mir sensor assay  (Addgene inc)

Journal: Oncology Reports

Article Title: POSTN + fibroblast-secreted small extracellular vesicles drive macrophage M2 polarization through BMP4/BMPR2/Smad signaling

doi: 10.3892/or.2026.9067

Figure Lengend Snippet: BMP4 activates BMPR2/Smad signaling to induce macrophage M2 polarization. (A) PCA analysis of macrophages were treated with sEV-derived CAF-S6 transfected with shNC and shPOSTN in three replicate times. (B) Venn-diagram of DEGs between shPOSTN sEV vs. CTRL and shPOSTN sEVs vs. shNC sEVs. (C) Heatmap showing the top DEGs in each group. (D) Volcano plot showing BMP4 downregulation in macrophages treated with shPOSTN sEVs vs. shNC sEVs. (E) GSEA analysis of hallmark pathways in macrophages treated with sEV-derived CAF-S6 transfected with either shPOSTN or shNC. (F) GO enrichment analysis was performed to identify pathways associated with the representative DEGs. (G) The bubble plot displays the top 20 markedly enriched KEGG pathways for the DEGs. (H) The mRNA expression of BMP4 in macrophages induced sEVs derived from CAF-S5/-S6 transfected with shNC and shPOSTN were analyzed by RT-qPCR. (I) The mRNA expression of TNF-α, IL-6, TGF-β and IL-10 in macrophages stimulated with BMP4 at concentrations of 0, 50 and 100 ng/ml. (J) The expression of CD163 and CD206 in macrophages treated with BMP4 at concentrations of 0, 100 ng/ml examined by flow cytometry. (K) The level of BMPR2, pSmad1/5/9, Smad 5 and Smad 9 in macrophages treated with BMP4 (100 ng/ml) for 48 h were examined by western blotting (n=3 per group). (L) The level of pSmad1/5/9, Smad5, Smad9, CD163 and CD206 in macrophages treatment with or without LDN193189 inhibitor (n=3 per group). Statistical significance was determined using a one-way ANOVA test, ns, not significant *P<0.05; **P<0.01; ****P<0.0001. Error bars represent the mean ± SEM. Source data for blotting assays, see . PCA, principal component analysis; sEV, small extracellular vesicles; sh, short hairpin; NC, negative control; DEGs, differentially expressed genes; POSTN, perostin; sEV, small extracellular vesicles; GSEA, gene set enrichment analysis; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; RT-qPCR, reverse transcription-quantitative PCR; pSmad, phosphorylated Smad.

Article Snippet: The primary antibodies included POSTN antibody (cat. no. ab14041; 1:500; Abcam), CD9 (cat. no. ab236630; 1:1,000; Abcam), CD81 (cat. no. ab79559; 1:1,000; Abcam), Calnexin (10427-2-AP; 1:1,000, Proteintech, Wuhan, China), CD80 (cat. no. ab134120; 1:1,000; Abcam), CD86 (cat. no. abs115477; 1:1,000; Absin Bioscience), CD163 (sc-33715; 1:1,000, Santa Cruz Biotechnology), CD206 (cat. no. ab64693; 1:1,000; Abcam), BMPR2 (cat. no. abs147034; 1:1,000; Absin Bioscience), Phospho-Smad 1 (Ser463/465)/Smad5(Ser463/465)/Smad9(Ser465/467) (cat. no. 13820; 1:1,000; Cell Signaling Technology, Inc.), Smad5 (12534; 1:1,000; Cell Signaling Technology, Inc.), Smad9 (cat. no. abs131190; 1:1,000; Absin Bioscience) and GAPDH (cat. no. 10494-1-AP; 1:5,000; Proteintech Group, Inc.), followed by incubation with a HRP-conjugated Goat anti-Rabbit IgG (H+L) as the secondary antibody (cat. no. SA00001-2; 1:3,000; Proteintech Group, Inc.) for 2 h. After washing the membrane three times with 1X TBST, the protein bands were visualized using an ECL detection system.

Techniques: Derivative Assay, Transfection, Expressing, Quantitative RT-PCR, Flow Cytometry, Western Blot, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Circulation

Article Title: Breast Cancer Reveals Latent BMPR2 -Related Susceptibility to Pulmonary Hypertension

doi: 10.1161/CIRCULATIONAHA.125.079067

Figure Lengend Snippet: BMPR2 (bone morphogenetic protein receptor type 2) is associated with pulmonary hypertension and breast cancer. A , Bioinformatic analysis reveals that BMPR2 is associated with 14 pulmonary arterial hypertension–related disease terms, 5 breast cancer–related disease terms, hyperandrogenism, and 2-oxo-hept-3-ene-1,7-dioate hydratase activity. B , Distribution of rare, predicted deleterious BMPR2 variants identified in breast tumors, including missense, nonsense, frameshift deletion, and insertion mutations from cBioPortal. The pulmonary arterial hypertension (PAH)–associated G83R variant is indicated. C , Copy number alteration (CNA) analysis of BMPR2 from cBioPortal breast cancer datasets (n=2599). Of 76 samples with copy number alterations, 92% were deep deletions and 8% amplifications. D , Violin plot depicting the distribution of BMPR2 log2 standardized mRNA levels across different breast cancer subtypes (basal-like, HER2-E, luminal A, and luminal B) and normal breast–like tissue. Embedded box plots represent the median (horizontal line) and interquartile range (box boundaries). Sample sizes for each group are indicated in parentheses. One-way ANOVA followed by Tukey-Kramer post hoc tests for multiple comparisons: ** P <0.01, **** P <0.0001. E , Quantification and representative immunofluorescence images showing decreased BMPRII protein expression in breast carcinoma compared with normal mammary gland tissue in human. Each dot represents a distinct patient, with values corresponding to the average signal from 8 mammary gland regions (n=4 non–breast cancer control, n=19 breast carcinoma). Unpaired t test. *** P <0.005. F through J , spontaneous mammary tumor development over time in Bmpr2-mutated ( Bmpr2 +/Δ71 ) and wild-type ( Bmpr2 +/+ ) rats. F , Kaplan-Meier curve showing the tumor-free cumulative risk in Bmpr2 +/Δ71 rats (red line) compared with Bmpr2 +/+ animals (black line) over time. Numbers at risk for each group are shown below the plot. G , Bar graph summarizing the percentage of rats with or without tumors at the end point. Bmpr2 +/Δ71 rats show a significantly higher tumor incidence (14%) compared with Bmpr2 +/+ controls (2%). Z score, n=71 Bmpr2 +/Δ71 and 45 Bmpr2 +/+ rats. H , Macroscopic and histological analysis of mammary tumors in Bmpr2 +/Δ71 rats. The representative macroscopic image ( left ) shows large tumor masses compared with healthy tissue. Histological sections depict healthy mammary tissue, fibroadenoma, and adenocarcinoma, stained with hematoxylin-eosin (H&E). Scale bars: 200 μm ( top ) and 50 μm ( bottom ). I , Correlation analysis between BMPR2 and BRCA1 expression in rat mammary tissue, showing a strong positive association (Pearson r=0.857, P <0.0001). J , Representative immunofluorescence staining of BMPR2 (green) and BRCA1 (magenta) in mammary glands from wild-type (WT) and Bmpr2 +/Δ71 rats. Scale bars: 50 μm. aa indicates amino acids; and A.U., arbitrary units.

Article Snippet: Seven-week-old (180 g) WT and Bmpr2 +/Δ71 female Sprague-Dawley rats were randomized into 4 groups.

Techniques: Activity Assay, Variant Assay, Immunofluorescence, Expressing, Control, Staining

Journal: Circulation

Article Title: Breast Cancer Reveals Latent BMPR2 -Related Susceptibility to Pulmonary Hypertension

doi: 10.1161/CIRCULATIONAHA.125.079067

Figure Lengend Snippet: Mammary tumors promote pulmonary hypertension development in Bmpr2 (bone morphogenetic protein receptor type 2)–mutated female rats. Mammary tumors were induced in female wild-type (WT) and Bmpr2 +/Δ71 rats by 7,12-dimethylbenz[a]anthracene (DMBA). Compared with wild-type, wild-type+tumor, and Bmpr2 +/Δ71 without tumors, Bmpr2 +/Δ71 +tumor rats exhibited increased ( A ) right ventricular systolic pressure (RVSP), ( B ) elevated total pulmonary resistance (TPR), ( C ) reduced stroke volume (SV), and ( D ) decreased cardiac output (CO). E , Elastic Van Gieson staining revealed greater pulmonary vascular remodeling in Bmpr2 +/Δ71 +tumor rats, which was associated with ( F ) increased pulmonary arterial smooth muscle cell proliferation, as shown by proliferating cell nuclear antigen (PCNA) (red) and smooth muscle actin (green) immunofluorescence, with representative images in G (scale bars: 10 μm for elastic Van Gieson stain and proliferating cell nuclear antigen, 50 μm for CD45 and CD68). Monocyte accumulation (CD68) ( H ) and leukocyte infiltration (CD45) ( I ) were also elevated in Bmpr2 +/Δ71 +tumor lungs, accompanied by increased IL-1β mRNA expression (droplet digital polymerase chain reaction, housekeeping gene HPRT1) ( J ) and NF-κB protein levels (Western blot) ( K ). Statistical comparisons were performed using 1-way ANOVA followed by Tukey-Kramer post hoc tests for multiple comparisons. α-SMA indicates alpha–smooth muscle actin; AB, amido black; and DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Seven-week-old (180 g) WT and Bmpr2 +/Δ71 female Sprague-Dawley rats were randomized into 4 groups.

Techniques: Staining, Immunofluorescence, Expressing, Digital PCR, Western Blot

Journal: Circulation

Article Title: Breast Cancer Reveals Latent BMPR2 -Related Susceptibility to Pulmonary Hypertension

doi: 10.1161/CIRCULATIONAHA.125.079067

Figure Lengend Snippet: Bmpr2 (bone morphogenetic protein receptor type 2) mutation is associated with proinflammatory mammary tumors and increased circulating inflammatory mediators. A , Volcano plot showing differentially expressed genes in mammary tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) in Bmpr2 +/Δ71 and wild-type (WT) female rats. Upregulated differentially expressed genes in Bmpr2 +/Δ71 tumors are shown in red, and downregulated differentially expressed genes are shown in blue. The dashed red line indicates the cutoff of adjusted P ( P adj)<0.05 and log 2 fold change (FC)>1. B , Gene Ontology (GO) analysis of upregulated differentially expressed genes was conducted using four databases: GO biological process (GO_BP, green), GO cellular component (GO_CC, blue), GO molecular function (GO_MF, mauve), and Kyoto Encyclopedia of Genes and Genomes (KEGG) (turquoise). The red arrowheads highlight GO terms related to inflammation. C , Bmpr2 +/Δ71 tumors exhibit increased monocyte accumulation (CD68 immunofluorescence) and ( D ) elevated leukocyte infiltration (CD45 immunofluorescence) compared with wild-type tumors. Representative images show positive cells, indicated by white arrows; scale bars: 50 µm. Bottom left insets display positive cells at higher magnification. E , the expression levels of 19 proinflammatory cytokines were measured in the blood of 15 Bmpr2 +/Δ71 +tumor animals and 15 wild-type+tumor animals. Bmpr2 +/Δ71 +tumor animals exhibit significantly increased levels of 5 proinflammatory cytokines: IL-6, LIX, IFN-γ, IL-1β, and IL-1α. F , Bmpr2 +/Δ71 +tumor animals show an overall increase in proinflammatory markers, quantified by a larger area under the curve (AUC). Statistical analysis was performed using unpaired t test. * P <0.05, ** P <0.01, *** P <0.005.

Article Snippet: Seven-week-old (180 g) WT and Bmpr2 +/Δ71 female Sprague-Dawley rats were randomized into 4 groups.

Techniques: Mutagenesis, Immunofluorescence, Expressing

Journal: Circulation

Article Title: Breast Cancer Reveals Latent BMPR2 -Related Susceptibility to Pulmonary Hypertension

doi: 10.1161/CIRCULATIONAHA.125.079067

Figure Lengend Snippet: Tumor-secreted IL-1β promotes proliferation of pulmonary arterial smooth muscle cells (PASMCs) in Bmpr2 +/Δ71 rats. A , Conditioned media (CM) harvested from Bmpr2 -mutated tumor cells (C.M.- Bmpr2 +/Δ71 ) increases proliferation of Bmpr2 +/Δ71 rat PASMCs ( Bmpr2 +/Δ71 r-PASMCs), as shown by Ki67 immunofluorescence (red). Representative images; scale bars: 50 µm. Elevated IL-1β levels in Bmpr2 +/Δ71 tumor cells compared with wild-type (WT) tumor cells were detected by ( B ) quantitative reverse transcription–polymerase chain reaction (qRT-PCR) (housekeeping gene HPRT1) and ( C ) IL-1β immunofluorescence (red). Representative images; scale bars: 5 µm. D , Quantification of Bmpr2 +/Δ71 r-PASMC proliferation: untreated Bmpr2 +/Δ71 r-PASMCs, Bmpr2 +/Δ71 r-PASMCs exposed to CM Bmpr2 +/Δ71 pretreated with IgG control antibody ( Bmpr2 +/Δ71 +IgG), and r Bmpr2 +/Δ71 -PASMCs exposed to CM Bmpr2 +/Δ71 pretreated with anti-IL-1β neutralizing monoclonal antibodies ( Bmpr2 +/Δ71 + IL-1β-ab). Ki67 immunofluorescence (red) was used for proliferation assessment. Representative images; scale bars: 50 µm. Statistical comparisons were performed using 1-way ANOVA followed by Tukey-Kramer post hoc tests for multiple comparisons and unpaired t test. A.U. indicates arbitrary units; DAPI, 4′,6-diamidino-2-phenylindole; IgG, immunoglobulin G; IL-1β, interleukin-1 beta; and r-PASMC, rat pulmonary arterial smooth muscle cell.

Article Snippet: Seven-week-old (180 g) WT and Bmpr2 +/Δ71 female Sprague-Dawley rats were randomized into 4 groups.

Techniques: Immunofluorescence, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Control, Bioprocessing

Journal: Circulation

Article Title: Breast Cancer Reveals Latent BMPR2 -Related Susceptibility to Pulmonary Hypertension

doi: 10.1161/CIRCULATIONAHA.125.079067

Figure Lengend Snippet: Schematic summary of the bidirectional relationship between BMPR2 (bone morphogenetic protein receptor type 2) deficiency, breast cancer, and pulmonary arterial hypertension (PAH). Preclinical setting: Bmpr2 +/Δ71 rats, characterized by reduced BMPR2 and BRCA1 expression, develop spontaneous mammary tumors with aging. Upon 7,12-dimethylbenz[a]anthracene (DMBA) exposure, Bmpr2 +/Δ71 rats exhibit exacerbated pulmonary hypertension, associated with increased lung inflammatory cell infiltration, elevated IL-1β levels, pulmonary vascular remodeling, and enhanced pulmonary arterial smooth muscle cell (PASMC) proliferation. Mammary tumors from Bmpr2 +/Δ71 rats show increased IL-1β expression and inflammatory infiltration, leading to higher circulating IL-1β and a proproliferative paracrine effect on pulmonary arterial smooth muscle cells. Human patients: Population-level analysis of the French National Healthcare Database revealed a bidirectional epidemiologic association between breast cancer and pulmonary arterial hypertension. The incidence of breast cancer was increased in patients with pulmonary arterial hypertension (215 vs 97 per 100 000 person-years), while the incidence of pulmonary arterial hypertension was higher in patients with breast cancer (66 vs 7 per 1 000 000 person-years). Transcriptomic and proteomic analyses of human samples showed reduced BMPR2 mRNA and protein levels in breast carcinoma compared with normal breast tissue. IL-1β indicates interleukin-1 beta.

Article Snippet: Seven-week-old (180 g) WT and Bmpr2 +/Δ71 female Sprague-Dawley rats were randomized into 4 groups.

Techniques: Expressing