Journal: PLoS ONE

Article Title: IGF-1-enhanced miR-513a-5p signaling desensitizes glioma cells to temozolomide by targeting the NEDD4L-inhibited Wnt/β-catenin pathway

doi: 10.1371/journal.pone.0225913

Figure Lengend Snippet: Volcano plot (A) and heat map of hierarchical gene clustering (B) demonstrating IGF-1-regulated microRNA profiles. After U87 MG cells were treated with 200 ng/ml IGF-1 for 48 h, total RNAs were extracted for a microarray analysis. For all array analyses, an adjusted (adj.) p value of <0.05 and a |log2 (ratio)| of ≥0.58 (±1.5 multiples of change) cutoff were applied. Volcano plots show the multiples of change (log 2 ratio) and probability (-log 10 adj. p values) of individual microRNAs from the microarray assay. The red dot shows the miR-513a-5p location. The heat map depicts the 241 microRNAs differentially expressed between IGF-1 subsets. A color was assigned to each microRNA based on its relative expression level across samples. (C) Detection of endogenous miR-513a-5p levels in human normal astrocytes and three different glioma cell lines. Endogenous miR-513a-5p levels were measured by a real-time PCR. The miR-191-5p level was used as an internal control. Data are the mean ± SD of three independent experiments. * p <0.05. Increased miR-513a levels in GBM patients and glioma stem cells (GSCs) compared to normal controls from TCGA miRNA microarray data (D), and GEO DataSets GSE61710 (E), GSE37366, and GSE41032 (F). (E and F) miR-513a-5p expression changes were analyzed by GEO2R software.

Article Snippet: SYBR® Green PCR Master Mix (cat. no. 4309155), the MultiScribe (tm) Reverse Transcriptase Kit (cat. no. N8080234), TaqMan Advanced miRNA cDNA Synthesis Kit (cat. no. A28007), TaqMan® pri-miR-513a-1 (cat. no. Hs03296262_pri), pri-miR-513a-2 (cat. no. Hs03295531_pri), TaqMan® Advanced miR-513a-5p (cat. no. 479483_mir), and TaqMan® Advanced miR-191-5p (cat. no. 477952_mir) were purchased from Applied Biosystems (Thermo Fisher Scientific).

Techniques: Microarray, Expressing, Real-time Polymerase Chain Reaction, Control, Software

Journal: PLoS ONE

Article Title: IGF-1-enhanced miR-513a-5p signaling desensitizes glioma cells to temozolomide by targeting the NEDD4L-inhibited Wnt/β-catenin pathway

doi: 10.1371/journal.pone.0225913

Figure Lengend Snippet: (A) Dose-dependent effects of IGF-1 on miR-513a-5p expression levels in U87-MG and M059K cells. (B) Effects of IGF-1 on primary (pri)-miR-513a-1 and pri-miR-513a-2 levels. The PI3K inhibitor, LY294002, but not the mitogen-activated protein kinase (MAPK) kinase inhibitor, U0126, attenuated IGF-1-stimulated miR-513a-5p (C) and pri-miR-513a-2 (D) levels. After cells were treated with the indicated doses of IGF-1 for 48 h or 200 ng/ml IGF-1 treatment for 48 h and pretreatment with 5 μM LY294002 and 10 μM U0126 for 1 h, endogenous miR-513a-5p, pri-miR-513a-1, and pri-miR-513a-2 levels were respectively measured by a real-time PCR. miR-191-5p and GAPDH levels were respectively used as internal controls for the mature and primary miR-513a-5p levels. Data are the mean ± SD of three independent experiments. * p <0.05.

Article Snippet: SYBR® Green PCR Master Mix (cat. no. 4309155), the MultiScribe (tm) Reverse Transcriptase Kit (cat. no. N8080234), TaqMan Advanced miRNA cDNA Synthesis Kit (cat. no. A28007), TaqMan® pri-miR-513a-1 (cat. no. Hs03296262_pri), pri-miR-513a-2 (cat. no. Hs03295531_pri), TaqMan® Advanced miR-513a-5p (cat. no. 479483_mir), and TaqMan® Advanced miR-191-5p (cat. no. 477952_mir) were purchased from Applied Biosystems (Thermo Fisher Scientific).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: IGF-1-enhanced miR-513a-5p signaling desensitizes glioma cells to temozolomide by targeting the NEDD4L-inhibited Wnt/β-catenin pathway

doi: 10.1371/journal.pone.0225913

Figure Lengend Snippet: (A) The inverse correlation between miR-513a and NEDD4L expression levels in TCGA microarray data. Using the median cutoff for miR-513a expression levels, patients were divided into two groups. A correlation was found in the group highly expressing miR-513a ( n = 259) by Pearson's correlation analysis. (B) Schematic diagram of potential miR-513a-5p-targeted sites in the NEDD4L 3’-untranslated region (UTR). (C and D) Effects of miR-513a-5p on NEDD4L 3’-UTR luciferase activity. To test for miR-513a-5p's effect, different doses of the miR-513a-5p plasmid were co-transfected with 500 ng of the pmiRGlo-NEDD4L 3’-UTR or mutant 3’-UTR. Luciferase activity was measured in these cells 24 h after transfection. Effects of miR-513a-5p overexpression on NEDD4L mRNA (E) and protein (F) expressions. After cells were transfected with the indicated dose of the miR-513a-5p plasmid for 24 h, relative mRNA and protein levels of NEDD4L were analyzed using a real-time PCR and an immunoblotting assay. (G) Dose-dependent effects of IGF-1 on reducing NEDD4L levels. (H) miR-513a-5p is involved in the IGF-1-reduced NEDD4L expression mechanism. After cells were treated with indicated doses of IGF-1 for 48 h or transfected with 1 μg of the miR-513a-5p plasmid combined with IGF-1 treatment for 48 h, the NEDD4L protein level was analyzed by an immunoblotting assay. Data are the mean ± SD of three experiments. * p <0.05. Inhibitory effects of miR-513a-5p on IGF-1-induced β-catenin nuclear accumulation (I) and -reduced NEDD4L levels (J). The protocol for nuclear protein extraction was showed in the method section. The β-catenin, lamni A/C, GAPDH, and NEDD4L protein levels were analyzed by immunoblotting assays.

Article Snippet: SYBR® Green PCR Master Mix (cat. no. 4309155), the MultiScribe (tm) Reverse Transcriptase Kit (cat. no. N8080234), TaqMan Advanced miRNA cDNA Synthesis Kit (cat. no. A28007), TaqMan® pri-miR-513a-1 (cat. no. Hs03296262_pri), pri-miR-513a-2 (cat. no. Hs03295531_pri), TaqMan® Advanced miR-513a-5p (cat. no. 479483_mir), and TaqMan® Advanced miR-191-5p (cat. no. 477952_mir) were purchased from Applied Biosystems (Thermo Fisher Scientific).

Techniques: Expressing, Microarray, Luciferase, Activity Assay, Plasmid Preparation, Transfection, Mutagenesis, Over Expression, Real-time Polymerase Chain Reaction, Western Blot, Protein Extraction

Journal: PLoS ONE

Article Title: IGF-1-enhanced miR-513a-5p signaling desensitizes glioma cells to temozolomide by targeting the NEDD4L-inhibited Wnt/β-catenin pathway

doi: 10.1371/journal.pone.0225913

Figure Lengend Snippet: (A) Effects of the overexpression of miR-513a-5p on downstream protein expressions of NEDD4L-inhibited WNT/β-catenin signaling. (B) Overexpression of miR-513a-5p enhanced the IGF-1-activated WNT/β-catenin pathway. (C) Overexpression of NEDD4L repressed the IGF-1-activated WNT/β-catenin pathway. After U87-MG cells were transfected with the indicated doses of the miR-513a-5p plasmid for 24 h, 1 μg of the miR-513a-5p plasmid combined with 200 ng/ml IGF-1 treatment for 48 h, or 2 μg NEDD4L plasmids combined with 200 ng/ml IGF-1 treatment for 48 h, WNT/β-catenin pathway downstream protein expressions were measured by an immunoblotting assay. Effects of miR-513a-5p (D) and NEDD4L (E) on IGF-1-reduced glioma sensitivity to TMZ. After U87-MG cells were transfected with 1 μg of the miR-513a-5p plasmid or 2 μg of the NEDD4L plasmid combined with 200 ng/ml IGF-1 and 200 μM TMZ treatment for 48 h, cell viability was measured by an MTT assay. Data are the mean ± SD of three independent experiments. * p <0.05. (F) Knockdown effects of miR-513a-5p on IGF-1-insensitized TMZ cytotoxicity. Cell viability was measured by a cell counting assay. Data are the mean ± SD of three independent experiments. * p <0.05.

Article Snippet: SYBR® Green PCR Master Mix (cat. no. 4309155), the MultiScribe (tm) Reverse Transcriptase Kit (cat. no. N8080234), TaqMan Advanced miRNA cDNA Synthesis Kit (cat. no. A28007), TaqMan® pri-miR-513a-1 (cat. no. Hs03296262_pri), pri-miR-513a-2 (cat. no. Hs03295531_pri), TaqMan® Advanced miR-513a-5p (cat. no. 479483_mir), and TaqMan® Advanced miR-191-5p (cat. no. 477952_mir) were purchased from Applied Biosystems (Thermo Fisher Scientific).

Techniques: Over Expression, Transfection, Plasmid Preparation, Western Blot, MTT Assay, Knockdown, Cell Counting

Journal: Oncology Letters

Article Title: Lectin-captured plasma microRNAs predict response and survival in patients with non-small cell lung cancer treated with nivolumab

doi: 10.3892/ol.2026.15669

Figure Lengend Snippet: Identification of circulating OAA1-captured miRNAs associated with lung cancer and resistance to nivolumab. (A) Lectin histochemistry using OAA1 on surgically resected lung cancer tissues (×200 magnification). The left panel shows OAA1 lectin staining in normal lung tissue, whereas the right panel shows OAA1 lectin staining in lung cancer tissue. (B) In the discovery phase, plasma samples from two patients with NSCLC with PD were collected before and after nivolumab treatment. After plasma purification using an OAA1 column, microarray analysis was performed to identify candidate miRNAs (miR-320a, miR-320b, and miR-3613-5p) associated with nivolumab resistance. In the validation phase, plasma samples from 48 patients with NSCLC (before nivolumab treatment) were analyzed with and without OAA1 column purification. The levels of the identified miRNAs were quantified using TaqMan qPCR, and statistical analyses were conducted to evaluate their predictive and prognostic values as biomarkers for nivolumab resistance. (C) Microarray analysis of OAA1-captured plasma miRNAs in patients with PD after nivolumab treatment reveals several upregulated candidate miRNAs, including miR-320a, miR-320b, and miR-3613-5p. (D) Relative levels of miR-320a, miR-320b, and miR-3613-5p in pre-treatment plasma from patients with NSCLC (n=48) and healthy controls (n=11) with and without OAA1 enrichment. Plasma miRNA levels were quantified by reverse transcription-qPCR. Relative miRNA levels were normalized to miR-16-5p and calculated using the 2 −ΔΔCq method, with the mean level of healthy volunteers serving as the calibrator. Statistical significance was determined using the Mann-Whitney U test. Data are presented as median with 95% confidence intervals. miRNA/miR, microRNA; NSCLC, non-small cell lung cancer; OAA1, Oscillatoria agardhii agglutinin 1; PD, progressive disease; qPCR, quantitative polymerase chain reaction.

Article Snippet: After thorough vortex mixing, the miRNA was purified using the NucleoSpin miRNA Plasma kit (Macherey-Nagel, Germany) with DNA digestion treatment, according to the manufacturer's protocol.

Techniques: Staining, Clinical Proteomics, Purification, Microarray, Biomarker Discovery, Reverse Transcription, MANN-WHITNEY, Real-time Polymerase Chain Reaction

Journal: Oncology Letters

Article Title: Lectin-captured plasma microRNAs predict response and survival in patients with non-small cell lung cancer treated with nivolumab

doi: 10.3892/ol.2026.15669

Figure Lengend Snippet: Differential expression of miR-320a, miR-320b, and miR-3613-5p in pre-treatment plasma from patients with non-small cell lung cancer with PR or SD + PD. (A) Relative levels of miR-320a, miR-320b, and miR-3613-5p without OAA1 enrichment. (B) With OAA1 column enrichment. Plasma miRNA levels were quantified by reverse transcription-quantitative polymerase chain reaction and are shown as relative abundance using the 2 −ΔCq method normalized to miR-16-5p. Statistical significance was determined using the Mann-Whitney U test. Data are presented as median with 95% confidence intervals. miR, microRNA; OAA1, Oscillatoria agardhii agglutinin 1; SD, stable disease; PD, progressive disease; PR, partial response.

Article Snippet: After thorough vortex mixing, the miRNA was purified using the NucleoSpin miRNA Plasma kit (Macherey-Nagel, Germany) with DNA digestion treatment, according to the manufacturer's protocol.

Techniques: Quantitative Proteomics, Clinical Proteomics, Reverse Transcription, Real-time Polymerase Chain Reaction, MANN-WHITNEY

Journal: Oncology Letters

Article Title: Lectin-captured plasma microRNAs predict response and survival in patients with non-small cell lung cancer treated with nivolumab

doi: 10.3892/ol.2026.15669

Figure Lengend Snippet: ROC curve analysis of miRNAs for predicting non-response to immune checkpoint inhibitor therapy in patients with non-small cell lung cancer treated with nivolumab. (A) ROC curves for circulating miR-320a, miR-320b, and miR-3613-5p without OAA1 column enrichment. (B) With OAA1 enrichment. AUC, area under the curve; CI, confidence interval; miR, microRNA; OAA1, Oscillatoria agardhii agglutinin 1; ROC, receiver operating characteristic.

Article Snippet: After thorough vortex mixing, the miRNA was purified using the NucleoSpin miRNA Plasma kit (Macherey-Nagel, Germany) with DNA digestion treatment, according to the manufacturer's protocol.

Techniques:

Journal: Oncology Letters

Article Title: Lectin-captured plasma microRNAs predict response and survival in patients with non-small cell lung cancer treated with nivolumab

doi: 10.3892/ol.2026.15669

Figure Lengend Snippet: Kaplan-Meier analysis for OS based on pre-treatment plasma levels of miR-320a, miR-320b, and miR-3613-5p in patients with non-small cell lung cancer treated with nivolumab (n=48). (A) Kaplan-Meier curves comparing the OS between the high- and low-expression groups for each miRNA without OAA1 column enrichment. (B) Kaplan-Meier curves for the same miRNAs with the OAA1 enrichment. The number of patients in each group is indicated in the legends. Cutoff values for high/low expression were determined using receiver operating characteristic analysis. P-values are shown; *P<0.05. miR, microRNA; OAA1, Oscillatoria agardhii agglutinin 1; OS, overall survival.

Article Snippet: After thorough vortex mixing, the miRNA was purified using the NucleoSpin miRNA Plasma kit (Macherey-Nagel, Germany) with DNA digestion treatment, according to the manufacturer's protocol.

Techniques: Clinical Proteomics, Expressing

Journal: PLoS ONE

Article Title: IGF-1-enhanced miR-513a-5p signaling desensitizes glioma cells to temozolomide by targeting the NEDD4L-inhibited Wnt/β-catenin pathway

doi: 10.1371/journal.pone.0225913

Figure Lengend Snippet: Volcano plot (A) and heat map of hierarchical gene clustering (B) demonstrating IGF-1-regulated microRNA profiles. After U87 MG cells were treated with 200 ng/ml IGF-1 for 48 h, total RNAs were extracted for a microarray analysis. For all array analyses, an adjusted (adj.) p value of <0.05 and a |log2 (ratio)| of ≥0.58 (±1.5 multiples of change) cutoff were applied. Volcano plots show the multiples of change (log 2 ratio) and probability (-log 10 adj. p values) of individual microRNAs from the microarray assay. The red dot shows the miR-513a-5p location. The heat map depicts the 241 microRNAs differentially expressed between IGF-1 subsets. A color was assigned to each microRNA based on its relative expression level across samples. (C) Detection of endogenous miR-513a-5p levels in human normal astrocytes and three different glioma cell lines. Endogenous miR-513a-5p levels were measured by a real-time PCR. The miR-191-5p level was used as an internal control. Data are the mean ± SD of three independent experiments. * p <0.05. Increased miR-513a levels in GBM patients and glioma stem cells (GSCs) compared to normal controls from TCGA miRNA microarray data (D), and GEO DataSets GSE61710 (E), GSE37366, and GSE41032 (F). (E and F) miR-513a-5p expression changes were analyzed by GEO2R software.

Article Snippet: SYBR® Green PCR Master Mix (cat. no. 4309155), the MultiScribe (tm) Reverse Transcriptase Kit (cat. no. N8080234), TaqMan Advanced miRNA cDNA Synthesis Kit (cat. no. A28007), TaqMan® pri-miR-513a-1 (cat. no. Hs03296262_pri), pri-miR-513a-2 (cat. no. Hs03295531_pri), TaqMan® Advanced miR-513a-5p (cat. no. 479483_mir), and TaqMan® Advanced miR-191-5p (cat. no. 477952_mir) were purchased from Applied Biosystems (Thermo Fisher Scientific).

Techniques: Microarray, Expressing, Real-time Polymerase Chain Reaction, Control, Software

Journal: PLoS ONE

Article Title: IGF-1-enhanced miR-513a-5p signaling desensitizes glioma cells to temozolomide by targeting the NEDD4L-inhibited Wnt/β-catenin pathway

doi: 10.1371/journal.pone.0225913

Figure Lengend Snippet: (A) Dose-dependent effects of IGF-1 on miR-513a-5p expression levels in U87-MG and M059K cells. (B) Effects of IGF-1 on primary (pri)-miR-513a-1 and pri-miR-513a-2 levels. The PI3K inhibitor, LY294002, but not the mitogen-activated protein kinase (MAPK) kinase inhibitor, U0126, attenuated IGF-1-stimulated miR-513a-5p (C) and pri-miR-513a-2 (D) levels. After cells were treated with the indicated doses of IGF-1 for 48 h or 200 ng/ml IGF-1 treatment for 48 h and pretreatment with 5 μM LY294002 and 10 μM U0126 for 1 h, endogenous miR-513a-5p, pri-miR-513a-1, and pri-miR-513a-2 levels were respectively measured by a real-time PCR. miR-191-5p and GAPDH levels were respectively used as internal controls for the mature and primary miR-513a-5p levels. Data are the mean ± SD of three independent experiments. * p <0.05.

Article Snippet: SYBR® Green PCR Master Mix (cat. no. 4309155), the MultiScribe (tm) Reverse Transcriptase Kit (cat. no. N8080234), TaqMan Advanced miRNA cDNA Synthesis Kit (cat. no. A28007), TaqMan® pri-miR-513a-1 (cat. no. Hs03296262_pri), pri-miR-513a-2 (cat. no. Hs03295531_pri), TaqMan® Advanced miR-513a-5p (cat. no. 479483_mir), and TaqMan® Advanced miR-191-5p (cat. no. 477952_mir) were purchased from Applied Biosystems (Thermo Fisher Scientific).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: IGF-1-enhanced miR-513a-5p signaling desensitizes glioma cells to temozolomide by targeting the NEDD4L-inhibited Wnt/β-catenin pathway

doi: 10.1371/journal.pone.0225913

Figure Lengend Snippet: (A) The inverse correlation between miR-513a and NEDD4L expression levels in TCGA microarray data. Using the median cutoff for miR-513a expression levels, patients were divided into two groups. A correlation was found in the group highly expressing miR-513a ( n = 259) by Pearson's correlation analysis. (B) Schematic diagram of potential miR-513a-5p-targeted sites in the NEDD4L 3’-untranslated region (UTR). (C and D) Effects of miR-513a-5p on NEDD4L 3’-UTR luciferase activity. To test for miR-513a-5p's effect, different doses of the miR-513a-5p plasmid were co-transfected with 500 ng of the pmiRGlo-NEDD4L 3’-UTR or mutant 3’-UTR. Luciferase activity was measured in these cells 24 h after transfection. Effects of miR-513a-5p overexpression on NEDD4L mRNA (E) and protein (F) expressions. After cells were transfected with the indicated dose of the miR-513a-5p plasmid for 24 h, relative mRNA and protein levels of NEDD4L were analyzed using a real-time PCR and an immunoblotting assay. (G) Dose-dependent effects of IGF-1 on reducing NEDD4L levels. (H) miR-513a-5p is involved in the IGF-1-reduced NEDD4L expression mechanism. After cells were treated with indicated doses of IGF-1 for 48 h or transfected with 1 μg of the miR-513a-5p plasmid combined with IGF-1 treatment for 48 h, the NEDD4L protein level was analyzed by an immunoblotting assay. Data are the mean ± SD of three experiments. * p <0.05. Inhibitory effects of miR-513a-5p on IGF-1-induced β-catenin nuclear accumulation (I) and -reduced NEDD4L levels (J). The protocol for nuclear protein extraction was showed in the method section. The β-catenin, lamni A/C, GAPDH, and NEDD4L protein levels were analyzed by immunoblotting assays.

Article Snippet: SYBR® Green PCR Master Mix (cat. no. 4309155), the MultiScribe (tm) Reverse Transcriptase Kit (cat. no. N8080234), TaqMan Advanced miRNA cDNA Synthesis Kit (cat. no. A28007), TaqMan® pri-miR-513a-1 (cat. no. Hs03296262_pri), pri-miR-513a-2 (cat. no. Hs03295531_pri), TaqMan® Advanced miR-513a-5p (cat. no. 479483_mir), and TaqMan® Advanced miR-191-5p (cat. no. 477952_mir) were purchased from Applied Biosystems (Thermo Fisher Scientific).

Techniques: Expressing, Microarray, Luciferase, Activity Assay, Plasmid Preparation, Transfection, Mutagenesis, Over Expression, Real-time Polymerase Chain Reaction, Western Blot, Protein Extraction

Journal: PLoS ONE

Article Title: IGF-1-enhanced miR-513a-5p signaling desensitizes glioma cells to temozolomide by targeting the NEDD4L-inhibited Wnt/β-catenin pathway

doi: 10.1371/journal.pone.0225913

Figure Lengend Snippet: (A) Effects of the overexpression of miR-513a-5p on downstream protein expressions of NEDD4L-inhibited WNT/β-catenin signaling. (B) Overexpression of miR-513a-5p enhanced the IGF-1-activated WNT/β-catenin pathway. (C) Overexpression of NEDD4L repressed the IGF-1-activated WNT/β-catenin pathway. After U87-MG cells were transfected with the indicated doses of the miR-513a-5p plasmid for 24 h, 1 μg of the miR-513a-5p plasmid combined with 200 ng/ml IGF-1 treatment for 48 h, or 2 μg NEDD4L plasmids combined with 200 ng/ml IGF-1 treatment for 48 h, WNT/β-catenin pathway downstream protein expressions were measured by an immunoblotting assay. Effects of miR-513a-5p (D) and NEDD4L (E) on IGF-1-reduced glioma sensitivity to TMZ. After U87-MG cells were transfected with 1 μg of the miR-513a-5p plasmid or 2 μg of the NEDD4L plasmid combined with 200 ng/ml IGF-1 and 200 μM TMZ treatment for 48 h, cell viability was measured by an MTT assay. Data are the mean ± SD of three independent experiments. * p <0.05. (F) Knockdown effects of miR-513a-5p on IGF-1-insensitized TMZ cytotoxicity. Cell viability was measured by a cell counting assay. Data are the mean ± SD of three independent experiments. * p <0.05.

Article Snippet: SYBR® Green PCR Master Mix (cat. no. 4309155), the MultiScribe (tm) Reverse Transcriptase Kit (cat. no. N8080234), TaqMan Advanced miRNA cDNA Synthesis Kit (cat. no. A28007), TaqMan® pri-miR-513a-1 (cat. no. Hs03296262_pri), pri-miR-513a-2 (cat. no. Hs03295531_pri), TaqMan® Advanced miR-513a-5p (cat. no. 479483_mir), and TaqMan® Advanced miR-191-5p (cat. no. 477952_mir) were purchased from Applied Biosystems (Thermo Fisher Scientific).

Techniques: Over Expression, Transfection, Plasmid Preparation, Western Blot, MTT Assay, Knockdown, Cell Counting

Journal: PLOS ONE

Article Title: A systematic review of non-coding RNA genes with differential expression profiles associated with autism spectrum disorders

doi: 10.1371/journal.pone.0287131

Figure Lengend Snippet: Studies examining ncRNA gene expression in ASD using tissue samples and laboratory methodologies that could be feasibly implemented into clinical practice (N = 33).

Article Snippet: 13:2 , 4.3 , Not stated: Sex and age matched , , 5 miRNA genes examined by qPCR on CFX96 BioRad , miR-16-5p.

Techniques: Gene Expression, Biomarker Discovery, Control, RNA Extraction, Gene Assay, Microarray, TLDA Assay, Sample Prep, Standard Deviation, SYBR Green Assay, Sequencing, Expressing, Isolation, Reverse Transcription, Clinical Proteomics, TaqMan microRNA Assay, Multiplex Assay, Construct, Fluorescence

Journal: Obesity Medicine

Article Title: Relationship between microRNA expression profiling and gestational diabetes: A systematic review

doi: 10.1016/j.obmed.2023.100487

Figure Lengend Snippet: Fig. 2. HeatMap and cluster patterns from gene expression pathways affected by miRNAs. A: Target of upregulated miRNAs in placental tissue, placenta-derived cells (Mononuclear cells), peripheral blood, plasma, plasma Evs, and serum B: Target of downregulated miRNAs in placental tissue and placenta-derived exosomes.

Article Snippet: RT-qPCR U6 n/a n/a HUANG et al., 2021* Trizol n/a RT-qPCR GAPDH n/a n/a Li 2015ɨ** RecoverAllTM Total Nucleic Acid Isolation kit (Ambion) y TRNzol reagent (TIANGEN Biotech, Beijing, China) miRNA first-strand cDNA kit (TIANGEN Biotech, Beijing, China) miRcute miRNA qPCR detection kit (TIANGEN Biotech, Beijing, China) U6 Agilent Human miRNA Microarray (Agilent) n/a Li et al., 2018** KingFisher Automated Purification Kits (Thermo Fisher) miRCURY LNATM microRNA Array Hi-Power Labeling kit (Qiagen) n/a Hairpin-itTMmiRNAs RealTime PCR Quantization Kit (GenePharma) n/a n/a n/a Li W 2022* TRIzol reagent (Magen Biotechnology Co.) TaKaRa PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa) miScript SYBR Green PCR Kit (TaKaRa) U6 n/a n/a Peng et al., 2019* Trizol (Ambion, USA) miRcute miRNA FirstStrand cDNA Synthesis Kit (TianGen, Beijing, China) miRcute miRNA qPCR Detection Kit (SYBR Green) (TianGen, Beijing China) U6 n/a n/a Sun et al., 2020* Trizol reagent (Invitrogen, Carlsbad, CA, USA) TaqMan MicroRNA Reverse Transcription Kit TaqMan Univel PCR Master Mix (Applied Biosystems, Foster City, CA, USA) U6 n/a n/a Tang 2020ᵻ* Trizol Reagent (Invitrogen, Carlsbad, CA, USA) M-MLV Reverse Transcriptase (Invitrogen) qRT–PCR (BIONEER, Daejeon, Korea) U6 n/a NEBNext Multiplex Small RNA Library Prep Set for Illumina_x0003_ (NEB, Ipswich, MA, USA) Wang S 2020* Trizol reagent (Invitrogen) Transcriptase Kit (Takara, Otsu, Japan) SYBR Green PCR Master Mix (Takara) U6 n/a n/a Zhang Lu 2021* Trizol n/a RT-qPCR U6 n/a n/a Zhao et al., 2014* Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) TaqMan MicroRNA Reverse Transcription kit TaqMan microRNA assay (Applied Biosystems, Branchburg, NJ, USA) U6 n/a n/a Serum Abdeltawab et al., 2020** miRVana PARIS kits (Life Technologies, Grand Island, NY, USA) TaqMan MicroRNA Reverse Transcription Kit TaqMan quantitative RTPCR.

Techniques: Gene Expression, Derivative Assay, Clinical Proteomics

Journal: Brain Pathology

Article Title: Role of micro RNA s Located on Chromosome Arm 10q in Malignant Gliomas

doi: 10.1111/bpa.12294

Figure Lengend Snippet: Expression of miR‐107, miR‐146b‐5p, miR‐346 and miR‐1287‐5p in astrocytic gliomas of different WHO grades. Relative miRNA expression levels were determined by real‐time RT‐PCR using let‐7a as reference. Box plots are depicted indicating normalized expression values according to Tukey's method. Asterisks indicate significant expression differences compared with non‐neoplastic brain tissue samples (*P < 0.05; **P < 0.01, ***P < 0.001; Kruskal–Wallis test and Dunn's post‐hoc test). AII, diffuse astrocytoma, WHO grade II; AAIII, anaplastic astrocytoma, WHO grade III; sGBIV, secondary glioblastoma, WHO grade IV; pGBIV, primary glioblastoma, WHO grade IV; NB, non‐neoplastic brain tissue; n, number of investigated tissue samples.

Article Snippet: To screen for DNA methylation‐ and histone acetylation‐dependent miRNA expression, cells were incubated in medium with 5‐aza‐2′‐deoxycytidine (5‐AZA‐dCR) and trichostatin A (TSA) under two different conditions 1 : 500 nM 5‐AZA‐dCR for 48 h, followed by 500 nM 5‐AZA‐dCR and 1 μM TSA for 24 h 2 ; 1 μM 5‐AZA‐dCR for 48 h, followed by 1 μM 5‐AZA‐dCR and 1 μM TSA for 24 h. RNA was extracted using Trizol (Life Technologies).

Techniques: Expressing, Quantitative RT-PCR

Journal: Brain Pathology

Article Title: Role of micro RNA s Located on Chromosome Arm 10q in Malignant Gliomas

doi: 10.1111/bpa.12294

Figure Lengend Snippet: miR‐146b‐5p expression in 5‐aza‐2′‐deoxycytidine (5‐AZA‐dCR) and trichostatin A (TSA) treated glioma cell lines (A) as well as methylation of CpG sites located in putative SP1 transcription factor‐binding sites associated with the miR‐146b‐5p locus (B). (A) Relative expression levels of miR‐146b‐5p were determined by real‐time RT‐PCR using let‐7a as reference gene. Expression of the miRNA in the 5‐AZA‐dCR/TSA samples was calculated relative to miRNA expression in the controls (Co). A + T1: cell lines were grown in medium supplemented with 500 nM 5‐AZA‐dCR for 48 h, washed and grown for another 24 h in medium with 500 nM 5‐AZA‐dcr and 1 µM TSA; A + T2: like A + T1, except for a higher 5‐AZA‐dCR concentration (1 µM). Asterisks indicate significant difference when compared with the respective non‐treated controls (*P < 0.05; **P < 0.01; paired t‐test). Bars represent the mean, and error bars represent the standard deviation of three independent experiments, except for U138MG A + T2 with two independent experiments. (B) Methylation analysis of the seven CpG sites located from nt 104,195,358 to nt 104,195,483 on chr10 (UCSC genome browser, GRCh37/hg19 assembly) was performed by pyrosequencing of two PCR products generated from sodium bisulfite‐modified DNA. The three CpG sites located within each of the three putative SP1‐binding sequences are labeled by hachures. The methylation status at each CpG site was gray scale‐coded as follows: white square, 0–25% methylated alleles; light gray square, 26–50% methylated alleles; gray square, 51–75% methylated alleles; black square, 76–100% methylated alleles. methCo, methylation positive control (CpGenome™ Universal Methylated DNA, Merck Millipore, Darmstadt, Germany); AII, diffuse astrocytoma WHO grade II; AAIII, anaplastic astrocytoma WHO grade III; sGBIV, secondary glioblastoma WHO grade IV; pGBIV, primary glioblastoma WHO grade IV; NB, non‐neoplastic brain tissue; Meth% (mean), mean of the percentages of methylated allele derived from pyrosequencing at the seven investigated CpG sites. IDH mut: gray square, IDH1 or IDH2 mutation detected; white square, no IDH1 or IDH2 mutation detected. TERT mut: gray square, TERT promoter mutation detected; white square, no TERT promoter mutation detected. Note a significant difference in the mean percentage of methylated alleles between primary glioblastoma (mean: 5% methylated alleles) and secondary glioblastoma (mean: 30% methylated alleles; P < 0.01; Student's t‐test, two‐sided).

Article Snippet: To screen for DNA methylation‐ and histone acetylation‐dependent miRNA expression, cells were incubated in medium with 5‐aza‐2′‐deoxycytidine (5‐AZA‐dCR) and trichostatin A (TSA) under two different conditions 1 : 500 nM 5‐AZA‐dCR for 48 h, followed by 500 nM 5‐AZA‐dCR and 1 μM TSA for 24 h 2 ; 1 μM 5‐AZA‐dCR for 48 h, followed by 1 μM 5‐AZA‐dCR and 1 μM TSA for 24 h. RNA was extracted using Trizol (Life Technologies).

Techniques: Expressing, Methylation, Binding Assay, Quantitative RT-PCR, Gene Expression, Concentration Assay, Standard Deviation, Generated, Modification, Labeling, Positive Control, Derivative Assay, Mutagenesis

Journal: Brain Pathology

Article Title: Role of micro RNA s Located on Chromosome Arm 10q in Malignant Gliomas

doi: 10.1111/bpa.12294

Figure Lengend Snippet: In vitro effects of miR‐107, miR‐146b‐5p, miR‐346 or miR‐1287‐5p overexpression on apoptotic activity (A) and proliferation (B) of glioma cells. A172 and T98G cells were transfected with either 10 nM, 25 nM or 50 nM pre‐miR™ precursor molecules (pre‐107, pre‐146‐5p, pre‐346, pre‐1287‐5p) or negative control pre‐miR™ miRNA precursors (pre‐NC1; Life Technologies). Apoptotic activity was determined by a luminescence‐based caspase‐3/7 assay. Proliferation was measured using a luminescence‐based BrdU incorporation assay. Dots represent the mean of five replicates. P‐values were calculated using paired t‐tests. Mean fold‐expression changes (FC) are also provided.

Article Snippet: To screen for DNA methylation‐ and histone acetylation‐dependent miRNA expression, cells were incubated in medium with 5‐aza‐2′‐deoxycytidine (5‐AZA‐dCR) and trichostatin A (TSA) under two different conditions 1 : 500 nM 5‐AZA‐dCR for 48 h, followed by 500 nM 5‐AZA‐dCR and 1 μM TSA for 24 h 2 ; 1 μM 5‐AZA‐dCR for 48 h, followed by 1 μM 5‐AZA‐dCR and 1 μM TSA for 24 h. RNA was extracted using Trizol (Life Technologies).

Techniques: In Vitro, Over Expression, Activity Assay, Transfection, Negative Control, BrdU Incorporation Assay, Expressing

Journal: Brain Pathology

Article Title: Role of micro RNA s Located on Chromosome Arm 10q in Malignant Gliomas

doi: 10.1111/bpa.12294

Figure Lengend Snippet: Validation of predicted miR‐107, miR‐146b‐5p, miR‐346 and miR‐1287‐5p target genes. (A) Results of mRNA analyses. Twenty‐four hours after seeding, cells were transfected with 25 nm pre‐miR™ precursor molecules (pre‐107, pre‐146b‐5p, pre‐346, pre‐1287‐5p; Life Technologies) or negative control pre‐miR™ miRNA precursors (pre‐NC1, Life Technologies). Total RNA was extracted 48 h after transfection and mRNA levels of the putative targets were determined by RT‐PCR using ARF1 (CDK6, EGFR), B2M (SEMA6A) or ACTG1 (TERT) as reference genes and universal human reference RNA (Stratagene) as calibrator. Data represent the mean of three independent transfection experiments. Error bars show standard deviations. Asterisks indicate significant difference as compared with the respective pre‐NC1 controls (paired t‐test, one‐sided). (B–C) Results of Western blot analyses. Cells were transfected with 50 nM pre‐miR™ precursor molecules (pre‐107, pre‐146b‐5p, pre‐1287‐5p) or negative control pre‐miR™ miRNA precursors (pre‐NC1). The expression levels of CDK6 (B), EGFR (C), α‐tubulin—as a loading control—were determined 72 h post‐transfection. Approximate sizes of the protein bands in kilo‐dalton (kDa) are indicated on the right. Below each exemplary Western blot, the results of protein quantification from three independent experiments are depicted. P‐values were calculated by a two‐sided paired t‐test. (D) Results of 3′‐UTR luciferase gene reporter assays. Relative luciferase activity in T98G cells were determined 48 h post‐transfection with 100 nm pre‐miR™ precursor molecules (pre‐107, pre‐146b‐5p, pre‐346, pre‐1287‐5p; Life Technologies) or negative control pre‐miR™ miRNA precursors (pre‐NC1, Life Technologies) in combination with either wild‐type (‐3′WT) or deleted/mutated (‐3'del, ‐3'mut) psiCHECK2 constructs. Renilla luciferase activity was normalized to F irefly luciferase activity. Vertical bars represent the mean of three independent experiments with each performed in quintuplicate. Asterisks indicate significant differences compared with pre‐NC1 transfected cells (paired t‐test, two‐sided).*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: To screen for DNA methylation‐ and histone acetylation‐dependent miRNA expression, cells were incubated in medium with 5‐aza‐2′‐deoxycytidine (5‐AZA‐dCR) and trichostatin A (TSA) under two different conditions 1 : 500 nM 5‐AZA‐dCR for 48 h, followed by 500 nM 5‐AZA‐dCR and 1 μM TSA for 24 h 2 ; 1 μM 5‐AZA‐dCR for 48 h, followed by 1 μM 5‐AZA‐dCR and 1 μM TSA for 24 h. RNA was extracted using Trizol (Life Technologies).

Techniques: Biomarker Discovery, Transfection, Negative Control, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Control, Luciferase, Activity Assay, Construct

Journal: Brain Pathology

Article Title: Role of micro RNA s Located on Chromosome Arm 10q in Malignant Gliomas

doi: 10.1111/bpa.12294

Figure Lengend Snippet: mRNA expression levels of the 10q‐miRNA targets CDK6, EGFR, SEMA6A, and TERT in astrocytic glioma. (A) The data for CDK6, EGFR, and SEMA6A expression were obtained from a published microarray dataset 42. Box plots are depicted indicating normalized expression values according to Tukey's method; n, number of tissue samples investigated. The mRNA expression levels of TERT were determined by real‐time RT‐PCR using ARF1 as reference. Asterisks indicate significant expression differences compared with non‐neoplastic brain tissue samples (*P < 0.05; **P < 0.01; Kruskal–Wallis test and Dunn's post‐hoc test). AII, diffuse astrocytoma WHO grade II; AAIII, anaplastic astrocytoma WHO grade III; sGBIV, secondary glioblastoma WHO grade IV; pGBIV, primary glioblastoma WHO grade IV; NB, non‐neoplastic brain tissue. (B) MiR‐346 expression in TERT‐mutant and wild‐type gliomas. MiR‐346 expression is significantly different between TERT promoter‐mutant compared with TERT promoter wild‐type glioma samples (Mann–Whitney test, ****P < 0.0001). (C) The two cases (GB113 and A151) with highest TERT expression in the absence of a detectable TERT promoter mutation showed evidence for a low‐level copy number gain of TERT (A151) or TERT amplification (GB113). APRT was used as a reference gene in the duplex PCR analysis. NB, non‐neoplastic brain tissue; NTC, no template control; bp, base pairs.

Article Snippet: To screen for DNA methylation‐ and histone acetylation‐dependent miRNA expression, cells were incubated in medium with 5‐aza‐2′‐deoxycytidine (5‐AZA‐dCR) and trichostatin A (TSA) under two different conditions 1 : 500 nM 5‐AZA‐dCR for 48 h, followed by 500 nM 5‐AZA‐dCR and 1 μM TSA for 24 h 2 ; 1 μM 5‐AZA‐dCR for 48 h, followed by 1 μM 5‐AZA‐dCR and 1 μM TSA for 24 h. RNA was extracted using Trizol (Life Technologies).

Techniques: Expressing, Microarray, Quantitative RT-PCR, Mutagenesis, MANN-WHITNEY, Amplification, Control

Journal: Brain Pathology

Article Title: Role of micro RNA s Located on Chromosome Arm 10q in Malignant Gliomas

doi: 10.1111/bpa.12294

Figure Lengend Snippet: Relationship between miRNA expression and IDH mutation status in our institutional glioma series (A) and the TCGA glioblastoma cohort (B). (A) MiR‐107 (P < 0.0001) and miR‐346 (P < 0.001) expression is increased, whereas the expression of miR‐146b‐5p (P < 0.01) and miR‐1287‐5p (P < 0.05) is decreased in astrocytic gliomas with IDH1/IDH2 mutation (IDH mut) compared with IDH1/IDH2 wild‐type (IDH wt) tumors (Mann–Whitney test). Box plots are depicted indicating normalized expression values according to Tukey's method; n, number of tissue samples investigated. (B) MiR‐146b‐5p expression is decreased in IDH1‐mutant (IDH1 mut) as compared with IDH1 wild‐type (IDH1 wt) glioblastomas of the TCGA cohort. MiRNA expression z‐scores and IDH1 mutation data were obtained from 158 TCGA glioblastoma cases via the cBio Cancer Genomics portal. P‐values were calculated using a two‐sided Student's t‐test and corrected for multiple testing (Bonferroni‐correction for 17 comparisons). IDH1 ‐mutant glioblastomas carry missense mutations affecting arginine 132; IDH1 wild‐type glioblastomas carry the regular arginine at codon 132; n, number of investigated cases.

Article Snippet: To screen for DNA methylation‐ and histone acetylation‐dependent miRNA expression, cells were incubated in medium with 5‐aza‐2′‐deoxycytidine (5‐AZA‐dCR) and trichostatin A (TSA) under two different conditions 1 : 500 nM 5‐AZA‐dCR for 48 h, followed by 500 nM 5‐AZA‐dCR and 1 μM TSA for 24 h 2 ; 1 μM 5‐AZA‐dCR for 48 h, followed by 1 μM 5‐AZA‐dCR and 1 μM TSA for 24 h. RNA was extracted using Trizol (Life Technologies).

Techniques: Expressing, Mutagenesis, MANN-WHITNEY

Journal: Cell Reports

Article Title: Endogenous Control of Immunity against Infection: Tenascin-C Regulates TLR4-Mediated Inflammation via MicroRNA-155

doi: 10.1016/j.celrep.2012.09.005

Figure Lengend Snippet: Tenascin-C Is Required for Induction of miR-155 in Activated BMDMs and in Septic Mice (A) Microarray analysis of miRNA profile in tnc +/+ and tnc − / − BMDMs stimulated for 8 hr with 100 ng/ml LPS. The heatmap shows suppression (green) and induction (red) of expression as fold change on a log 2 scale, relative to nonstimulated BMDMs (n = 3 mice per genotype). (B and C) Expression of TLR-induced miRNAs in tnc +/+ and tnc − / − BMDMs (identified in A) classified as early (B) and late (C) response genes (n = 3 per genotype; mean ± SEM). ∗∗ p < 0.01; ns, not significant. (D) Time course analysis of miR-155, pri-miR-155, miR-146a, and miR-21 expression in tnc +/+ and tnc − / − BMDMs stimulated for 4–72 hr (miR-155), 0.5–48 hr (pri-miR-155), or 4–96 hr (miR-146a, miR-21) with 100 ng/ml LPS. RNA used in (A) (n = 3 mice per group) and RNA isolated from BMDMs derived from two (miR-146a, miR-21) and four (miR-155, pri-miR-155) additional mice per genotype was analyzed by quantitative RT-PCR (mean ± SEM). ∗∗ p < 0.01 compared to tnc − / − BMDMs at the same time point. (E) Correlation of miR-155 expression with TNF-α synthesis in tnc +/+ and tnc − / − BMDMs nonstimulated or stimulated with 100 ng/ml LPS for 4, 8, and 24 hr (n = 5 mice per genotype; R = 1.0; p < 0.05). (F) Quantitative RT-PCR analysis of miR-155 expression in spleen from tnc +/+ and tnc − / − mice 1.5 hr after LPS injection (n = 3 per genotype; mean ± SEM). ∗ p < 0.05. Data are from two independent experiments.

Article Snippet: For pri-miRNA detection, RNA was reverse transcribed with AffinityScript reverse transcriptase and random primers (Stratagene), followed by real-time PCR with TaqMan primer sets for mouse pri-miR155 (Mm03306395_pri) and HPRT1 (Applied Biosystems).

Techniques: Microarray, Expressing, Isolation, Derivative Assay, Quantitative RT-PCR, Injection

Journal: Cell Reports

Article Title: Endogenous Control of Immunity against Infection: Tenascin-C Regulates TLR4-Mediated Inflammation via MicroRNA-155

doi: 10.1016/j.celrep.2012.09.005

Figure Lengend Snippet: miR-155 Overexpression Rescues TNF-α Release in Tnc − / − BMDMs in Response to LPS (A) Quantitative RT-PCR analysis of miR-155 expression in tnc +/+ untransfected BMDMs and tnc − / − untransfected, mock transfected, and transfected with control precursor or miR-155 precursor BMDMs stimulated for 8 hr with 100 ng/ml LPS. Results are presented relative to those of nonstimulated BMDMs (n = 3; mean ± SEM). ∗ p < 0.05; ns, not significant. Data are representative of four independent experiments each with cells obtained from three mice per genotype. (B) ELISA of TNF-α secreted by tnc +/+ untransfected BMDMs and tnc − / − untransfected, mock transfected, and transfected with control precursor or miR-155 precursor BMDMs stimulated for 8 hr with 100 ng/ml LPS (n = 6; mean ± SEM). ∗∗∗ p < 0.001, ∗∗ p < 0.01; ns, not significant. Data are from two independent experiments each with cells obtained from three mice per genotype. See also Figure S4 .

Article Snippet: For pri-miRNA detection, RNA was reverse transcribed with AffinityScript reverse transcriptase and random primers (Stratagene), followed by real-time PCR with TaqMan primer sets for mouse pri-miR155 (Mm03306395_pri) and HPRT1 (Applied Biosystems).

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Transfection, Control, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports

Article Title: Endogenous Control of Immunity against Infection: Tenascin-C Regulates TLR4-Mediated Inflammation via MicroRNA-155

doi: 10.1016/j.celrep.2012.09.005

Figure Lengend Snippet: TNF-α Production in Tnc − / − BMDMs Is Rescued by miR-155 Overexpression but Not Soluble Tenascin-C, Related to Figure 7 (A) Quantitative RT-PCR analysis of miR-155 expression in tnc +/+ untransfected BMDMs and tnc −/− untransfected, mock transfected and transfected with control precursor or miR-155 precursor BMDMs, all in the absence of any stimulation with LPS (n = 3; mean ± SEM). Data are representative of four independent experiments each with cells obtained from three mice per genotype. (B) ELISA of TNF-α secreted by tnc +/+ untransfected BMDMs and tnc −/− untransfected, mock transfected and transfected with control precursor or miR-155 precursor BMDMs, all in the absence of any stimulation with LPS (n = 6; mean ± SEM). Data are from two independent experiments each with cells obtained from three mice per genotype. (C) ELISA of TNF-α and IL-6 secreted by tnc +/+ and tnc −/− BMDMs stimulated for 24 hr with 100ng/ml LPS without (-) or with recombinant purified tenascin-C (TN-C) (n = 3; mean ± SEM). ∗∗∗ = p < 0.001 compared to tnc +/+ BMDMs stimulated with LPS. Data are representative of three independent experiments each with cells obtained from three mice per genotype. The same results were obtained when we added equivalent amounts of commercially purified human tenascin-C.

Article Snippet: For pri-miRNA detection, RNA was reverse transcribed with AffinityScript reverse transcriptase and random primers (Stratagene), followed by real-time PCR with TaqMan primer sets for mouse pri-miR155 (Mm03306395_pri) and HPRT1 (Applied Biosystems).

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Transfection, Control, Enzyme-linked Immunosorbent Assay, Recombinant, Purification