Journal: Oncotarget

Article Title: Up-regulation of matrix metalloproteinases in a mouse model of chemically induced colitis-associated cancer: the role of microRNAs

doi:

Figure Lengend Snippet: (A) Venn diagram displaying the prediction of murine miRNAs targeting Mmp3 , Mmp10 , and Mmp13 mRNAs, respectively. (B) qRT-PCR was performed to confirm the expression of the selected miRNAs in mouse colitis tissue, CAC tissue, and normal colon tissue. Several miRNAs including the seven shown here were downregulated in mouse colitis tissue and CAC tissue compared with normal colon tissue. Relative expression of each miRNA was determined with the 2 −ΔΔCt method. U6 was used as the internal control, and the expression values for “normal” tissue were set as 1. (C) MiRNA mimics or inhibitors were transfected into CT26.WT cells, and the expression of Mmps was determined by qRT-PCR. Gapdh was used as the internal control, and the expression values of “mimics or inhibitor control” were set as 1. (D) Transfection with miR-128, miR-134, or miR-330 mimic decreased Mmp3, Mmp10, and Mmp13 protein levels, respectively, whereas transfection with the miR-128, miR-134, or miR-330 inhibitor increased Mmp3, Mmp10, and Mmp13 levels, respectively, in CT26.WT cells. (E) Relative expression of Mmp3 , Mmp10 , and Mmp13 and of miR-128, miR-134, and miR-330 in LPS-stimulated murine macrophages (RAW264.7) revealed a negative correlation based on Spearman analysis (r = –0.58, p < 0.01; r = −0.65, p < 0.001; r = −0.67, p < 0.001). All data are shown as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control.

Article Snippet: Transfection with the miRNA mimics or miRNA inhibitors was carried out using HiPerFect Transfection Reagent (Qiagen, Valencia, CA, USA).

Techniques: Quantitative RT-PCR, Expressing, Control, Transfection

Journal: Oncotarget

Article Title: Up-regulation of matrix metalloproteinases in a mouse model of chemically induced colitis-associated cancer: the role of microRNAs

doi:

Figure Lengend Snippet: (A) Scheme for the potential binding site of miR-128, miR-134, and miR-330 in the 3′-UTR of Mmp3 , Mmp10 , and Mmp13 and the sequence of each intact miR-128, miR-134, and miR-330 binding site (wild-type, wt) and its mutant (Mut) within the luciferase reporter vector. (B) Luciferase assay with HEK293 cells, which were cotransfected with the indicated miRNA mimics (or control) and a luciferase reporter containing the 3′-UTR (wild type or mutant) of the indicated Mmps. An empty luciferase reporter construct was used as a negative control (Blank). Luciferase activities were measured 36 h post-transfection. miR-128, miR-134, and miR-330 suppressed the luciferase activity of Mmp3 , Mmp10 , and Mmp13 , respectively, in luciferase wild-type reporter constructs. The data are the mean ± SD for separate transfections (n = 3). * p < 0.05, ** p < 0.01 compared with control. (C) Based on an analysis with the DAVID 2008 Functional Annotation Bioinformatics Microarray Analysis Tool, the pie chart shows the percentage of predicted target genes that may be involved in different pathways.

Article Snippet: Transfection with the miRNA mimics or miRNA inhibitors was carried out using HiPerFect Transfection Reagent (Qiagen, Valencia, CA, USA).

Techniques: Binding Assay, Sequencing, Mutagenesis, Luciferase, Plasmid Preparation, Control, Construct, Negative Control, Transfection, Activity Assay, Functional Assay, Microarray

Journal: Oncotarget

Article Title: Up-regulation of matrix metalloproteinases in a mouse model of chemically induced colitis-associated cancer: the role of microRNAs

doi:

Figure Lengend Snippet: (A) Wound-healing assay showing that miR-128, miR-134, and miR-330 suppressed the migration of CT26.WT cells. (B) Transwell migration assay showing that miR-128, miR-134, and miR-330 decreased the invasion ability of CT26.WT cells. Representative figures of the migrated stained cells are shown. The cells in five randomly selected areas were counted. The data are shown as the mean ± SD. (C) Ectopic expression of miR-128, miR-134, and miR-330 inhibits the proliferation of murine colon cancer cells. CT26.WT cells were plated in 96-well plates after transfection with miRNA-specific mimics, miRNA inhibitors, mimics control, or inhibitor control. Cell proliferation was assessed on the indicated days using the MTT assay. (D) Flow cytometry analysis of the cell cycle distribution of CT26.WT cells transfected with the miR-128, miR-134, and miR-330 mimics or mimics control. Bar graph indicates the percentage of cells remaining in phases G 0 –G 1 , S, and G 2 . (E) Tube formation of HUVECs was suppressed by treatment with medium preconditioned with CT26.WT cells overexpressing miR-128, miR-134, or miR-330 when compared with that of the control group cells. Transfection of cells with Mmp3 , Mmp10 , or Mmp13 rescued the angiogenic capabilities of cells overexpressing miR-128, miR-134, or miR-330. All data are shown as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control.

Article Snippet: Transfection with the miRNA mimics or miRNA inhibitors was carried out using HiPerFect Transfection Reagent (Qiagen, Valencia, CA, USA).

Techniques: Wound Healing Assay, Migration, Transwell Migration Assay, Staining, Expressing, Transfection, Control, MTT Assay, Flow Cytometry

Journal: Oncotarget

Article Title: Up-regulation of matrix metalloproteinases in a mouse model of chemically induced colitis-associated cancer: the role of microRNAs

doi:

Figure Lengend Snippet: (A) Effects of transfection with miRNA mimics on miRNA expression in CT26.WT cells. (B) The tail vein of nude mice was intravenously injected with murine colon cancer cells (CT26.WT) transfected with miR-128, miR-134, or miR-330 mimics or mimics control. After 14 days, the mice were sacrificed, necropsies were performed, and images of whole lung tissue were acquired. (C) The weight of the nude mice was measured every 3 days. After sacrifice, lung tissue was excised and weighed, and the ratio of (weight of lung tissue)/(weight of whole body) was calculated. (D) Representative images of hematoxylin and eosin–stained lung sections, which revealed the metastatic nodules. (E) Immunohistochemical staining revealed that transfection of CT26.WT cells with miR-128, miR-134, or miR-330 mimics resulted in decreased expression of Mmp3, Mmp10, or Mmp13, respectively, within the resulting tumors. All data are shown as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control.

Article Snippet: Transfection with the miRNA mimics or miRNA inhibitors was carried out using HiPerFect Transfection Reagent (Qiagen, Valencia, CA, USA).

Techniques: Transfection, Expressing, Injection, Control, Staining, Immunohistochemical staining

Journal: PLoS Biology

Article Title: Identifying novel strategies for treating human hair loss disorders: Cyclosporine A suppresses the Wnt inhibitor, SFRP1, in the dermal papilla of human scalp hair follicles

doi: 10.1371/journal.pbio.2003705

Figure Lengend Snippet: ( A ) Microarray analysis of CsA-treated (6 hours) human HFs ( n = 3 male patient samples). ( B–E ) Characterisation of SFRP1 mRNA (in situ hybridisation; B–D ) and protein (immunofluorescence; E ) expression in the human HF bulb. ( F ) Schematic of SFRP1 in the human HF bulb. ( G–J ) SFRP1 protein analysis after CsA treatment (48 hours) in the human HF bulb ( n = 10 HFs Control, 11 HFs CsA; from 2 male patient samples). (K–N) In situ hybridisation of SFRP1 mRNA with CsA treatment (48 hours). ( O ) qRT-PCR analysis of SFRP1 after CsA treatment (48 hours) ( n = 3 male patient samples). (H and J) Two-tailed unpaired t test, (I) Mann-Whitney test, and (O) one-sample t test. Data are expressed as mean ± SEM. Scale bars = 50 μm. Underlying data can be found in . ANP32E , acidic nuclear phosphoprotein 32 family member E; APOBEC3A , apolipoprotein B mRNA editing enzyme catalytic subunit 3A; BDP1 , B double prime 1, subunit of RNA polymerase III transcription initiation factor IIIB; CCND2 , cyclin D2; CsA, Cyclosporine A; CLCN5 , chloride voltage-gated channel 5; CXCL8 , C-X-C motif chemokine ligand 8; DAB2 , DAB2, clathrin adaptor protein; DP, dermal papilla; EEA1 , early endosome antigen 1; FDCSP , follicular dendritic cell secreted protein; FLG2 , filaggrin family member 2; HF, hair follicle; HM, hair matrix; KISS1R , KISS1 receptor; MALAT1 , metastasis associated lung adenocarcinoma transcript 1; NLRP2 , NLR family pyrin domain containing 2; Pre-Cx, pre-cortex; qRT-PCR, quantitative real-time PCR; PPIB , peptidylprolyl isomerase B; PTX3 , pentraxin 3; RTCA , RNA 3′-terminal phosphate cyclase; SFRP1, secreted frizzled related protein 1; SLFN11 , schlafen family member 11; SPP1 , secreted phosphoprotein 1; TET2 , tet methylcytosine dioxygenase 2.

Article Snippet: Quantitative PCR was performed in triplicate using Taqman probes (Life Technologies, Taqman assay ID: SFRP1: Hs00610060_m1, AXIN2: Hs01063170_m1, LEF1: Hs01547250_m1, GAPDH: Hs02758991_g1).

Techniques: Microarray, In Situ, Hybridization, Immunofluorescence, Expressing, Control, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY, Real-time Polymerase Chain Reaction

Journal: PLoS Biology

Article Title: Identifying novel strategies for treating human hair loss disorders: Cyclosporine A suppresses the Wnt inhibitor, SFRP1, in the dermal papilla of human scalp hair follicles

doi: 10.1371/journal.pbio.2003705

Figure Lengend Snippet: (A–F) The human HF bulb expresses the core components of the canonical β-catenin pathway. Immunofluorescence of β-catenin ( A ), in situ hybridisation of AXIN2 ( B ), LEF1 ( C ), and WLS ( D ). (G–O) qRT-PCR analysis of SFRP1 and the direct β-catenin target genes AXIN2 and LEF1 in the human HF with IWP-2 ( G, J, M ), rhSFRP1 ( H, K, N ), and WAY-316606 ( I, L, O ) treatment ( n = 4 male patient samples). ( P–T ) Analysis of AXIN2 mRNA by in situ hybridisation after WAY-316606 treatment ( n = 15 HFs control, 14 HFs WAY-316606; from 3 male patient samples). (G–O) One-sample t test, (Q and S) two-tailed unpaired t test (R and T), and Mann-Whitney test. Data are expressed as mean ± SEM. Scale bars = 50 μm. Underlying data can be found in . AXIN2 , axis inhibition protein 2; DP, dermal papilla; HF, hair follicle; IWP-2, inhibitor of Wnt production-2; LEF1 , lymphoid enhancer binding factor 1; n.s, not significant; PPIB , peptidylprolyl isomerase B; Pre-Cx, pre-cortex, qRT-PCR, quantitative real-time PCR; rhSFRP1, recombinant human SFRP1; SFRP1, secreted frizzled related protein 1; WAY, WAY-316606; WLS , Wntless.

Article Snippet: Quantitative PCR was performed in triplicate using Taqman probes (Life Technologies, Taqman assay ID: SFRP1: Hs00610060_m1, AXIN2: Hs01063170_m1, LEF1: Hs01547250_m1, GAPDH: Hs02758991_g1).

Techniques: Immunofluorescence, In Situ, Hybridization, Quantitative RT-PCR, Control, Two Tailed Test, MANN-WHITNEY, Inhibition, Binding Assay, Real-time Polymerase Chain Reaction, Recombinant

Journal: PLoS Biology

Article Title: Identifying novel strategies for treating human hair loss disorders: Cyclosporine A suppresses the Wnt inhibitor, SFRP1, in the dermal papilla of human scalp hair follicles

doi: 10.1371/journal.pbio.2003705

Figure Lengend Snippet: (A) Hair shaft elongation of human HFs ex vivo with WAY-316606 treatment ( n = 31 HFs Control, 30 HFs WAY-316606; from 3 male patient samples). ( B ) K85 protein quantification using immunofluorescence after 48 hours of treatment with WAY-316606 ( n = 16 HFs control, 14 HFs WAY-316606; from 3 male patient samples). ( C ) Macroscopic quantification of hair cycle stage on day 6 with WAY-316606 treatment. ( D–H ) Validation of HF cycle stage with Ki-67/TUNEL analysis ( n = 21 HFs control, 20 HFs WAY-316606; from 3 male patient samples) and Masson's-Fontana ( n = 17 HFs control, 20 HFs WAY-316606; from 3 male patient samples). (I) Working hypothesis. Data are expressed as mean ± SEM. (A, B, E, G, and H) Two-tailed unpaired t test and (F) Mann-Whitney test. Scale bars, A = 1 mm; B, D, and H = 50 μm. Underlying data can be found in . AL, Auber’s line; Axin2, axis inhibition protein 2; CsA, Cyclosporine A; DC, differentiating cell; DP, dermal papilla; DPC, dermal papilla cell; HF, hair follicle; K85, Keratin 85; LEF1, lymphoid enhancer binding factor 1; SFRP1, secreted frizzled related protein 1; TAC, transient amplifying cell; WAY, WAY-316606.

Article Snippet: Quantitative PCR was performed in triplicate using Taqman probes (Life Technologies, Taqman assay ID: SFRP1: Hs00610060_m1, AXIN2: Hs01063170_m1, LEF1: Hs01547250_m1, GAPDH: Hs02758991_g1).

Techniques: Ex Vivo, Control, Immunofluorescence, Biomarker Discovery, TUNEL Assay, Two Tailed Test, MANN-WHITNEY, Inhibition, Binding Assay

Journal: Nature communications

Article Title: DNA methylation directs microRNA biogenesis in mammalian cells.

doi: 10.1038/s41467-019-13527-1

Figure Lengend Snippet: Fig. 3 Drosha genomic occupancy is sensitive to DNA methylation. a Plot of expression of transcripts encoding miRNA biogenesis proteins (n = 40) and other RNA-binding proteins (n = 166) in WT vs. TKO cells. R- and p-values of Pearson’s product-moment correlation are given. b Drosha mRNA levels in WT and TKO mouse ESCs quantified by qRT-PCR. Bars represent means of three independent experiments. Values were normalized to tubulin. c Relative Drosha occupancy over miRNA genomic regions in WT and TKO mouse ESCs determined by ChIP with an antibody against Drosha. Immunoprecipitated DNA was quantified by qRT-PCR with primers spanning the indicated pre-miRNA sequences. Data were normalized to input DNA. d Efficiency of miRNA biogenesis measured as the ratio of mature miRNA to pri-miRNA expression levels for the indicated miRNAs in WT mouse ESCs upon treatment with 2.5 µM 5-Aza relative to vehicle control. Data were normalized to RPLP0 for pri-miRNAs and RNU6 for mature miRNAs. e Drosha protein levels in untreated control WT mouse ESCs and cells treated with 2.5 µM 5-Aza. GAPDH was used as the loading control. f Relative Drosha occupancy over miRNA genomic regions in untreated WT mouse ESCs and cells treated with 2.5 µM 5-Aza determined by ChIP with an antibody against Drosha. Immunoprecipitated DNA was quantified by qRT-PCR with primers spanning the indicated pre-miRNA sequences of the methylated and unmethylated groups. Data were normalized to input DNA. All Error bars represent ± SEM (n = 3). * represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001; NS = not significant; t-test. Source data are provided as a Source Data file.

Article Snippet: Expression of genes in fragments per kilobase of transcript per million mapped reads (FPKM) was calculated via cufflinks82 with default parameters. miRNAs expression in mouse WT R1 ESCs and TKO R1 cells was evaluated in house using a NanoString microarray (full microarray data at the GEO server: GSE124879).

Techniques: DNA Methylation Assay, Expressing, RNA Binding Assay, Quantitative RT-PCR, Immunoprecipitation, Control, Methylation

Journal: Nature communications

Article Title: DNA methylation directs microRNA biogenesis in mammalian cells.

doi: 10.1038/s41467-019-13527-1

Figure Lengend Snippet: Fig. 4 MeCP2 binding at methylated miRNA loci slows Pol II allowing miRNAs biogenesis. a Pol II-pSer2 occupancies for each group of miRNAs across 100 bp of miRNA region and 100 bp of the flanking regions. Regions upstream of the 5‘ ends of the pre-miRNAs between all methylated groups vs. the unmethylated groups were tested. All tests were statistically significant. ***p < 0.001; t-test. b Relative Pol II-pSer2 to Pol II-pSer5 (Ser2/Ser5) occupancies analyzed by ChIP over select miRNA regions in WT and TKO mouse ESCs. Immunoprecipitated DNA was quantified by qRT-PCR using primers spanning the indicated pre-miRNA sequences. Data were normalized to input DNA. c MeCP2 occupancies in regions upstream of start sites between all methylated groups vs. the two unmethylated groups were tested. All tests were statistically significant except for the test between the bilateral and the depleted groups. ***p < 0.001; t-test. d Relative MeCP2 occupancy over miRNA genomic regions in WT and TKO ESCs determined by ChIP analysis. Immunoprecipitated DNA was quantified by qRT-PCR using primers spanning the indicated pre-miRNA sequences. Data were normalized to input DNA. e SP1 occupancy for each group of miRNAs. The regions upstream of the start sites between all methylated groups vs. the unmethylated groups were tested. All tests were statistically significant. ***p < 0.001; t-test. f MeCP2 protein levels in WT mouse ESCs upon treatment with siMeCP2 or siControl. GAPDH was used as a loading control. g Efficiency values for the methylated miRNAs and unmethylated miRNAs from WT mouse ESCs upon treatment with siMeCP2 or siControl. Data were normalized to RPLP0 for pri-miRNAs and U6 for mature miRNAs. h Relative Pol II-pSer2 to Pol II-pSer5 (Ser2/Ser5) occupancy analyzed by ChIP in WT mouse ESCs upon treatment with siMeCP2 or siControl. Immunoprecipitated DNA was quantified by qRT-PCR. Data were normalized to input DNA. All error bars represent ± SEM (n = 3); * represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001; NS = not significant; t-test. Source data are provided as a Source Data file.

Article Snippet: Expression of genes in fragments per kilobase of transcript per million mapped reads (FPKM) was calculated via cufflinks82 with default parameters. miRNAs expression in mouse WT R1 ESCs and TKO R1 cells was evaluated in house using a NanoString microarray (full microarray data at the GEO server: GSE124879).

Techniques: Binding Assay, Methylation, Immunoprecipitation, Quantitative RT-PCR, Control

Journal: Biomedical reports

Article Title: Lipolysis by downregulating miR-92a activates the Wnt/β-catenin signaling pathway in hypoxic rats.

doi: 10.3892/br.2020.1340

Figure Lengend Snippet: Figure 1. Analysis of miR‑92a expression in the epididymal fat of hypoxic rats. (A) B3L, B6L and B9L were from normoxic rats. (B) D2L, D6L, and D9L were from hypoxic rats. (C) Relative expression of miRNAs in the normoxic and hypoxic rats. miR‑92a expression levels in the hypoxic rats were reduced compared with the normoxic rats based on the miRNA micro array analysis. miR, microRNA; Rno, Rattus norvegicus.

Article Snippet: The following reagents and instruments were used: miRcute miRNA Isolation kit (Tiangen Biotech Co., Ltd.), miRcute miRNA cDNA First Strand Synthesis kit (Tiangen Biotech Co., Ltd.), miRcute MiRNA Quantitative Fluorescence Detection kit (cat. no. FP401; Tiangen Biotech Co., Ltd.), SuperReal PreMix SYBR‐Green (cat. no. FP204; Tiangen Biotech Co., Ltd.), TIANScript II cDNA First Strand Synthesis kit (cat. no. KR107; Tiangen Biotech Co., Ltd.) and RT‐qPCR amplifier (BIOER FQD‐96A; Hangzhou Bioer Co., Ltd.).

Techniques: Expressing, Microarray

Journal: Biomedical reports

Article Title: Lipolysis by downregulating miR-92a activates the Wnt/β-catenin signaling pathway in hypoxic rats.

doi: 10.3892/br.2020.1340

Figure Lengend Snippet: Figure 3. Analysis of miR‑92a binding with Fzd10. (A) Wild‑type and mutant‑type binding sequences of miR‑92a with Fzd10. (B) Dual luciferase reporter assay. Wild‑type miR‑92a bound and degraded wild‑type Fzd10 mRNA in the Fzd10+miR92a group. Single Fzd10 or mutated Fzd10 mRNA did not result in altered fluorescence values of the empty carrier pRL‑TK, and miR‑92a was not significantly altered the fluorescence values of the mutated Fzd10 mRNA in the mFzd10+miR92a group. miR/miRNA, microRNA; Fzd10, frizzled 10; mFzd10, mutated Fzd10.

Article Snippet: The following reagents and instruments were used: miRcute miRNA Isolation kit (Tiangen Biotech Co., Ltd.), miRcute miRNA cDNA First Strand Synthesis kit (Tiangen Biotech Co., Ltd.), miRcute MiRNA Quantitative Fluorescence Detection kit (cat. no. FP401; Tiangen Biotech Co., Ltd.), SuperReal PreMix SYBR‐Green (cat. no. FP204; Tiangen Biotech Co., Ltd.), TIANScript II cDNA First Strand Synthesis kit (cat. no. KR107; Tiangen Biotech Co., Ltd.) and RT‐qPCR amplifier (BIOER FQD‐96A; Hangzhou Bioer Co., Ltd.).

Techniques: Binding Assay, Luciferase, Reporter Assay, Fluorescence

Journal: PLoS ONE

Article Title: Profiling Circulating MicroRNA Expression in Experimental Sepsis Using Cecal Ligation and Puncture

doi: 10.1371/journal.pone.0077936

Figure Lengend Snippet: Clustering of differentially expressed circulating microRNAs in the whole blood of C567BL/6 mice at 4, 8, and 24 h after cecal ligation and puncture (A) and subcutaneous bacteria injection with 1×10 8 E. coli. or S. aureus (B).

Article Snippet: Mouse genome-wide miRNA microarray analysis and statistical analysis was performed by Phalanx Biotech.

Techniques: Ligation, Bacteria, Injection

Journal: PLoS ONE

Article Title: Profiling Circulating MicroRNA Expression in Experimental Sepsis Using Cecal Ligation and Puncture

doi: 10.1371/journal.pone.0077936

Figure Lengend Snippet: Venn diagram showing the common and specific up-regulated miRNAs in the cecal ligation and puncture experiment without and after subcutaneous injection of gram-negative or gram-positive bacteria.

Article Snippet: Mouse genome-wide miRNA microarray analysis and statistical analysis was performed by Phalanx Biotech.

Techniques: Ligation, Injection, Bacteria