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MicroRNA-7 inhibits epithelial-to-mesenchymal transition and metastasis of breast cancer cells via targeting FAK expression - PubMed

MicroRNA-7 inhibits epithelial-to-mesenchymal transition and metastasis of breast cancer cells via targeting FAK expression

Xiangjun Kong et al. PLoS One. 2012.

Abstract

Focal adhesion kinase (FAK) is an important mediator of extracellular matrix integrin signaling, cell motility, cell proliferation and cell survival. Increased FAK expression is observed in a variety of solid human tumors and increased FAK expression and activity frequently correlate with metastatic disease and poor prognosis. Herein we identify miR-7 as a direct regulator of FAK expression. miR-7 expression is decreased in malignant versus normal breast tissue and its expression correlates inversely with metastasis in human breast cancer patients. Forced expression of miR-7 produced increased E-CADHERIN and decreased FIBRONECTIN and VIMENTIN expression in breast cancer cells. The levels of miR-7 expression was positively correlated with E-CADHERIN mRNA and negatively correlated with VIMENTIN mRNA levels in breast cancer samples. Forced expression of miR-7 in aggressive breast cancer cell lines suppressed tumor cell monolayer proliferation, anchorage independent growth, three-dimensional growth in Matrigel, migration and invasion. Conversely, inhibition of miR-7 in the HBL-100 mammary epithelial cell line promoted cell proliferation and anchorage independent growth. Rescue of FAK expression reversed miR-7 suppression of migration and invasion. miR-7 also inhibited primary breast tumor development, local invasion and metastatic colonization of breast cancer xenografts. Thus, miR-7 expression is decreased in metastatic breast cancer, correlates with the level of epithelial differentiation of the tumor and inhibits metastatic progression.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. miR-7 expression is decreased in breast cancer and is associated with tumor metastasis.

(A) The expression level of mature miR-7 in breast cancer (n = 42) or normal breast tissues (n = 27) was determined using quantitative PCR. Both breast cancer and normal breast are fresh tissues. Box-plot lines represent medians and interquartile ranges of the normalized threshold values; whiskers and spots indicate 10–90 percentiles and the remaining data points. (B) The relative expression of mature miR-7 in 19 non-metastatic breast cancer tissue and 23 metastatic breast cancer tissue samples. The expression level of mature miR-7 is normalized to U6 small nuclear RNA.

Figure 2
Figure 2. miR-7 decreases FAK expression by directly targeting its 3′-UTR.

(A) The potential binding sequences for miR-7 within the FAK 3′-UTR of human (H.s), chimpanzee (P.t), mouse (M.m), guinea pig (C.p), rabbit (O.c), rat (R.n), and dog (C.f). Seed sequences are highlighted and underlined. (B) Luciferase reporter plasmids were constructed by the insertion of full length FAK 3′-UTR into the region immediately downstream of the luciferase gene. The sequences of two predicted miR-7 binding sites within the FAK 3′-UTR, including wild-type full-length UTR or mutant (highlighted and underlined) binding site are shown. (C) Relative luciferase activity was analyzed after the above reporter plasmids or control reporter plasmid were cotransfected with miR-7 mimics or control mimics in MDA-MB-435s cells. (D) Relative expression of miR-7 by quantitative PCR (top) and immunoblot for FAK expression (bottom) in the indicated cell lines. NT, non-tumorigenic. (E) Immunoblot assays of endogenous FAK protein levels in MDA-MB-435s and MDA-MB-231 cells transfected with miR-7 mimics or control mimics and those in HBL-100 cells transfected with miR-7 inhibitor or control inhibitor.

Figure 3
Figure 3. miR-7 promotes an epithelial phenotype in breast cancer cells.

(A) Morphology of MDA-MB-435s and MDA-MB-231 cells transiently transfected with mature miR-7 or the control miRNA. (B) Immunoblot analysis of FIBRONECTIN, VIMENTIN, E-CADHERIN, N-CADHERIN and SNAIL in MDA-MB-435s and MDA-MB-231 cells transfected with mature miR-7 or control miRNA. (C) The correlation of mature miR-7 with E-CADHERIN mRNA in 42 breast cancer samples. (D) Correlation of mature miR-7 with VIMENTIN mRNA in 42 breast cancer samples. Pearson correlation coefficients (R) and P-values (p) are indicated.

Figure 4
Figure 4. Restoration of FAK attenuates miR-7 mediated cell migration and invasion inhibition.

(A) Transwell migration and invasion assays of MDA-MB-435s and MDA-MB-231 cells transfected with miR-7 mimics and control mimics. (B) Transwell migration and invasion assays of MDA-MB-435s and MDA-MB-231 cells transfected with miR-7 mimics and control mimics with or without FAK restoration. (C) Immunoblot analysis of FAK expression in MDA-MB-435s and MDA-MB-231 cells transfected with miR-7 mimics or control mimics with or without FAK restoration. ** p<0.01, *** p<0.001.

Figure 5
Figure 5. miR-7 inhibits breast cancer cell tumorigenesis in vitro.

(A) miR-7 decreases MDA-MB-435s and MDA-MB-231 cell proliferation in vitro and inhibition of endogenous miR-7 expression promotes HBL-100 cell monolayer proliferation. (B) miR-7 inhibits MDA-MB-435s anchorage independent growth in soft agar. (C) Inhibition of miR-7 promotes HBL-100 cell anchorage independent growth in soft agar. (D) Morphology of MDA-MB-435s and MDA-MB-231 cells cultured in three-dimensional Matrigel after transfection with mature miR-7 or the control miRNA.

Figure 6
Figure 6. miR-7 inhibits breast cancer cell growth, local invasion in vivo.

(A) Primary tumor growth upon orthotopic injection of 1.0×106 MDA-MB-435s cells infected as indicated. n = 12 per group per time point. (B) H&E staining of primary tumors and mice lungs after orthotopic injection. Arrows: tumor cells venous (a), muscle (b) invasion and lung metastasis foci (d). (scale bar: 100 µm) (C) FAK, VIMENTIN, FIBRONECTIN, E-CADHERIN stained primary tumors after orthotopic injection. Arrow indicate metastatic foci (scale bar: 50 µm). (D) Evaluation of nuclear BrdU incorporation and TUNEL positive (apoptotic) nuclei in MDA-MB-435s tumors. ***, p<0.001.

Figure 7
Figure 7. miR-7 inhibits breast cancer cell metastasis to mice lungs.

(A) H&E stained sections of lungs isolated from mice that received tail vein injection of miR-7 or control infected MDA-MB-435s cells (scale bar: 100 µm). (B) Numbers of lung micrometastases per section in individual mice (each data point represents a different mouse; n = 7 mice per group). (C) Expression of human HPRT mRNA relative to mouse 18S rRNA in the lungs of the tumor-bearing mice.

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This work was supported by the National Key Scientific Program of China (2010CB912804,2012CB934002), National Natural Science Foundation of China (30971492), Cancer Science Institute of Singapore and the Chinese Academy of Sciences Visiting Professorship for Senior International Scientists (2010T2SO3). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.