miR-149 expression affects cell adhesion and cell spreading

Cell adhesion and spreading are prerequisites for matrix-dependent cell migration. To study this in more detail, we transiently transfected MDA-MB-231 cells with control miRNA and miR-149, respectively, and plated the cells onto collagen-coated dishes for different periods of time (Figure 26A). While control cells were fully spread 4 hours after plating, miR-149 ex-pressing cells had a rounder morphology and after 24 hours they were still less spread (Figure 26A). To measure cell adhesion and spreading in real-time, we plated cells onto col-lagen-coated E-plates and analyzed the impedance (cell index) using the xCELLigence de-vice. The cell index is proportional to coverage of the surface area by spreading cells. Figure 26B shows that the plateau reached by miR-149 expressing cells was lower than that of the

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control cells. The number of adherent cells was unaffected by miR-149 expression (data not shown), indicating a defect in cell spreading rather than cell adhesion.

Figure 26: miR-149 interferes with cell spreading on collagen. MDA-MB-231 cells were transiently transfected with a control miRNA (miR-con) or miR-149. Three days post transfection cells were har-vested and plated onto collagen in 0.5% FCS. (A) Cells were fixed and stained with crystal violet at the indicated times. (B) Cell adhesion and spreading on collagen-coated E-plates was measured using the xCELLigence device. Values correspond to the mean ± SEM of duplicate samples.

For a better understanding of the molecular details we analyzed cellular architecture of MDA-MB-231 cells at early times of adhesion by phalloidin and vinculin staining. Phalloidin binds to filamentous actin thereby visualizing the cytoskeleton, while the focal adhesion protein vinculin connects integrins with the actin cytoskeleton. Compared with the control, miR-149 expressing cells exhibited a depolarized actin cytoskeleton and failed to establish prominent cell protrusions and lamellipodia (Figure 27).

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Figure 27: miR-149 affects polarization of actin cytoskeleton during cell spreading on collagen.

MDA-MB-231 cells were transiently transfected with a control miRNA (miR-con) or miR-149. Three days post transfection cells were harvested and plated onto collagen-coated coverslips for the indicat-ed times in 0.5% FCS followindicat-ed by staining with phalloidin and an anti-vinculin antibody. The confocal images shown are stacks of three sections taken from the bottom of the cell.

We next examined biochemically how miR-149 expression affected the activation of key sig-naling molecules downstream of integrin engagement by analyzing Src and paxillin phos-phorylation. The focal adhesion protein paxillin and the intracellular non-receptor tyrosine kinase Src are involved in the regulation of focal adhesions and cytoskeletal remodeling. The activity of both proteins is regulated by phosphorylation. In miR-149 overexpressing cells Src phosphorylation was strongly reduced especially at early times of adhesion and spreading, whereas paxillin phosphorylation, an indicator of focal adhesion formation and turnover, was still low 4 hours after plating onto collagen (Figure 28). This is in line with the impaired spreading phenotype in miR-149 expressing cells.

Figure 28: miR-149 interferes with cell spreading on collagen. MDA-MB-231 cells were transiently transfected with a control miRNA (miR-con) or miR-149. Three days post transfection cells were har-vested and seeded onto collagen-coated dishes in the presence of 0.5% FCS. After the indicated times cells were lysed and lysates were immunoblotted using the indicated antibodies. The blots were cropped and in each case the corresponding panels are from the same gel.

3.2.4 Molecular targets of miR-149

To obtain insight into the potential signaling molecules affected by miR-149, we used the miRecords online tool (http://mirecords.biolead.org/) that integrates eleven miRNA prediction programs, resulting in 325 unique targets predicted by at least four different algorithms. The-se genes were mapped on the Kyoto Encyclopedia of Genes and Genomes (KEGG) way database, enabling the annotation of 79 genes. The pie chart in Figure 29 depicts path-ways that contain at least three annotated genes and comprises 49 genes in total (see Table 23).

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Figure 29: miR-149 targets molecules downstream of the integrin pathway. The pie chart depicts the mapping of 325 unique miR-149 target genes onto KEGG pathways (Version 2.5). Human path-ways containing at least three target genes are shown, comprising 49 genes in total.

Notably, apart from ‘cytokine cytokine receptor interaction’, ‘MAPK signaling pathway’ and

‘regulation of actin cytoskeleton’, ‘focal adhesion’ contained the highest number of predicted miR-149 target genes.

Table 23: List of predicted miR-149 target genes according to their KEGG pathway annotation.

KEGG pathway gene symbol

HSA04510 focal adhesion PAK3, PDGFRA, PGF, TNXB, PDGFC, KDR, VAV2, PXN, RAP1B, RAP1A, BCL2, TLN2, SRC

HSA04060 cytokine cytokine receptor pathway

CLCF1, PDGFRA, EDA, TNFRSF19, PDGFC, KDR, INHBB, IL6, ACVR1B

HSA04010 MAPK signaling pathway PRKACG, PDGFRA, DUSP16, SRF, NTRK2, RAP1B, RAP1A, ACVR1B

HSA04810 regulation of actin cyto-skeleton

PAK3, PIP4K2B, BAIAP2, PDGFRA, VAV2, PXN, ARPC4

HSA04540 GAP junction ADCY1, PRKACG, PDGFRA, PDGFC, PRKG1, SRC HSA04310 WNT siganling pathway PRKACG, CAMK2G, FZD5, SFRP1, TBL1X, FOSL1, HSA04350 TGFβ signaling pathway SP1, ID4, INHBB, ACVRL1, ACVR1B

HSA04360 axon giudance SEMA4G, PAK3, EPHB3, UNC5C, SEMA5A HSA04720 long term potentiation ADCY1, PRKACG, CAMK2G, RAP1B, RAP1A HSA04916 melanogenesis ADCY1, PRKACG, CREB3L3, CAMK2G, FZD5 HSA04020 Ca signaling pathway ADCY1, PRKACG, PDGFRA, ATP2A2, CAMK2G HSA05211 renal cell carconoma PAK3, PGF, EGLN3, RAP1B, RAP1A

HSA04210 Apoptosis PRKAR2A, PRKACG, BCL2, IRAK4

HSA04670 leukocyte transendothelial migration

VAV2, PXN, RAP1B, RAP1A

HSA04912 GNRH signaling pathway ADCY1, PRKACG, CAMK2G, SRC HSA05210 colorectal cancer PDGFRA, FZD5, BCL2, ACVR1B

FOCAL ADHESION (13)

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HSA04012 ErbB signaling pathway PAK3, CAMK2G, SRC HSA04370 VEGF signaling pathway KDR, PXN, SRC HSA04640 hematopoetic cell lineage CD59, MS4A1, IL6

HSA04740 olfactory transduction PRKACG, CAMK2G, PRKG1 HSA05215 prostate cancer PDGFRA, CREB3L3, BCL2 HSA04520 adherens junction BAIAP2, ACVR1B, SRC HSA04530 tight junction EPB41L1, EXOC4, SRC

The focal adhesion pathway includes the Ras related small GTPases Rap1A and Rap1B and the guanine nucleotide exchange factor (GEF) Vav2. To assess whether Rap1a/b and Vav2 were targeted by miR-149, we performed qRT-PCR analyses of mRNA samples extracted from MDA-MB-231 cells transfected with control miRNA and miR-149, respectively. Indeed, compared with the control, the transcript levels of Rap1A/B and Vav2 were significantly re-duced by miR-149 expression (Figure 30). Of note, we did not observe any changes in the expression levels of PAK3, Src or paxillin, which were also predicted as targets and are as-sociated with the focal adhesion pathway.

Figure 30: mir-149 targets molecules downstream of the integrin pathway. MDA-MB-231 cells were transiently transfected with a control miRNA (miR-con) or miR-149, respectively. Two days post transfection, RNA was extracted and Vav2, Rap1A and Rap1B transcript levels were determined by qRT-PCR. Values were normalized to GAPDH. Data are shown as the mean ± SEM of four independ-ent experimindepend-ents and were analyzed using two way Anova with Bonferroni posttests. ***p < 0.001.