Journal: International Journal of Molecular Sciences

Article Title: Breast Cancer-Derived Extracellular Vesicle miR-425-5p (miR-425) Promotes Brain Metastasis via Activating Astrocytes Through the Novel miR-425-ZNF24-CCL8 Signaling Axis

doi: 10.3390/ijms27073197

Figure Lengend Snippet: miR-425 suppresses transcription factor ZNF24 in astrocytes. ( A ) Predicted miR-425 binding sites within the CREB1, BCOR, and ZNF24 3′-UTRs from TargetScan. ( B ) ZNF24 mRNA expression is significantly decreased in astrocytes overexpressing miR-425. CREB1 and BCOR mRNA expression is unchanged as measured by RT-qPCR. ( C ) ZNF24 protein expression is decreased in astrocytes transfected with miR-425 mimic as indicated by Western blot analysis. ( D ) miR-425 suppresses ZNF24 3′-UTR activity as measured by dual luciferase reporter assay. ( E ) Ectopic expression of ZNF24 significantly decreases CCL8 and VEGFA mRNA expression, while KITLG is unchanged. mRNA expression measured by RT-qPCR. ( F ) Ectopic expression of ZNF24 suppresses CCL8 protein expression, but not SCF as indicated by Western blot analysis. ( G ) ZNF24 knockdown significantly increases CCL8 and VEGFA mRNA expression, but not KITLG. mRNA expression as indicated by RT-qPCR. ( H ) ZNF24 knockdown increases CCL8 and SCF protein expression as measured by Western blot analysis. Fold change was calculated in panels ( B , D , E , G ). Student’s t -test was used in panels ( B , D , E , G ). N = 3 experimental replicates unless otherwise indicated.

Article Snippet: Astrocytes were transfected with control or miR-425 mimic, control vector or ZNF24 3′-UTR (GeneCopoeia; Cs-HmiT119980-MT05-01), control vector or ZNF24 overexpression (GeneCopoeia; cat. #B221017-1240ueu), and control promoter or CCL8 promoter (GeneCopoeia; cat. #HPRM46246-PG02) constructs using X-tremeGENE HP DNA Transfection Reagent (Millipore Sigma; cat. #6366244001).

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Transfection, Western Blot, Activity Assay, Luciferase, Reporter Assay, Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Breast Cancer-Derived Extracellular Vesicle miR-425-5p (miR-425) Promotes Brain Metastasis via Activating Astrocytes Through the Novel miR-425-ZNF24-CCL8 Signaling Axis

doi: 10.3390/ijms27073197

Figure Lengend Snippet: ZNF24 suppresses astrocyte activation and decreases CCL8 expression. ( A ) Human astrocytes activated by a cocktail of IL-1α, TNFα, and C1q. Astrocytes were serum-starved overnight before stimulation. Nuclei were stained with DAPI (blue), and GFAP was visualized in red. Representative 20× images. Scale bar indicates 100 µm. ( B ) Validation of GFAP mRNA expression in activated astrocytes as indicated by RT-qPCR. ( C ) Activated astrocytes transfected with ZNF24 plasmid have significantly reduced GFAP expression as indicated by GFAP IF. All slides were imaged at 4× or 20× using the Keyence BZ-X810 microscopes (cat. # BZ-X810 Keyence). At least 300 cells per slide were evaluated to identify GFAP+ cells. Representative 20× images. Scale bar indicates 100 µm. ( D ) Validation of GFAP reduction and ZNF24 overexpression with ectopic expression of ZNF24. mRNA expression measured by RT-qPCR. ( E ) ZNF24 binding to the CCL8 promoter in two regions (-69 and -205 bases upstream of the TSS) measured by ChIP-qPCR. ( F ) Ectopic expression of ZNF24 suppresses CCL8 promoter activity as indicated by dual luciferase reporter assays. ( G , H ) ZNF24 and CCL8 mRNA expression remains unchanged in breast cancer cells transfected with control or miR-425 mimic. mRNA expression measured by RT-qPCR. Fold change was calculated in panels ( B , D – H ). Student’s t -test was used in panels ( A – H ). N = 3 experimental replicates unless otherwise indicated.

Article Snippet: Astrocytes were transfected with control or miR-425 mimic, control vector or ZNF24 3′-UTR (GeneCopoeia; Cs-HmiT119980-MT05-01), control vector or ZNF24 overexpression (GeneCopoeia; cat. #B221017-1240ueu), and control promoter or CCL8 promoter (GeneCopoeia; cat. #HPRM46246-PG02) constructs using X-tremeGENE HP DNA Transfection Reagent (Millipore Sigma; cat. #6366244001).

Techniques: Activation Assay, Expressing, Staining, Biomarker Discovery, Quantitative RT-PCR, Transfection, Plasmid Preparation, Over Expression, Binding Assay, ChIP-qPCR, Activity Assay, Luciferase, Control

Journal: International Journal of Molecular Sciences

Article Title: Breast Cancer-Derived Extracellular Vesicle miR-425-5p (miR-425) Promotes Brain Metastasis via Activating Astrocytes Through the Novel miR-425-ZNF24-CCL8 Signaling Axis

doi: 10.3390/ijms27073197

Figure Lengend Snippet: The miR-425-ZNF24-CCL8 signaling pathway is upregulated in brain metastases and activated astrocytes of mice intracardially injected with breast cancer cells overexpressing miR-425. ( A ) Increased EV-miR-425 expression in mouse serum from the SKBR3-Luc-miR-425 group compared to the control group. miR-425 expression measured by RT-qPCR ( N = 5 per group). ( B ) SKBR3-Luc-miR-425 group mouse serum has significantly higher levels of mouse CCL8 as measured by ELISA ( N = 7 per group). ( C ) No significant difference in mouse SCF levels in SKBR3-Luc-miR-425 or control mice serum as measured by ELISA ( N = 5 per group). ( D , E ) GFAP H-score and intratumoral astrocytes are significantly higher in brain metastases from the SKBR3-Luc-miR-425 mouse group than the control in mouse brain sections as measured by IHC ( N = 5 per group). ( F ) Brain metastases from the SKBR3-Luc-miR-425 group have significantly increased Ki-67 positive cells as measured by IHC ( N = 5 per group). ( G ) Tumor-adjacent and infiltrative astrocytes in brain metastases from the SKBR3-Luc-miR-425 group have decreased ZNF24 staining measured by IHC ( N = 5 per group). ( H ) Representative IHC images at 20× magnification. Scale bar indicates 100 µm. ( I ) Co-staining IF of ZNF24 (green) and GFAP (red) in mouse brain sections containing brain metastases. Nuclei were stained with DAPI (blue). Representative images at 20× magnification. Scale bar indicates 100 µm. ( J ) Schematic of described mechanism by which breast cancer-derived EV-miR-425 activates astrocytes by suppressing ZNF24, increasing CCL8, and thereby promoting BCBM. Fold change was calculated in panel A. Student’s t -test used in panels ( A – G , I ).

Article Snippet: Astrocytes were transfected with control or miR-425 mimic, control vector or ZNF24 3′-UTR (GeneCopoeia; Cs-HmiT119980-MT05-01), control vector or ZNF24 overexpression (GeneCopoeia; cat. #B221017-1240ueu), and control promoter or CCL8 promoter (GeneCopoeia; cat. #HPRM46246-PG02) constructs using X-tremeGENE HP DNA Transfection Reagent (Millipore Sigma; cat. #6366244001).

Techniques: Injection, Expressing, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Derivative Assay

Journal: Diabetologia

Article Title: Human adipose microRNA-221 is upregulated in obesity and affects fat metabolism downstream of leptin and TNF-α

doi: 10.1007/s00125-013-2950-9

Figure Lengend Snippet: miR-221 targets the 3′ UTRs of ADIPOR1 and ETS1 and decreases their protein levels. ( a,b ) TargetScan conserved predicted miR-221 binding site on the 3′ UTRs of ADIPOR1 and ETS1 . ( c,d ) Quantification of dual-luciferase assay in HEK 293 cells co-transfected with miR-221 mimic (black bars) or control oligonucleotide (white bars) and ADIPOR1/2 ( c ) or ETS1 ( d ) 3′ UTR reporter plasmids. ( d ) Two overlapping fragments of the ETS1 3′-UTR were subcloned into separate vectors, indicated (A) and (B). Firefly/renilla luciferase ratio (luc ratio) was normalised to no-UTR controls. Error bars indicate SD ( n = 5). * p < 0.05 (two-tailed Mann–Whitney U test). ( e ) Quantification of QRT-PCR for ETS1 mRNA in human pre-adipocytes transfected with miR-221 mimic (black bars) or control oligonucleotide (white bars). Error bars indicate SD ( n = 3). ( f ) Immunoblots for ADIPOR1, ETS1 and β-actin in human pre-adipocytes transfected with miR-221 mimic (+) or control oligonucleotide. ( g ) Quantification of the immunoblots shown in ( f ). Background-subtracted mean signal ( n = 2) for ADIPOR1 and ETS1, normalised to the loading control (β-actin). Black bars, miR-221 mimic; white bars, control oligonucleotide

Article Snippet: For 3′ untranslated (UTR) reporter assays, JetPrime (Polyplus-transfection SA, Illkirch, France) was used to co-transfect HEK 293 cells (ATCC) with miRNA 3′ UTR target expression vectors for human ETS1 , ADIPOR1 , ADIPOR2 or control (Genecopoeia, Germantown, MD, USA), and human miR-221 mimic (Dharmacon, Lafayette, CO, USA) or scrambled control oligonucleotide, in 24-well plates.

Techniques: Binding Assay, Luciferase, Transfection, Control, Two Tailed Test, MANN-WHITNEY, Quantitative RT-PCR, Western Blot

Journal: Gut

Article Title: microRNA overexpression in slow transit constipation leads to reduced Na V 1.5 current and altered smooth muscle contractility

doi: 10.1136/gutjnl-2019-318747

Figure Lengend Snippet: Thirteen of 372 miRNA examined are differentially expressed in slow transit constipation. (A) Heatmap plotting negative Ct values from the miRNA qPCR array organised by increasing p values. The box indicates the ones with p<0.05. The expression level of miRNAs is colour-coded as indicated in the legend. (B) Blowup of highlighted group of miRNAs, with hierarchical clustering analysis on the left. (C) Map of SCN5A mRNA 3′-UTR with binding sites for miRNAs obtained from multiple prediction algorithms and literature. In the boxes are position and sequence of target region complementary to each miRNA seed. In red are miRNAs identified by qPCR array screening. (D) SCN5A mRNA expression is reduced in STC smooth muscle. Data are median±IQR. n=10–11. *p<0.05, Mann-Whitney test. CTRL, control; miRNA, microRNA; qPCR, quantitative PCR; STC, slow transit constipation.

Article Snippet: HEK293 cells were plated in six-well plates for 72 hours in minimum essential medium (MEM), supplemented with 10% fetal bovine serum and penicillin/streptomycin), then transfected by Lipofectamine 3000 (Invitrogen) with SCN5A full-length 3′ UTR (2259 bp) reporter vector (HmiT016601-MT05, Genecopoeia) and/or 50 nM of let-7f miRNA mimic (hsa-let7f-5p, Life Technologies) in a total volume of 2.4 mL per well.

Techniques: Expressing, Binding Assay, Sequencing, MANN-WHITNEY, Real-time Polymerase Chain Reaction

Journal: Gut

Article Title: microRNA overexpression in slow transit constipation leads to reduced Na V 1.5 current and altered smooth muscle contractility

doi: 10.1136/gutjnl-2019-318747

Figure Lengend Snippet: HuSMCs have characteristics similar to GI smooth muscle cells. (A) Representative images of cell tractions’ colour maps generated by the same HuSMC in basal conditions (left) and response to carbachol (right). The colours correspond to magnitudes of traction forces generated as indicated in the colour bar. The RMS traction forces generated are quantified in the graph. Data are median±IQR, n=7, *p<0.05, Mann-Whitney test. (B) HuSMCs (passage 6, P6) are highly enriched for mRNA for smooth muscle cell marker genes, including SCN5A and CACNA1C, compared with later passages (>10, P10), which lose smooth muscle phenotype (n=4). (C) HuSMCs express Na V 1.5 protein by western blot. (D) Representative traces of Na + currents recorded from HuSMCs. (E) I/V plot with Na + currents recorded from HuSMCs. Time to peak (F) and time constants of inactivation (G) calculated from currents recorded at different voltages. ACTA2 , smooth muscle actin; CACNA1C, L-type calcium channel α1C; CARB, carbachol; CTRL, control; GAPDH , glyceraldehyde 3-phosphate dehydrogenase; HuSMC, human smooth muscle cells; MYH11 , myosin-11; pA, peak Na + currents; pF, whole cell capacitance; RMS, root mean square; SCN5A , sodium channel Na V 1.5; SMTN , smoothelin.

Article Snippet: HEK293 cells were plated in six-well plates for 72 hours in minimum essential medium (MEM), supplemented with 10% fetal bovine serum and penicillin/streptomycin), then transfected by Lipofectamine 3000 (Invitrogen) with SCN5A full-length 3′ UTR (2259 bp) reporter vector (HmiT016601-MT05, Genecopoeia) and/or 50 nM of let-7f miRNA mimic (hsa-let7f-5p, Life Technologies) in a total volume of 2.4 mL per well.

Techniques: Generated, MANN-WHITNEY, Marker, Western Blot

Journal: Gut

Article Title: microRNA overexpression in slow transit constipation leads to reduced Na V 1.5 current and altered smooth muscle contractility

doi: 10.1136/gutjnl-2019-318747

Figure Lengend Snippet: Overexpression of Let-7f significantly reduced Na + current density in HuSMCs and resulted in changes in the cells’ properties. (A) RT-qPCR show efficient delivery of miRNA after transfection of mimics in HuSMCs (means±SDs, n=6). (B) Representative patch-clamp traces for HuSMC±miRNA mimics. (C) Peak Na + current densities were reduced after transfection with let-7f (means±SEMs, n=5–17 cells, *p<0.05 by a one-way analysis of variance with Dunnett’s post-test). (D) NLUs of HEK-293 cells transfected with SCN5A 3′UTR vector alone ( control ) or cotransfected with the 3′UTR vector and miRNA mimic ( let-7f ) (means±SEMs, n=6 transfections, *p<0.05 to same-plate controls by a two-tailed paired t-test). GLuc, Gaussia luciferase; HuSMC, human smooth muscle cells; miRNA, microRNA; NLU, normalised luciferase unit; NT, non-targeted; pA, peak Na + currents; pF, whole cell capacitance; SEAP, secreted alkaline phosphatase.

Article Snippet: HEK293 cells were plated in six-well plates for 72 hours in minimum essential medium (MEM), supplemented with 10% fetal bovine serum and penicillin/streptomycin), then transfected by Lipofectamine 3000 (Invitrogen) with SCN5A full-length 3′ UTR (2259 bp) reporter vector (HmiT016601-MT05, Genecopoeia) and/or 50 nM of let-7f miRNA mimic (hsa-let7f-5p, Life Technologies) in a total volume of 2.4 mL per well.

Techniques: Over Expression, Quantitative RT-PCR, Transfection, Patch Clamp, Plasmid Preparation, Two Tailed Test, Luciferase

Journal: Cells

Article Title: Modification of Mesenchymal Stem/Stromal Cell-Derived Small Extracellular Vesicles by Calcitonin Gene Related Peptide (CGRP) Antagonist: Potential Implications for Inflammation and Pain Reversal

doi: 10.3390/cells13060484

Figure Lengend Snippet: ( A ) Gene construct of adenovirus vector containing the gene for CGRP 8–37 . The CGRP 8–37 fragment was inserted downstream of the signal sequence of peptidylglycine-amidating monooxygenase (ssPAM/pGEMT). This arrangement facilitates the amidation and secretion of CGRP 8–37 . ( B ) MSC were isolated from human infrapatellar fat pad (IFP), transduced with gene construct, and sorted by FACS cell sorting to generate aCGRP IFP-MSC. aCGRP IFP-MSC sEVs were isolated and characterized for their miRNA cargo. Functional assessment of aCGRP IFP-MSC sEVs was performed by macrophage polarization and cortical neurons neuroinflammation assays.

Article Snippet: Pre-designed human MSC exosome 166 miRNA qPCR arrays (GeneCopoeia; ) were performed using 1000 ng cDNA per IFP-MSC sample (n = 2), and processed using StepOne Real-time thermocycler (Applied Biosystems, LLC, Waltham, MA, USA).

Techniques: Construct, Plasmid Preparation, Sequencing, Isolation, Transduction, FACS, Functional Assay

Journal: Cells

Article Title: Modification of Mesenchymal Stem/Stromal Cell-Derived Small Extracellular Vesicles by Calcitonin Gene Related Peptide (CGRP) Antagonist: Potential Implications for Inflammation and Pain Reversal

doi: 10.3390/cells13060484

Figure Lengend Snippet: ( A ) 147 distinct miRNA were present in aCGRP IFP-MSC sEVs (n = 2). Nineteen highly present miRNAs are included in black-bordered box. ( B ) miRNAs present in aCGRP IFP-MSC sEVs were involved in the regulation of numerous genes and pathways. Predominantly, miRNAs present in sEVs are involved in the regulation of TGF-β/Wnt/FGFR pathways. Putative miRNA interactomes were generated using a miRNet centric network visual analytics platform. The miRNA target gene data were collected from well-annotated database miRTarBase v8.0 and miRNA-gene interactome network refining was performed with 2.0 betweenness cut-off. Values (with 34 cycles cut-off point) were represented in a topology miRNA-gene interactome network using force atlas layout and hypergeometric test algorithm.

Article Snippet: Pre-designed human MSC exosome 166 miRNA qPCR arrays (GeneCopoeia; ) were performed using 1000 ng cDNA per IFP-MSC sample (n = 2), and processed using StepOne Real-time thermocycler (Applied Biosystems, LLC, Waltham, MA, USA).

Techniques: Generated, Refining

Journal: Cells

Article Title: Modification of Mesenchymal Stem/Stromal Cell-Derived Small Extracellular Vesicles by Calcitonin Gene Related Peptide (CGRP) Antagonist: Potential Implications for Inflammation and Pain Reversal

doi: 10.3390/cells13060484

Figure Lengend Snippet: ( A ) In aCGRP IFP-MSC sEVs (n = 2), 19 miRNAs cargos were highly present. These distinct miRNAs regulate genes involved in the production of cytokines, the recruitment of monocytes, and cartilage homeostasis. From these miRNAs, 7 miRNAs were associated in previous studies with significant anti-inflammatory/immunomodulatory effects in vitro and in vivo. Putative miRNA interactomes were generated using a miRNet centric network visual analytics platform. The miRNA target gene data were collected from well-annotated database miRTarBase v8.0 and miRNA-gene interactome network refining was performed with 2.0 betweenness cut-off. Values (with 34 cycles cut-off point) were represented in a topology miRNA-gene interactome network using force atlas layout and hypergeometric test algorithm. ( B ) In silico analysis revealed a functional correlation of identified miRNAs in aCGRP IFP-MSC sEVs with genes involved in M2 macrophage polarization, immunomodulatory and pain signaling, and cartilage homeostasis. The miRDB online database for prediction of functional miRNA targets has been used to correlate highly expressed target genes in macrophages with specific miRNAs identified by aCGRP IFP-MSC sEV miRNA profiling. MirTarget prediction scores are in the range of 0–100% probability, and candidate transcripts with scores ≥ 50% are presented as predicted miRNA targets in miRDB.

Article Snippet: Pre-designed human MSC exosome 166 miRNA qPCR arrays (GeneCopoeia; ) were performed using 1000 ng cDNA per IFP-MSC sample (n = 2), and processed using StepOne Real-time thermocycler (Applied Biosystems, LLC, Waltham, MA, USA).

Techniques: In Vitro, In Vivo, Generated, Refining, In Silico, Functional Assay

Journal: Cells

Article Title: Modification of Mesenchymal Stem/Stromal Cell-Derived Small Extracellular Vesicles by Calcitonin Gene Related Peptide (CGRP) Antagonist: Potential Implications for Inflammation and Pain Reversal

doi: 10.3390/cells13060484

Figure Lengend Snippet: ( A , B ) Multiple immunomodulatory and reparative molecules secreted as a cargo of aCGRP IFP-MSC sEVs (n = 2). sEVs showed presence of key immunomodulatory molecules including TIMP-2, IL-8, MCP-1, IL-6, ICAM-1, sTNF-RI, MIP-1β, IL-10, and IP-10. In parallel, key reparative molecules including HGF, VEGF, EGFR, IGFBP-1, βFGF, and IGFBP-6 showed presence in aCGRP IFP-MSC sEVs. These proteins are listed in descending order based on their presence levels within sEVs. The miRDB online database for prediction of functional miRNA targets has been used to correlate highly expressed target genes in macrophages with specific miRNAs identified by aCGRP IFP-MSC sEV miRNA profiling. In terms of biological processes, various categories were highly affected and presented as % of proteins involved in a category to the total proteins detected. aCGRP IFP-MSC sEVs protein cargo have effects on PI3K-Akt signaling pathway (59%), MAPK signaling pathway (55%), Ras signaling pathway (52%), Rap1 signaling pathway (43%), cytokine-cytokine receptor interaction (24%), and Jak-STAT signaling pathway (19%). MirTarget prediction scores are in the range of 0–100% probability, and candidate transcripts with scores ≥ 50% are presented as predicted miRNA targets in miRDB.

Article Snippet: Pre-designed human MSC exosome 166 miRNA qPCR arrays (GeneCopoeia; ) were performed using 1000 ng cDNA per IFP-MSC sample (n = 2), and processed using StepOne Real-time thermocycler (Applied Biosystems, LLC, Waltham, MA, USA).

Techniques: Functional Assay

Journal: Cell Death & Disease

Article Title: Cell differentiation versus cell death: extracellular glucose is a key determinant of cell fate following oxidative stress exposure

doi: 10.1038/cddis.2014.52

Figure Lengend Snippet: Sirtuin 3 is a novel target of miR28-5p. RNA levels of ( a ) sirtuin 3 but not ( b ) sirtuin 1 were lower in peroxide-treated cells cultured in high glucose compared with untreated controls. Similarly, protein levels (as measured by relative quantification of the band density from western blots) of ( c ) sirtuin 3 were significantly lower in peroxide-treated cells cultured in high glucose compared with untreated controls or cells cultured in low glucose with or without peroxide treatment. ( d ) However, protein levels of sirtuin 1 were not significantly different between treatments. ( e ) No increase in the level of acetylated p53 was observed in peroxide-treated cells cultured in high glucose and infected with a sirtuin 3-bearing adenoviral vector (adSirt3). A GFP-bearing adenoviral vector (adGFP) was used as a control. ( f ) Levels of miR28-5p were significantly higher in peroxide-treated cells cultured in high but not low glucose compared with untreated controls. ( g ) Graph highlighting the difference in levels of miR28-5p in untreated cells in high and low glucose (note the y -axis scale differs from that in f ). Levels of miR28-5p were higher in untreated cells cultured in high glucose compared with in cells cultured in low glucose. ( h ) In cells cultured in high glucose, RNA levels of sirtuin 3 were significantly higher in peroxide-treated cells transfected with a miR28-5p inhibitor compared with peroxide-treated controls. ( i ) Conversely, in cells cultured in low glucose, RNA levels of sirtuin 3 were significantly lower in cells expressing a miR28-5p mimic compared with peroxide-treated controls. ( j ) Activity of a 3′-UTR sirtuin 3 reporter in which the 3′ UTR sequence of sirtuin 3 had been inserted downstream of the secreted GLuc reporter gene was significantly lower in cells transfected with a miR28-5p mimic compared with non-mimic expressing cells. A secreted alkaline phosphatase (SEAP) reporter driven by the CMV promoter cloned into the same vector served as an internal control for transfection efficiency. The miR28-5p mimic had no effect on the negative control 3′UTR construct, demonstrating its specificity for the sirtuin 3 3′-UTR. Statistically significant ( P ≤0.05) differences between treatments are indicated by *. Results are expressed as mean±S.D. Experiments were performed on cells isolated from a minimum of three different patients. Western blot images shown are representative of those obtained for all patients

Article Snippet: Cells were then transfected with 100 ng of either negative control 3′-UTR (cat. no. CmiT000001-MT05) or Sirt3 3′UTR (cat no. HmiT006079-MT05) GLuc-SEAP vector (GeneCopoeia, Rockville, MD, USA) with 30 nM of either miR28-5p mimic or mimic control using lipofectamine 2000 (all from Life Technologies).

Techniques: Cell Culture, Western Blot, Infection, Plasmid Preparation, Transfection, Expressing, Activity Assay, Sequencing, Clone Assay, Negative Control, Construct, Isolation

Journal: Cell Death & Disease

Article Title: Cell differentiation versus cell death: extracellular glucose is a key determinant of cell fate following oxidative stress exposure

doi: 10.1038/cddis.2014.52

Figure Lengend Snippet: Proposed mechanism for the differential response of tenocytes to oxidative stress under different extracellular glucose concentrations. Oxidative stress results in upregulation of both FOXO1 as well as HIF1 α . Under high-glucose conditions, miR28-5p levels are also upregulated, particularly in oxidative stress-exposed cells. miR28-5p directly inhibits expression of the p53 deacetylase sirtuin 3, allowing accumulation of acetylated p53. FOXO1 promotes transcription of bim , the gene product of which is a proapoptotic protein. p53 inhibits expression of miR17-92, a cluster of miRNAs including the bim repressor miR17-5p. Downregulation of miR17-92 by p53 coupled with increased bim transcription by FOXO1 allows accumulation of bim RNA levels and increased bim-mediated apoptosis. Under low-glucose conditions, however, the miR28-5p–sirt3-p53 pathway is not stimulated. Instead, p38 MAPK is activated and acts on both FOXO1 and HIF1 α , resulting in the inhibition of FOXO1 transcriptional activity and activation of HIF1 α . HIF1 α promotes the expression of sox9 and scleraxis , two genes whose products are essential for tenocyte differentiation

Article Snippet: Cells were then transfected with 100 ng of either negative control 3′-UTR (cat. no. CmiT000001-MT05) or Sirt3 3′UTR (cat no. HmiT006079-MT05) GLuc-SEAP vector (GeneCopoeia, Rockville, MD, USA) with 30 nM of either miR28-5p mimic or mimic control using lipofectamine 2000 (all from Life Technologies).

Techniques: Expressing, Histone Deacetylase Assay, Inhibition, Activity Assay, Activation Assay

Journal: Cell Stress

Article Title: MiR-200c reprograms fibroblasts to recapitulate the phenotype of CAFs in breast cancer progression

doi: 10.15698/cst2024.03.293

Figure Lengend Snippet: (A) UMAP visualization of assigning normal fibroblast and CAF identity to cluster 4 and 5 in normal breast tissue (NT) and breast cancer tumor tissue (TT) by scRNAseq. Circled clusters are fibroblasts. All cell type profiles are listed in SFig. 1A. (B) Expression of markers in fibroblast cluster 4 from NT and TT by DotPlot. (C) VinPlot of expression of collagen, activated fibroblast, EMP and stemness markers in cluster 4 from NT and TT. *Seurat adjusted p < 0.05. (D) MIR200CHG level in cluster 4 from NT and TT. (E) Level of miR-200c in NT and TT from the TCGA breast cancer miRNAseq dataset. (F) Correlation between MIR200CHG and miR-200c from the TCGA breast cancer dataset. (G) Methylation status of miR-200c promoter region. Six sites are hypo-methylated in the tumor versus normal group. (H) Plot of the correlation between miR200c CNV and promoter methylation revealing a negative correlation. (A-D) are based on publicly available scRNA-seq data sets (GEO accession code GSE161529: GSM4909254 and GSM4909296). Data analysis was done in R using the Seurat package v4.3 with default parameters, padj<0.05.

Article Snippet: Precursor miR-200c (HmiR0180-MR03-10), anti-miR-200c (HmiR-AN0302-AM04) and control (CmiR0001-MR03, CmiR-AN0001-AM04) lentiviral plasmids were purchased from GeneCopoeia.

Techniques: Expressing, Methylation

Journal: Cell Stress

Article Title: MiR-200c reprograms fibroblasts to recapitulate the phenotype of CAFs in breast cancer progression

doi: 10.15698/cst2024.03.293

Figure Lengend Snippet: (A) CD24 and CD44 expression patterns by flow cytometry in BJ1 fibroblasts under homotypic and co-culture conditions with MCF7 carcinoma cells. (B) Fold change of miR-200c and miR-205 expression in BJ1 fibroblast cells co-cultured with carcinoma cells compared to homotypic culture. (C) Flow plots showed CD24 and CD44 level change in BJ1 cells overexpressing miR-200c and scramble control. (D) Images of cell morphology and K8/18 (Red) expression of BJ1 with precursor miR-200c expression and scramble control (scale bar: 20 μm). (E) Senescence β-galactosidase staining in BJ1 cells overexpressing miR-200c and scramble control. (F, G) Fold change in miR-200c and miR-205 expression in BJ1 and HS5 fibroblast cells with overexpression of miR-200c or control. *p<0.05.

Article Snippet: Precursor miR-200c (HmiR0180-MR03-10), anti-miR-200c (HmiR-AN0302-AM04) and control (CmiR0001-MR03, CmiR-AN0001-AM04) lentiviral plasmids were purchased from GeneCopoeia.

Techniques: Expressing, Flow Cytometry, Co-Culture Assay, Cell Culture, Control, Staining, Over Expression

Journal: Cell Stress

Article Title: MiR-200c reprograms fibroblasts to recapitulate the phenotype of CAFs in breast cancer progression

doi: 10.15698/cst2024.03.293

Figure Lengend Snippet: (A-D) RNAseq data for BJ1miR-200c versus control. (A) Volcano plot showed the most differentially expressed genes. (B) Boxplots for the relevant markers expression of senescence, HDMs, cytokine, glycolysis and growth factors. (C) GSEA plot enrichment of Reactome pathways driven by miR200c (D) GSEA of Hallmark pathways driven by miR200c. (E) Protein levels of BJ1 cells overexpressing or inhibiting miR-200c and control under normoxic and hypoxic conditions. (F) Barplot from proteomic assay showing genes with fold change >2 between BJ1miR-200c and control. (G) COMMD1 level in fibroblasts with miR-200c overexpression or inhibition.

Article Snippet: Precursor miR-200c (HmiR0180-MR03-10), anti-miR-200c (HmiR-AN0302-AM04) and control (CmiR0001-MR03, CmiR-AN0001-AM04) lentiviral plasmids were purchased from GeneCopoeia.

Techniques: Control, Expressing, Proteomic Assay, Over Expression, Inhibition

Journal: Cell Stress

Article Title: MiR-200c reprograms fibroblasts to recapitulate the phenotype of CAFs in breast cancer progression

doi: 10.15698/cst2024.03.293

Figure Lengend Snippet: (A) Phospho-NFκB p65 expression of fibroblasts with miR-200c downregulation or overexpression. (B) NFκB activity in NIH3T3 NFκB luciferase reporter cells with miR-200c overexpression or downregulation under normoxic (21% O2) and hypoxic (1% O2) conditions (Random Luciferase Units-RLU). *p<0.05. (C) Cytokine, chemokine and receptor expression profile in fibroblast conditioned media. BJ1 F200c or control were cultured in serum-free DMEM media. The levels of various factors in the cell-free culture media were measured by antibody arrays at day 2 and 4. Data was showed as fold change ± SD. *p<0.05. (D) HIF and MCT4 expression in fibroblasts with miR-200c modulation. (E) HIF activity in NIH3T3 HIF luciferase reporter cells with miR-200c overexpression or downregulation under normoxic and hypoxic conditions. *p<0.05. (F) Lactate level in BJ1miR-200c and control under normoxic and hypoxic conditions. *p<0.05.

Article Snippet: Precursor miR-200c (HmiR0180-MR03-10), anti-miR-200c (HmiR-AN0302-AM04) and control (CmiR0001-MR03, CmiR-AN0001-AM04) lentiviral plasmids were purchased from GeneCopoeia.

Techniques: Expressing, Over Expression, Activity Assay, Luciferase, Control, Cell Culture

Journal: Cell Stress

Article Title: MiR-200c reprograms fibroblasts to recapitulate the phenotype of CAFs in breast cancer progression

doi: 10.15698/cst2024.03.293

Figure Lengend Snippet: (A-C) Flow data showed functional mitochondrial mass, apoptosis and proliferation in cancer cells co-cultured with F200c. (D) S phase of MCF7 cells in homotypic culture or co-culture with miR-200c BJ1 fibroblasts and controls, with and without Tamoxifen treatment for 24 hours. (E) Wound healing. Wound gap area of MCF7 cells exposed to conditioned media from BJ1 miR-200c or control at time points 0 and 16 hours post-exposure. (F) PD-L1 expression in MCF7 and AT3 cancer cells under homotypic and co-culture with miR200c fibroblasts or controls. *p<0.05. (G) Correlation between CCL5 and PD-L1.

Article Snippet: Precursor miR-200c (HmiR0180-MR03-10), anti-miR-200c (HmiR-AN0302-AM04) and control (CmiR0001-MR03, CmiR-AN0001-AM04) lentiviral plasmids were purchased from GeneCopoeia.

Techniques: Functional Assay, Cell Culture, Co-Culture Assay, Control, Expressing

Journal: Cell Stress

Article Title: MiR-200c reprograms fibroblasts to recapitulate the phenotype of CAFs in breast cancer progression

doi: 10.15698/cst2024.03.293

Figure Lengend Snippet: (A-F) Co-injection of AT3-RFP cells with MEFs-GFP miR-200c or controls. Mice were sacrificed 6 weeks post-injection. (A) Tumor Z-stack images. (B) Tumor volume. (C) Immunoblot to assess the metabolic markers in the generated tumors. (D) Prevalence of metastatic disease. (E, F) Representative images for lung India ink staining and peritoneal metastasis are shown. (G-I) Co-injection of 4T1 cells and miR-200c NIH3T3 fibroblasts or controls. Mice were sacrificed 18 days post-co-injection. (G) Tumor volume. (H) Liver weight and metastatic percent. (I) Lung metastatic colonies. 6-TG resistant lung colonies quantified using image J.

Article Snippet: Precursor miR-200c (HmiR0180-MR03-10), anti-miR-200c (HmiR-AN0302-AM04) and control (CmiR0001-MR03, CmiR-AN0001-AM04) lentiviral plasmids were purchased from GeneCopoeia.

Techniques: Injection, Western Blot, Generated, Staining

Journal: Cell Stress

Article Title: MiR-200c reprograms fibroblasts to recapitulate the phenotype of CAFs in breast cancer progression

doi: 10.15698/cst2024.03.293

Figure Lengend Snippet: (A) Representative PD-L1 staining in tumor sections. Co-injection of 4T1 cells and miR-200c NIH3T3 fibroblasts or controls (scale bar: 20μm). Immunohistochemistry quantification for PD-L1 in random units as per ImageJ quantification. (B-D) Co-injection with AT3 cells and MEFs miR-200c or control. (B) PD-L1 immunoblot. (C, D) Tumor growth with chemo-immunotherapy. Mice were treated by IP with Paclitaxel and anti-PD-L1 versus DMSO and control IgG after tumor implantation.

Article Snippet: Precursor miR-200c (HmiR0180-MR03-10), anti-miR-200c (HmiR-AN0302-AM04) and control (CmiR0001-MR03, CmiR-AN0001-AM04) lentiviral plasmids were purchased from GeneCopoeia.

Techniques: Staining, Injection, Immunohistochemistry, Control, Western Blot, Tumor Implantation

Journal: Cell Stress

Article Title: MiR-200c reprograms fibroblasts to recapitulate the phenotype of CAFs in breast cancer progression

doi: 10.15698/cst2024.03.293

Figure Lengend Snippet: (A-C) Flow data showing functional mitochondrial mass, PFBF and NBDG in MEF overexpressing anti-miR-200c versus control. (D) Flow data showing NBDG in AT3 cancer cells co-cultured with fibroblasts overexpressing anti-miR-200c versus control. (E) MCF7 cells co-injected with BJ1 anti-miR-200c or control. Tumor volume was measured at over time and at time of sacrifice (n=8-10, *p<0.05). (F, G) Liver and Spleen weight from mice co-injected with 4T1 and NIH 3T3 cells anti-miR-200c or control. (n=5-13, *p<0.05). (H, I) Co-injection of AT3 cells with MEFs with COMMD1 KD/WT overexpressing anti-miR-200c or controls. Mice were sacrificed 24 days post-injection. (H)Tumor volume. (n=8-10, *p<0.05). (I)Immunoblot to assess PD-L1, TOMM20 and MYC in the generated tumors.

Article Snippet: Precursor miR-200c (HmiR0180-MR03-10), anti-miR-200c (HmiR-AN0302-AM04) and control (CmiR0001-MR03, CmiR-AN0001-AM04) lentiviral plasmids were purchased from GeneCopoeia.

Techniques: Functional Assay, Control, Cell Culture, Injection, Western Blot, Generated

Journal: Cell Stress

Article Title: MiR-200c reprograms fibroblasts to recapitulate the phenotype of CAFs in breast cancer progression

doi: 10.15698/cst2024.03.293

Figure Lengend Snippet: (A-H) RNAseq data for mice tumors of co-injection with AT3 and MEF anti-miR-200c/COMMD1 KD versus control. (A) Heatmap showed differentially expressed genes in four groups. (B) OPLS-DA of G2 versus G1 and G3 versus G1. (C) G2 versus G1. Schematic and barplot showed the differentially expressed genes regarding highly connected networks. (D) Fold change of MYC and JUN in G2 versus G1. (E) Plot for immune assay of antimiR-200c vs control group. (F) GSEA hallmark pathways in G3 versus G1. (G) Fold change of TGFB1, TGFB3 expression in G3 versus G1. (H)Gene alteration related to check point effect in COMMD1 KD vs WT group. (I) Immunophenoscore analysis comparing immunosuppressive cells in COMMD1 KD vs WT group.

Article Snippet: Precursor miR-200c (HmiR0180-MR03-10), anti-miR-200c (HmiR-AN0302-AM04) and control (CmiR0001-MR03, CmiR-AN0001-AM04) lentiviral plasmids were purchased from GeneCopoeia.

Techniques: Injection, Control, Expressing

Journal: Cell Stress

Article Title: MiR-200c reprograms fibroblasts to recapitulate the phenotype of CAFs in breast cancer progression

doi: 10.15698/cst2024.03.293

Figure Lengend Snippet: (A) ROS level in BJ1 cells under homotypic and coculture condition. *p<0.05. (B) ROS level in BJ1miR-200c or control. (C) Histone demethylation mediated by KDM1A demethylase through an FAD-dependent amine oxidase reaction, releasing one molecule of H 2 0 2 . (D, E) Global DNA methylation was measured by 5-mC percentage in sorted co-culture and homotypic BJ1 cells or with redox modulators. (F) Fold change of miR-200c and miR-205 in BJ1 fibroblasts exposed to drugs regulating the epigenetic repressive state. (G, H) Promoter DNA methylation of miR-200c in fibroblasts. BJ1 cells under homotypic or co-culture condition or exposure to H 2 O 2 for 4 days . *p<0.05. (I) Model of fibroblasts and carcinoma cells cross talk regulated by miR-200c. In the TME, ROS causes DNA hypomethylation of fibroblasts, which promotes miR-200c transcription. Reprogramming fibroblasts via miR-200c towards MET/senescence reduces stemness, induces aberrant chromatin changes and histone demethylation mediated by KDM1A demethylase through an FAD-dependent amine oxidase reaction with H 2 0 2 release. Meanwhile it induces miR-205 and reduces COMMD1 expression along with NFκB and HIF activation. Furthermore, these reprogrammed fibroblasts promote cancer aggressiveness.

Article Snippet: Precursor miR-200c (HmiR0180-MR03-10), anti-miR-200c (HmiR-AN0302-AM04) and control (CmiR0001-MR03, CmiR-AN0001-AM04) lentiviral plasmids were purchased from GeneCopoeia.

Techniques: Control, DNA Methylation Assay, Co-Culture Assay, Expressing, Activation Assay

Journal: Cell Stress

Article Title: MiR-200c reprograms fibroblasts to recapitulate the phenotype of CAFs in breast cancer progression

doi: 10.15698/cst2024.03.293

Figure Lengend Snippet: In the TME, the interaction between fibroblasts and carcinoma cells stimulates oxidative stress with reactive oxygen species (ROS) release. ROS in fibroblasts triggers DNA hypomethylation leading to miR-200c transcriptional enrichment. MET in fibroblasts via miR-200c induces senescence, miR-205 and suppresses COMMD1 that activates NFκB and HIF signaling and recapitulates the phenotype of CAFs with downregulation of CAV1 and upregulation of MCT4 as well as cytokines, growth factors and lactate release. Paramountly, these reprogrammed fibroblasts promote cancer aggressiveness.

Article Snippet: Precursor miR-200c (HmiR0180-MR03-10), anti-miR-200c (HmiR-AN0302-AM04) and control (CmiR0001-MR03, CmiR-AN0001-AM04) lentiviral plasmids were purchased from GeneCopoeia.

Techniques: