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: Experimental and Therapeutic Medicine

Article Title: miR‑137 is a diagnostic tumor‑suppressive miRNA that targets SPHK2 to promote M1‑type tumor‑associated macrophage polarization

doi: 10.3892/etm.2023.12096

Figure Lengend Snippet: miR-137 inhibits the release of IL-13, and promotes the release of TNFα and IFNγ. HK was used as control group, and SH1, SH2, SH3 as knockdown groups. (A) miR-137 overexpression in glioma cells reduced IL-13 production. (B) miR-137 overexpression in glioma cells promoted TNFα production. (C) miR-137 overexpression in glioma cells promoted IFNγ production. * P<0.05, ** P<0.01 and *** P<0.001 vs. empty vector. (D and E) After transduction with lentiviral vectors, SPHK2 mRNA was detected using reverse transcription-quantitative PCR. ** P<0.01 vs. U373-HK. (F) SPHK2 protein level was assessed using western blot analysis. (G-I) The supernatant from NC and sh-SPHK2 was collected, and IL-13, TNF-α and IFN-γ were detected using ELISA. * P<0.05, ** P<0.01, and **** P<0.0001 vs. NC. miR, microRNA; SPHK2, sphingosine kinase 2; sh-, short hairpin; NC, negative control.

Article Snippet: The wild-type (p-WT) and mutant-type (p-MT) reporter vectors of the SPHK2 3'-UTR were constructed using the pEZX-MT01 Luciferase miRNA Expression Reporter Vector (GeneCopoeia, Inc.).

Techniques: Control, Knockdown, Over Expression, Plasmid Preparation, Transduction, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: miR‑137 is a diagnostic tumor‑suppressive miRNA that targets SPHK2 to promote M1‑type tumor‑associated macrophage polarization

doi: 10.3892/etm.2023.12096

Figure Lengend Snippet: SPHK2 is a direct target of miR-137. (A) miR-137 binding site in SPHK2 3'-UTR was predicted using TargetScan. SPHK2-3'-UTR-WT and SPHK2-3'-UTR-MT carried in recombinant luciferase mRNAs transcribed by p-WT and p-MT. (B) Reverse transcription-quantitative PCR analysis of SPHK2 mRNA expression in the cells as indicated. Their relative expression levels were normalized against GAPDH. The ratios of SPHK2/GAPDH in cells transfected with the scramble sequence were set to 1.0. (C) Western blot analysis of SPHK2 protein expression in the cells as indicated. (D and E) Dual-luciferase reporter assays were performed using (D) U373 and (E) U251 cells co-transfected with p-WT or p-MT and scramble sequence or miR-137 mimics. All experiments were performed at least in triplicate and the data in (B-E) are presented as the mean ± SD. * P<0.05 and ** P<0.01 vs. Scramble. SPHK2, sphingosine kinase 2; miR, microRNA; UTR, untranslated region; WT, wild-type; MUT, mutant.

Article Snippet: The wild-type (p-WT) and mutant-type (p-MT) reporter vectors of the SPHK2 3'-UTR were constructed using the pEZX-MT01 Luciferase miRNA Expression Reporter Vector (GeneCopoeia, Inc.).

Techniques: Binding Assay, Recombinant, Luciferase, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Transfection, Sequencing, Western Blot, Mutagenesis

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: PLoS ONE

Article Title: Downregulation of miR-205 Modulates Cell Susceptibility to Oxidative and Endoplasmic Reticulum Stresses in Renal Tubular Cells

doi: 10.1371/journal.pone.0041462

Figure Lengend Snippet: PCR primers used for detection of mRNAs.

Article Snippet: The EGLN2 3′-UTR miRNA target sequence expression clone in pEZX-MT01 vector with fLuc was purchased from GeneCopoeia (miTarget TM miRNA 3′-UTR Target Clone, Rockville, MD).

Techniques:

Journal: PLoS ONE

Article Title: Downregulation of miR-205 Modulates Cell Susceptibility to Oxidative and Endoplasmic Reticulum Stresses in Renal Tubular Cells

doi: 10.1371/journal.pone.0041462

Figure Lengend Snippet: (A) Quantitative real-time RT-PCR analysis revealed that the expression of EGLN2 was increased by miR-205 inhibition, but not significantly altered by overexpression of miR-205 compared to negative control transfected HK-2. No changes were seen in expression levels of EGLN1 (PHD2) or EGLN3 (PHD3). The data represent the means ± S.E. of triplicate analysis from three independent experiments. *P<0.05 by Dunnett’s multiple comparison test. (B) Western blot analysis showed that EGLN2 was also increased at protein level by miR-205 inhibition. The result of densitmetry represent the means ± S.E. of triplicate analysis from two independent experiments.**P<0.01 by standard t -test. (C) Quantitative real-time RT-PCR analysis revealed that the expression level of EGLN2 was increased in HK-2 exposed to hypoxia-reoxygenation or TUN, but these increases were supressed by miR-205 overexpression (▪) compared to negative control transfected HK-2 (□). *P<0.05 by Tukey’s multiple comparison test. (D) EGLN2 (transcript variant 1) contains the predicted binding site for miR-205 in its 3′-UTR. The predicted pairing of the mRNA target region (top) and miRNA (bottom) is as indicated, wherein solids line indicates hydrogen base pairing and the dotted line indicates Watson-Crick/wobble base pairing. The shaded sequence indicates the seed region of miR-205. (E) Mutant 3′-UTR sequence that abolished binding to miR-205. (F) HK-2 cells were transiently cotransfected with a luciferase reporters expressing EGLN2 3′-UTR or mutant EGLN2 3′-UTR miRNA target sequence and miR-205 mimic, miR-205 inhibitor, or negative control. At 24 hours posttransfection, firefly luciferase activities were measured and normalized with Renilla luciferase activities. Relative luciferase activity was increased by miR-205 inhibition when compared with control, while mutant EGLN2 3′-UTR had no effect on luciferase activity, indicating that miR-205 acts on the 3′-UTR of the EGLN2. Though transfection of HK-2 with EGLN2 3′-UTR vector along with miR-205 overexpression tended to decrease luciferase activity, this change was not sicnificant. The data represent the means ± S.E. of triplicate measurements from two independent experiments. *P<0.05 by Tukey’s multiple comparison test. (G) When transiently transfected HK-2 with EGLN2 3′-UTR vector were exposed to oxidative or ER stress, the relative luciferase activities were significantly increased, and these increases were canceled with mutant EGLN2 3′-UTR vectors. Furthermore, co-transfection of EGLN2 3′-UTR vector with miR-205 mimic partially restrained these stress-induced increases of luciferase activities. The data represent the means ± S.E. of triplicate measurements from two independent experiments. *P<0.05 by Tukey’s multiple comparison test.

Article Snippet: The EGLN2 3′-UTR miRNA target sequence expression clone in pEZX-MT01 vector with fLuc was purchased from GeneCopoeia (miTarget TM miRNA 3′-UTR Target Clone, Rockville, MD).

Techniques: Quantitative RT-PCR, Expressing, Inhibition, Over Expression, Negative Control, Transfection, Comparison, Western Blot, Variant Assay, Binding Assay, Sequencing, Mutagenesis, Luciferase, Activity Assay, Control, Plasmid Preparation, Cotransfection

Journal: PLoS ONE

Article Title: Downregulation of miR-205 Modulates Cell Susceptibility to Oxidative and Endoplasmic Reticulum Stresses in Renal Tubular Cells

doi: 10.1371/journal.pone.0041462

Figure Lengend Snippet: When cells are exposed to oxidative stress or ER stress, maladaptive down-regulation of miR-205 and subsequent up-regulation of EGLN2 occurs. ATF4 and HIF, which are negatively regulated by EGLN2, are supposed to contribute to the downstream suppression of anti-oxidant enzymes.

Article Snippet: The EGLN2 3′-UTR miRNA target sequence expression clone in pEZX-MT01 vector with fLuc was purchased from GeneCopoeia (miTarget TM miRNA 3′-UTR Target Clone, Rockville, MD).

Techniques: