Journal: International Journal of Molecular Sciences

Article Title: MicroRNA Signature in an In Vitro Keratinocyte Model of Diabetic Wound Healing

doi: 10.3390/ijms251810125

Figure Lengend Snippet: Differentially expressed miRNAs with significant variances under high-glucose and hypoxic environment.

Article Snippet: Small RNAs (<200 nucleotides) were enriched using cutoff concentrators (Sartorius, Gottingen, Germany). miRNAs were labeled with Cy5 dye (ULS ® microRNA Labeling Kit, Kreatech Diagnostics, Amsterdam, Netherlands) and hybridized to human miRNA OneArray ® 4.1 microarray chips (Phalanx Biotech Group, Hsinchu, Taiwan).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: MicroRNA Signature in an In Vitro Keratinocyte Model of Diabetic Wound Healing

doi: 10.3390/ijms251810125

Figure Lengend Snippet: Differential expression of miRNAs in HaCaT cells under high-glucose/hypoxia versus normal conditions. ( A ) Heat map and cluster dendrogram of 143 differentially expressed miRNAs. Columns represent miRNA expression at 24 h and 48 h under normal (24 hr1 and 48 hr1) and high-glucose/hypoxia (24 hr4 and 48 hr4) conditions. ( B ) Heat map and cluster dendrogram of 11 miRNAs with the highest changes on the expression levels at 24 and 48 h post-wounding (labeled with red asterisks in ( A )). This heat map shows miRNA changes after the normalization of the high-glucose/hypoxia condition over the normal condition. Subsequent qRT-PCR verification on these miRNAs is shown in .

Article Snippet: Small RNAs (<200 nucleotides) were enriched using cutoff concentrators (Sartorius, Gottingen, Germany). miRNAs were labeled with Cy5 dye (ULS ® microRNA Labeling Kit, Kreatech Diagnostics, Amsterdam, Netherlands) and hybridized to human miRNA OneArray ® 4.1 microarray chips (Phalanx Biotech Group, Hsinchu, Taiwan).

Techniques: Quantitative Proteomics, Expressing, Labeling, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: MicroRNA Signature in an In Vitro Keratinocyte Model of Diabetic Wound Healing

doi: 10.3390/ijms251810125

Figure Lengend Snippet: Verification of the expression patterns of the cDNA microarray by qRT-PCR. ( A ) Differentially expressed miRNAs at 24 h post-wounding. ( B ) Differentially expressed miRNAs at 48 h post-wounding. A total of 11 miRNAs were selected for further verification by qRT-PCR in this study. In the figures, the relative miRNA expression levels are all expressed on a log2 scale, with data from the cDNA microarray shown in blue, and data from three independent experiments of qRT-PCR shown in red.

Article Snippet: Small RNAs (<200 nucleotides) were enriched using cutoff concentrators (Sartorius, Gottingen, Germany). miRNAs were labeled with Cy5 dye (ULS ® microRNA Labeling Kit, Kreatech Diagnostics, Amsterdam, Netherlands) and hybridized to human miRNA OneArray ® 4.1 microarray chips (Phalanx Biotech Group, Hsinchu, Taiwan).

Techniques: Expressing, Microarray, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: MicroRNA Signature in an In Vitro Keratinocyte Model of Diabetic Wound Healing

doi: 10.3390/ijms251810125

Figure Lengend Snippet: Effects of miR-3138 mimic and miR-3679-5p inhibitor transfection on keratinocyte migration. The present experiments were performed under normal ( A – C ) and high-glucose/hypoxic conditions ( D – F ). ( A , D ) Relative miR-3138 and miR-3679-5p levels post-transfection. ( B , E ) Representative images of keratinocyte migration after the transfection with miRNA mimic and inhibitor. ( C , F ) Average wound closure percentages from three independent experiments after the transfection with miRNA mimic and inhibitor. Different letters labeled at the top of columns indicate statistically significant differences ( p < 0.05).

Article Snippet: Small RNAs (<200 nucleotides) were enriched using cutoff concentrators (Sartorius, Gottingen, Germany). miRNAs were labeled with Cy5 dye (ULS ® microRNA Labeling Kit, Kreatech Diagnostics, Amsterdam, Netherlands) and hybridized to human miRNA OneArray ® 4.1 microarray chips (Phalanx Biotech Group, Hsinchu, Taiwan).

Techniques: Transfection, Migration, Labeling

Journal: International Journal of Molecular Sciences

Article Title: MicroRNA Signature in an In Vitro Keratinocyte Model of Diabetic Wound Healing

doi: 10.3390/ijms251810125

Figure Lengend Snippet: Bioinformatic prediction of the target genes for miR-3138 ( A ) and miR-3679-5p ( B ). Venn diagrams show overlapped target numbers predicted by TargetScan (red), miRDB (green), and DIANA (blue) databases. The pairing patterns between the sequences of target gene 3’UTR and miRNA are shown below each diagram.

Article Snippet: Small RNAs (<200 nucleotides) were enriched using cutoff concentrators (Sartorius, Gottingen, Germany). miRNAs were labeled with Cy5 dye (ULS ® microRNA Labeling Kit, Kreatech Diagnostics, Amsterdam, Netherlands) and hybridized to human miRNA OneArray ® 4.1 microarray chips (Phalanx Biotech Group, Hsinchu, Taiwan).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: MicroRNA Signature in an In Vitro Keratinocyte Model of Diabetic Wound Healing

doi: 10.3390/ijms251810125

Figure Lengend Snippet: Primers used in this study.

Article Snippet: Small RNAs (<200 nucleotides) were enriched using cutoff concentrators (Sartorius, Gottingen, Germany). miRNAs were labeled with Cy5 dye (ULS ® microRNA Labeling Kit, Kreatech Diagnostics, Amsterdam, Netherlands) and hybridized to human miRNA OneArray ® 4.1 microarray chips (Phalanx Biotech Group, Hsinchu, Taiwan).

Techniques: Sequencing

Journal: Journal of Nanobiotechnology

Article Title: Effective delivery of miR-150-5p with nucleus pulposus cell-specific nanoparticles attenuates intervertebral disc degeneration

doi: 10.1186/s12951-024-02561-x

Figure Lengend Snippet: Knockout of miR-150-5p potentially relieves intervertebral disc aging and degeneration. A Selection strategy for miRNAs in nucleus pulposus (NP) tissues based on microarray-based sequencing. B , C Nucleus pulposus cells (NPCs) were obtained from intervertebral discs with and without degeneration (Co6/Co7 vs. Co5/Co6). A heatmap and volcano plot were generated to show the differences in the expression profiles of miRNAs between the intervertebral disc degeneration (IDD) and normal control (NC) groups. There were 22 significantly dysregulated miRNAs in the IDD group (13 miRNAs upregulated and 9 miRNAs downregulated). D Principal component analysis (PCA) of miRNA-normalized expression data from NPCs. PCA of the data revealed strong separation between the two groups and good homogeneity within each group for the miRNA arrays. E Histological examination of NP tissues from NCs and IDD patients. (Scale bar, 100 μm.) F Compared with those in controls (n = 30), miR-150-5p expression levels were increased in NP cells and tissues from IDD patients (n = 67). ***P < 0.001 by the Mann‒Whitney U test. G FISH analysis demonstrated that the levels of miR-150-5p were greater in the NPCs of IDD patients than in those of controls. (Scale bar, 50 μm.) H Targeting strategy for generating miR-150-5p knockout (KO) mice. I The genotypes of the miR-150-5p KO mice were confirmed by Southern blot analysis. J Intervertebral discs were harvested from wild-type (WT) and miR-150-5p KO littermate embryos (E16.5 and E18.5). Hematoxylin and eosin (HE) staining was used to visualize the intervertebral discs of WT and miR-150-5p KO littermate embryos (E16.5 and E18.5). The development of intervertebral discs was not significantly different between WT and miR-150-5p KO littermates. (Scale bar, 100 μm.) K WT and miR-150-5p KO littermates were subjected to needle puncture-induced IDD surgery. Representative images of 6- and 12-week post-IDD surgery intervertebral disc sections. (Scale bar, 100 μm.) L Histological score indicating that miR-150-5p KO could attenuate IDD development (n = 6 per group). P < 0.01 was determined by two-tailed unpaired Student′s t test. M HE and Safranin O staining of intervertebral discs from WT or miR-150-5p KO mice at 6, 15 and 22 months. (Scale bar, 100 μm.) N Histological analysis indicated that miR-150-5p KO could ameliorate age-related IDD in mice. (n = 6 per group) P < 0.01 by two-tailed unpaired Student’s t test

Article Snippet: Intervertebral discs were collected 14 days after needle puncture, and the differentially expressed miRNAs were subsequently screened via a miRNA microarray chip (Affymetrix® 4.0 miRNA Array, USA) (Fig. A).

Techniques: Knock-Out, Selection, Microarray, Sequencing, Generated, Expressing, Southern Blot, Staining, Two Tailed Test

Journal: Journal of Biomedical Science

Article Title: The RNA-binding protein KSRP aggravates malignant progression of clear cell renal cell carcinoma through transcriptional inhibition and post-transcriptional destabilization of the NEDD4L ubiquitin ligase

doi: 10.1186/s12929-023-00949-9

Figure Lengend Snippet: KH-type splicing regulatory protein (KSRP) negatively regulates neural precursor cell-expressed developmentally downregulated 4 like (NEDD4L) mRNA stability via inducing miR-629-5p upregulation and targeting AU-rich elements (AREs) of the 3' untranslated region (3’UTR). A Decay rate of NEDD4L mRNA after treatment with 5 μg/mL actinomycin D for the indicated times in Caki-1 cells with or without KSRP-knockdown. B Flowchart for identifying miRNA induced by KSRP. The left part shows that TCGA miRNA sequencing data were retrieved from UCSC Xena. TCGA KIRC patients with low expression of miRNA (> 10% patients with reads per million mapped reads (RPM) of < 1) were excluded. The remaining 401 miRNAs were used to perform a differentially expressed (DE)-miRNA analysis between tumor ( n = 241) and normal tissues ( n = 70). Forty miRNAs were identified as upregulated miRNAs (with fold change (FC) of > 2 and a false discovery rate (FDR) of < 0.01) in KIRC tumor tissues. Right part shows 29 miRNAs with an FC of < 0.5 as identified in KSRP-depleted Caki-1 cells. By intersecting miRNAs upregulated in TCGA KIRC tissues with miRNAs induced by KSRP in ccRCC cells, miR-629-5p was identified as a critical candidate. C Real-time qPCR analysis of miR-629-5p in Caki-1 cells transfected with a KSRP shRNA, pEGFP-KSRP, or their corresponding control vector. D, E Caki-1 cells were transfected with an miR-629-5p mimic ( D ) or miR-629-5p inhibitor ( E ) for 24 h. NEDD4L protein levels and invasive ability of cells were respectively determined by Western blot (WB) (left panel) and Matrigel invasion (right panel) assays. F Expressions of KSRP and NEDD4L (left panel) and invasive ability of cells (right panel) were determined in Caki-1 cells transfected with pEGFP-KSRP with or without an miR-629-5p inhibitor. G Correlation between miR-629-5p and NEDD4L expression in TCGA KIRC patients as demonstrated in dot plots. Correlation coefficients and p values were evaluated by a Pearson correlation analysis. H GSEA of TCGA KIRC patients with high (top 5%) versus low (bottom 5%) expressions of miR-629-5p. A gene set of the epithelial-mesenchymal transition (EMT) derived from HALLMARK was used. The normalized enrichment score (NES) and FDR are shown in the enrichment plot. I Correlations of miR-629-5p expression with EMT markers were demonstrated in a correlation plot using miRNA sequencing data of TCGA KIRC patients. Correlation coefficients and p values were evaluated by a Pearson correlation analysis. J WB analysis of E-cadherin, N-cadherin, and vimentin expressions in Caki-1 and 786-O cells transfected with an miR-629-5p mimic. K Left panel, Schematic of the human NEDD4L-3' untranslated region (3'UTR) inserted in the luciferase reporter vector. Positions of AU-rich elements (AREs) are indicated by two gray oblongs, and the corresponding sequences are shown underneath. Right panel, Relative luciferase activities of Caki-1 cells transfected with the NEDD4L luciferase 3’UTR reporter vector containing wild-type or mutant ARE domains with or without KSRP shRNA. Values are presented as the mean ± SD of three independent experiments. ** p < 0.01, compared to the respective control groups

Article Snippet: To identify which miRNAs are regulated by KSRP, a high-throughput and specific miRNA microarray (human miRNA OneArray ® miRNA profiling chip) using Caki-1 cells with/without KSRP-KD was conducted by the Phalanx Biotech Group (Hsinchu, Taiwan).

Techniques: Knockdown, Sequencing, Expressing, Transfection, shRNA, Control, Plasmid Preparation, Western Blot, Derivative Assay, Luciferase, Mutagenesis

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Integration of Different “-omics” Technologies Identifies Inhibition of the IGF1R-Akt-mTOR Signaling Cascade Involved in the Cytotoxic Effect of Shikonin against Leukemia Cells

doi: 10.1155/2013/818709

Figure Lengend Snippet: Results and relations of mRNA microarray, miRNA microarray, and stable-isotope dimethyl labeling for quantitative proteomics. Only molecules from the omics datasets that met the expression fold change cutoff of ≥±1.2 are shown in this diagram. The intersections of the Venn diagram indicate the number of corresponding molecules deregulated in different assays independently from the direction of their deregulated expression.

Article Snippet: Human miRNA microarray chips (8 × 60 K, Agilent Technologies) were used.

Techniques: Microarray, Labeling, Expressing

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Integration of Different “-omics” Technologies Identifies Inhibition of the IGF1R-Akt-mTOR Signaling Cascade Involved in the Cytotoxic Effect of Shikonin against Leukemia Cells

doi: 10.1155/2013/818709

Figure Lengend Snippet: Top up- and downregulated molecules in U937 cells after treatment with shikonin. Quantitative changes in the proteome were studied by stable-isotope dimethyl labeling. Microarray experiments were used to analyze changes in the mRNA and miRNA expressione.

Article Snippet: Human miRNA microarray chips (8 × 60 K, Agilent Technologies) were used.

Techniques: Labeling, Microarray, Cell Differentiation