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. 2011 Nov;9(11):1500-8.
doi: 10.1158/1541-7786.MCR-11-0311. Epub 2011 Aug 29.

miR-31 is a broad regulator of β1-integrin expression and function in cancer cells

Katarzyna Augoff  1 , Mitali Das, Katarzyna Bialkowska, Brian McCue, Edward F Plow, Khalid Sossey-Alaoui
Affiliations

miR-31 is a broad regulator of β1-integrin expression and function in cancer cells

Katarzyna Augoff et al. Mol Cancer Res. 2011 Nov.

Abstract

Integrins are adhesion receptors involved in bidirectional signaling that are crucial for various cellular responses during normal homeostasis and pathologic conditions such as cancer progression and metastasis. Aberrant expression of noncoding microRNAs (miRNA) has been implicated in the deregulation of integrin expression and activity, leading to the development and progression of cancer tumors, including their acquisition of the metastatic phenotype. miR-31 is a key regulator of several critical genes involved in the invasion-metastasis cascade in cancer. Using diverse cell-based, genetic, biochemical, flow cytometry, and functional analyses, we report that miR-31 is a master regulator of integrins as it targets multiple α subunit partners (α2, α5, and αV) of β1 integrins and also β3 integrins. We found that expression of miR-31 in cancer cells resulted in a significant repression of these integrin subunits both at the mRNA and protein levels. Loss of expression of α2, α5, αV, and β3 was a direct consequence of miR-31 targeting conserved seed sequences in the 3' untranslated region of these integrin subunits leading to their posttranscriptional repression, which was reflected in their diminished surface expression in live cells. The biological consequence of decreased the cell surface of these integrins was a significant inhibition of cell spreading in a ligand-dependent manner. Although different reports have shown that a single integrin can be regulated by several miRNAs, here we show that a single miRNA, miR-31, is able to specifically target several integrin subunits to regulate key aspects of cancer cell invasion and metastasis.

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Figures

Fig. 1
Fig. 1. miR-31 inhibits expression of specific α and β integrin subunits
(A) Alignment of miR-31 seed sequence with candidate target sequences within the 3’UTRs of the indicated integrin subunits. (B) semi-quantitative RT-PCR, (C) Real-time qt RT-PCR and (D) immunoblot of the indicated integrin subunits from MDA-MB-231 cells or MDA-MB-231 cells with stable expression of either control or miR-31 vector. Densitometry and statistical analyses of the Western Blot data is shown in Fig. S2.
Fig. 2
Fig. 2. Surface expression of integrin α-subunits and their β1 subunit partner is suppressed by miR-31
Expression profiles of different integrin α subunits in MDA-MB-231 cells overexpressing GFP-pBABE vector control or GFP-miR-31. Surface expression of different α subunit partners of the β1 integrin subunit is quantitated as the relative fluorescence intensity (RFI) with subunit specific mAbs by flow cytometry. Values are means± S.E. and are representative of 3 independent experiments. Representative histograms are provided below in which the empty histograms are for the vector control and the filled histograms are for miR-31 overexpressing cells.
Fig. 3
Fig. 3. Expression profile of different integrin β subunits in MDA-MB-231 cells expressing and GFP-miR-31 or control GFP-pBABE
(A) Surface expression of different β integrin subunits (other than β1) as determined by flow cytometry with representative histograms given below. Empty histogram is for vector and filled histogram is for miR-31 overexpressing cells. Values are means ± S.E. and are representative of 3 independent experiments. (B). Surface levels of αVβ3, αVβ5 and αVβ6 reveal differences between vector and miR-31 cells for αVβ3 and αVβ5 integrins. The integrin αV level has been used as the control. Empty histogram is for vector and filled histogram is for miR-31 overexpressing cells. Values are means± S.E. and are representative of 3 independent experiments.
Fig. 4
Fig. 4. Targeting of α2, α5 and αV subunits by miR-31 decreases β1 integrin activation
MDA-MB-231 cells stably overexpressing GFP-miR-31 show reduced β1 integrin activation due to a quantitative reduction in β1 integrin surface expression. (A) Activation of β1 integrins was monitored with HUTS-4 antibody, specific for activated β1 integrins, by flow cytometry. Spontaneous HUTS-4 binding or that induced by MnCl2 are shown. Cells were either left untreated or treated with 3.5mM MnCl2 and incubated with HUTS-4 antibody followed by antimouse Alexa Fluor 647 IgG to monitor β1 integrin activation status. (B) HUTS-4 binding normalized to total β1 integrin expression measured with mAb MAB2000 (Millipore) on control vector- or the miR-31-expressing cells in the resting and Mn2+-activated states. HUTS-4 binding was done as in (A) and integrin expression was monitored using the same protocol as for HUTS-4 staining, using the expression-specific β1 antibody. Values in both panels are means±S.E. and representative of 3 independent experiments.
Fig. 5
Fig. 5. miR-31 directly targets the 3’UTR of ITGA2, ITGA5, ITGAV and ITGB3 integrins and represses their expression in MDA-MB-231 cells
MDA-MB-231 cells were transfected with the luciferase reporter plasmids containing the 3’UTR of the indicated integrin subunit or with deletion of the miR31 seed sequences, and co-transfected with or without miR31. Luciferase activities were measured after 24 h. For all luciferase activity assays, renilla luciferase activity was used for normalization. The normalized firefly luciferase activity of each construct was plotted as a ratio of the control microRNA over the miR-31 treatment. The data are the mean ± s. d. of at least 3 independent transfections.
Fig. 6
Fig. 6. miR-31 affects cell spreading in a ligand-dependent manner
Wild-type and miR-31-expressing MDA-MB-231 cells spread on collagen, Matrigel, fibronectin and laminin for 1 h, were fixed, permeabilized and stained for 30 min with Alexa 568-phalloidin to visualize actin filaments. (A) In comparison to vector control cells, miR-31-transfected cells showed significant inhibition of spreading and actin organization on collagen and Matrigel; lesser perturbation on laminin, and no difference on fibronectin. The images shown are representative micrographs of 12-15 independent scans for each integrin ligand. Bar = 40 μm. (B) Quantitation of cell spreading presented as the total surface area covered by cells, in square micrometer, averaged to 300 cells.
Fig. 7
Fig. 7. A function blocking mAb to α2β1 completely inhibits cell spreading in both the control cells and the miR31-expressing MDA-MB-231 cells
Wild-type and miR-31-expressing MDA-MB-231 cells where incubated with either an α2β1 monoclonal blocking antibody (LS-C247646) or control IgG and spread on collagen for 1 h. Cells were then fixed, permeabilized and stained for 30 min with Alexa 568-phalloidin to visualize actin filaments. A. The images shown are representative micrographs of 12-15 independent scans for each treatment. Bar = 40 μm. B. Quantitation of cell spreading presented as the total surface area covered by cells, in square micrometer, from 300 cells.
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References

    1. Johnson MS, Lu N, Denessiouk K, Heino J, Gullberg D. Integrins during evolution: evolutionary trees and model organisms. Biochim Biophys Acta. 2009 April;1788(4):779–89. - PubMed
    1. Humphries JD, Byron A, Humphries MJ. Integrin ligands at a glance. J Cell Sci. 2006 October 1;119(Pt 19):3901–3. - PMC - PubMed
    1. Cavallaro U, Dejana E. Adhesion molecule signalling: not always a sticky business. Nat Rev Mol Cell Biol. 2011 March;12(3):189–97. - PubMed
    1. Schmidt S, Friedl P. Interstitial cell migration: integrin-dependent and alternative adhesion mechanisms. Cell Tissue Res. 2010 January;339(1):83–92. - PMC - PubMed
    1. Desgrosellier JS, Cheresh DA. Integrins in cancer: biological implications and therapeutic opportunities. Nat Rev Cancer. 2010 January;10(1):9–22. - PMC - PubMed

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