for 3-5 days, and miRNA and total RNA was extracted and subjected to qRT-PCR analyses. F. Association of miR-133a expression with relapse-free survival of the patients with breast cancer.
Figure 2
Figure 2
Regulation of breast cancer cell growth, invasion and migration by miR-133a. A. MCF-7 cells were grown and transiently transfected with miR-133a mimic or negative control, and cell proliferation was then assessed afterwards. The experiments were performed in triplicate and repeated thrice. B. MCF-7 cells were grown and transiently transfected with miR-133a ASO or negative control, and cell proliferation was assessed afterwards. The experiments were performed in triplicate and repeated thrice. C. Transwell migration and Matrigel invasion assays. MDA-MB-231 and MCF-7 cells were grown and transiently transfected with miR-133a mimic or miR-133a ASO for 2 days and subjected to migration and invasion assays. Representative photographs (upper) and quantification (lower) are shown. Magnification: × 200. D. Wound healing assay. Cells were transfected with ASO NC or miR-133a ASO for 72 h. Images were taken at 0, 24, and 48 h. Quantification of cell motility by measuring the distance between the invading front of cells in three random selected microscopic fields for each condition and time point. The degree of motility is expressed as percent of wound closure as compared with the zero time point. Magnification: × 100. * P < 0.05; ** P < 0.01.
Figure 3
Figure 3
FSCN1 is the _target gene of miR-133a in breast cancer cells. A. Putative conserved _target site in the FSCN1 3'UTR was identified with the _targetScan database: The conserved 7-bp seed sequence of miR-133a is aligned to FSCN1 mRNA. B. QRT-PCR analysis of FSCN1 mRNA expression. Breast cancer MCF-7 cells were grown and transiently transfected with miR-133a mimic and then subjected to RNA extraction and qRT-PCR analysis. C. Western blot analysis of FSCN1 protein expression. MCF-7 cells were grown and transfected with miR-133a mimic or negative control. Total cellular protein was isolated and subjected to Western blot analysis of FSCN1 expression. ß-actin was used as an internal control. D. Luciferase reporter assay. MCF-7 cells were transfected with a reporter vector psiCHECK2-FSCN1 3'UTR or psiCHECK2 control vector and miR-133a mimic or negative control. Each transfection was carried out in triplicate. Luciferase assay was performed 48 h after gene transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. E. Transwell migration and matrigel invasion assays. MCF-7 cells were grown and transiently transfected with miR-133a ASO, miR-133a ASO plus FSCN1 siRNA, or scrambled sequence oligonucleotides as negative control for 2 days and subjected to migration and invasion assays. Magnification: ×200.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Carthew RW, Sontheimer EJ. Origins and mechanisms of miRNAs and siRNAs. Cell. 2009;136:642–655. - PMC - PubMed
    1. Zhao Y, Samal E, Srivastava D. Serum response factor regulates a muscle-specific microRNA that _targets Hand2 during cardiogenesis. Nature. 2005;436:214–220. - PubMed
    1. Costinean S, Zanesi N, Pekarsky Y, Tili E, Volinia S, Heerema N, Croce CM. Pre-B cell proliferation and lymphoblastic leukemia/high-grade lymphoma in E(mu)-miR155 transgenic mice. Proc Natl Acad Sci USA. 2006;103:7024–7029. - PMC - PubMed
    1. Kawasaki H, Taira K. Retraction: Hes1 is a _target of microRNA-23 during retinoic-acid-induced neuronal differentiation of NT2 cells. Nature. 2003;426:100. - PubMed
    1. Chan JA, Krichevsky AM, Kosik KS. MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. Cancer Res. 2005;65:6029–6033. - PubMed

Publication types

MeSH terms

  NODES
Association 1
INTERN 1
twitter 2
Verify 1