TGFβ1-induced EPCs underwent EndMT.

<p>(A) Flow cytometry analysis showed that 95% of cells were positive for both CD34 and <i>KDR</i>. (B) After the treatment with TGFβ1 (5 ng/mL) for 7 days, the morphology changed in EPCs (original magnification ×200, Bar=200 μm). (C) The mRNA expression levels of endothelial cell markers (CD31 and vWF) and mesenchymal cell markers (α-SMA and myocardin) in TGFβ1-induced EPCs were determined by using qRT-PCR. (D) After the treatment with TGFβ1 (5 ng/mL) for 7 days, miR-126 expression in EPCs was determined by using qRT-PCR. EPCs without any treatment were used as the normal control. Data are shown as mean ± S.D. (n = 3). **, <i>P</i> < 0.01 compared with the normal control.. The number of observations (n) represents the number of independent cell preparations. </p

microRNA 126 Inhibits the Transition of Endothelial Progenitor Cells to Mesenchymal Cells via the PIK3R2-PI3K/Akt Signalling Pathway

<div><p>Aims</p><p>Endothelial progenitor cells (EPCs) are capable of proliferating and differentiating into mature endothelial cells, and they have been considered as potential candidates for coronary heart disease therapy. However, the transition of EPCs to mesenchymal cells is not fully understood. This study aimed to explore the role of microRNA 126 (miR-126) in the endothelial-to-mesenchymal transition (EndMT) induced by transforming growth factor beta 1 (TGFβ1). </p> <p>Methods and Results</p><p>EndMT of rat bone marrow-derived EPCs was induced by TGFβ1 (5 ng/mL) for 7 days. miR-126 expression was depressed in the process of EPC EndMT. The luciferase reporter assay showed that the PI3K regulatory subunit p85 beta (PIK3R2) was a direct target of miR-126 in EPCs. Overexpression of miR-126 by a lentiviral vector (lenti-miR-126) was found to downregulate the mRNA expression of mesenchymal cell markers (α-SMA, sm22-a, and myocardin) and to maintain the mRNA expression of progenitor cell markers (CD34, CD133). In the cellular process of EndMT, there was an increase in the protein expression of PIK3R2 and the nuclear transcription factors FoxO3 and Smad4; PI3K and phosphor-Akt expression decreased, a change that was reversed markedly by overexpression of miR-126. Furthermore, knockdown of PIK3R2 gene expression level showed reversed morphological changes of the EPCs treated with TGFβ1, thereby giving the evidence that PIK3R2 is the target gene of miR-126 during EndMT process. </p> <p>Conclusions</p><p>These results show that miR-126 targets <i>PIK3R2</i> to inhibit EPC EndMT and that this process involves regulation of the PI3K/Akt signalling pathway. miR-126 has the potential to be used as a biomarker for the early diagnosis of intimal hyperplasia in cardiovascular disease and can even be a therapeutic tool for treating cardiovascular diseases mediated by the EndMT process.</p> </div

PIK3R2 was a direct target of miR-126 in EPCs.

<p>(A) Diagram of <i>PIK3R2</i>-3′UTR-containing luciferase reporter gene construct and the 22-bp target site of miR-126 in <i>PIK3R2</i>-3′UTR. The mutated nucleotides in <i>PIK3R2</i>-3′UTR fragments are underlined. (B) Luciferase reporter assays. The wild-type or mutant reporter plasmids were cotransfected into EPCs, which were infected by control-lentivirus or miR-126-lentivirus. The relative luciferase values shown were normalized to transfections with the wild-type reporter plasmids. Values are the average ± S.D. of 3 replicates. (C)The scheme of the luciferase assay to evaluate the direct inhibition of mir-126 on PIK3R2 protein expression. (D) The expression levels of PIK3R2 mRNA in miR-126 EPCs, miR-126 control, and the negative control—all under TGFβ1 treatment—were examined by qRT-PCR. β-actin was used as an internal control. (E) The protein expression of PIK3R2 was examined by western blotting. β-actin was used as an internal control. ** represents <i>P</i> < 0.01 with an LSD t-test. </p

miR-126 activated the PI3K/Akt and inhibited the FoxO3/Smad4 signalling pathways in TGFβ1-induced EPCs.

<p>After the treatment with TGFβ1 (5 ng/mL) for 7 days, the protein in EPCs were prepared. Immunoblotting assays were performed using specific antibodies against PI3K, phosphor-Akt, Akt, FoxO3, and Smad4. β-actin and lamin A were used as internal controls for total cells and the nuclear proteins assay separately. The relative protein levels of these proteins were determined by densitometry analysis (n = 3). Data are shown as mean ± S.D. values. * and ** represent P < 0.05 and P < 0.01 with an LSD t-test, respectively. The number of observations (n) represents the number of independent cell preparations. </p

miR-126 inhibits EPC EndMT via targeting PIK3R2.

<p>(A) PIK3R2 expression was down-regulated by PIK3R2siRNA. (B) After the pre-treatment with PIK3R2siRNA, EPCs were treated TGFβ1 (5 ng/mL) for 7 days to induce EndMT. The αSMA expressions were detected by using immunofluorescence staining (Bar=50 μM; Blue for DAPI; Red for αSMA). ** represents <i>P</i> < 0.01 with an LSD t-test.</p

Overexpression of miR-126 inhibited the EndMT process of EPCs induced by TGFβ1.

<p>(A) The mRNA expression of progenitor cell markers (CD31, CD34, CD133, and <i>KDR</i>), (B) endothelial cell markers (VEGF, <i>Flt-1</i>, eNOS, and iNOS), and (C) mesenchymal cell markers (α-SMA, <i>sm22-α</i>, and myocardin) was determined by using qRT-PCR. (D) Protein expression of α-SMA was detected by immunofluorescence staining. EPCs induced by TGFβ1 were transfected with a miR-126-expressing lentiviral vector, an empty vector (lentivector), or neither, and EPCs without any treatment were used as the normal control. EPCs infected with an empty vector were used as the Lenti-miR control. Data shown are representative of 3 independent experiments. **, <i>P</i> < 0.01 compared with the Lenti-miR control. </p

Altered expression of heparan sulfate in <i>Ndst1</i>^f/f<i>MMTVCre</i>⁺ mammary epithelia.

<p>(<b>A</b>) Frozen sections of mammary glands were incubated with biotinylated FGF2, which binds to heparan sulfate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010691#pone.0010691-Bai1" target="_blank">[55]</a>. Binding of FGF2 was detected with streptavidin-HRP (brown stain). In wildtype <i>Ndst1</i>^f/f<i>MMTVCre</i>^– glands FGF2 binds to the basement membrane surrounding the epithelial ducts (arrowheads). The right panel magnifies the boxed region, revealing the sharp staining of the basement membrane underlying the epithelial cells. FGF2 also binds to the matrix surrounding the fat pad adipocytes (white arrowheads). Mutant <i>Ndst1</i>^f/f<i>MMTVCre</i>⁺ glands retain FGF2 binding around fat pad adipocytes, but binding to the basement membrane was greatly reduced. The lower right panel magnifies the boxed region. The bar in the left panels  = 50 µm, right panels 12.5 µm. (<b>B</b>) Heparan sulfate was isolated from [6-³H]glucosamine labeled mammary epithelial cells derived from <i>Ndst1</i>^f/f<i>MMTVCre</i>^– and <i>Ndst1</i>^f/f<i>MMTVCre</i>⁺ mice and degraded with nitrous acid <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010691#pone.0010691-Shively1" target="_blank">[58]</a>. The individual oligosaccharides were separated by gel filtration chromatography and the area under the peaks was used to determine the extent of N-sulfation of the chains <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010691#pone.0010691-Bame1" target="_blank">[59]</a>. dp, degree of polymerization. Inset: Graph of comparison of areas under the curves.</p

Characterization of the lactational defect in <i>Ndst1</i>-deficient mammary glands.

<p>Quantitative RT-PCR and Western blotting were used to characterize lactational capacity of <i>Ndst1</i>^f/f<i>MMTVCre</i>⁺ females. (<b>A</b>) RNA isolated from d1L <i>Ndst1</i>^f/f<i>MMTVCre</i>^– and <i>Ndst1</i>^f/f<i>MMTVCre</i>⁺ mammary glands was analyzed by quantitative RT-PCR for the expression of whey acidic protein (WAP), β-casein, and keratin 18, genes specifically expressed by mammary epithelial cells. Data was normalized to the expression of GAPDH transcripts present in the sample. (<b>B</b>) Western blotting with antibodies to keratin 7 in d1L glands further confirmed that the <i>Ndst1</i>^f/f<i>MMTVCre</i>⁺ mammary glands have a reduced population of epithelia. (<b>C</b>) Western blotting with antibodies to mouse milk from d1L glands shows that milk production was diminished in <i>Ndst1</i>^f/f<i>MMTVCre</i>⁺ glands. Blotting of d14P extracts showed that the antibodies were selective for milk protein except for a minor band at 67 kDa.</p

Hematoxylin/eosin stained sections of mammary glands.

<p>Mammary glands from D8P, D15P, and D1L <i>Ndst1</i>^f/f<i>MMTVCre</i>⁻ (<b>A–C, respectively</b>) and <i>Ndst1</i>^f/f<i>MMTVCre</i>⁺ (<b>D–F, respectively</b>) mice were stained with hematoxylin/eosin. No difference in glandular morphology was noted at D8P, but differences in ductal density occurred at D15P, which is quantified in (<b>G</b>). The difference in density between mutant and wildtype increased dramatically by D1L. Bars  = 125 µm.</p

Long Chain Fatty Acyl-CoA Synthetase 4 Is a Biomarker for and Mediator of Hormone Resistance in Human Breast Cancer

<div><p>The purpose of this study was to determine the role of long-chain fatty acyl-CoA synthetase 4 (ACSL4) in breast cancer. Public databases were utilized to analyze the relationship between ACSL4 mRNA expression and the presence of steroid hormone and human epidermal growth factor receptor 2 (HER2) in both breast cancer cell lines and tissue samples. In addition, cell lines were utilized to assess the consequences of either increased or decreased levels of ACSL4 expression. Proliferation, migration, anchorage-independent growth and apoptosis were used as biological end points. Effects on mRNA expression and signal transduction pathways were also monitored. A meta-analysis of public gene expression databases indicated that ACSL4 expression is positively correlated with a unique subtype of triple negative breast cancer (TNBC), characterized by the absence of androgen receptor (AR) and therefore referred to as quadruple negative breast cancer (QNBC). Results of experiments in breast cancer cell lines suggest that simultaneous expression of ACSL4 and a receptor is associated with hormone resistance. Forced expression of ACSL4 in ACSL4-negative, estrogen receptor α (ER)-positive MCF-7 cells resulted in increased growth, invasion and anchorage independent growth, as well as a loss of dependence on estrogen that was accompanied by a reduction in the levels of steroid hormone receptors. Sensitivity to tamoxifen, triacsin C and etoposide was also attenuated. Similarly, when HER2-positive, ACSL4-negative, SKBr3 breast cancer cells were induced to express ACSL4, the proliferation rate increased and the apoptotic effect of lapatinib was reduced. The growth stimulatory effect of ACSL4 expression was also observed <i>in vivo</i> in nude mice when MCF-7 control and ACSL4-expressing cells were utilized to induce tumors. Our data strongly suggest that ACSL4 can serve as both a biomarker for, and mediator of, an aggressive breast cancer phenotype. </p> </div