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LC Sciences lc murine mirna microarray chips
Neurogenic Transcription Factors that Are Potentially Targeted by the miR-466/669 Cluster
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1) Product Images from "Arsenic Induces Members of the mmu-miR-466-669 Cluster Which Reduces NeuroD1 Expression"

Article Title: Arsenic Induces Members of the mmu-miR-466-669 Cluster Which Reduces NeuroD1 Expression

Journal: Toxicological Sciences

doi: 10.1093/toxsci/kfx241

Neurogenic Transcription Factors that Are Potentially Targeted by the miR-466/669 Cluster
Figure Legend Snippet: Neurogenic Transcription Factors that Are Potentially Targeted by the miR-466/669 Cluster

Techniques Used:

The miR-466-669 cluster share sequence similarities. Sequences of mature miRNA from the miR-466-669 cluster were aligned using Clustal Omega. All mature sequences were initially aligned together and then were subdivided into 4 major groups which have high similarity. The highlighted nucleotides were used to derive the 4 consensus sequences (as light blue). An asterisk indicates the sequence identity among all miRNAs within the group.
Figure Legend Snippet: The miR-466-669 cluster share sequence similarities. Sequences of mature miRNA from the miR-466-669 cluster were aligned using Clustal Omega. All mature sequences were initially aligned together and then were subdivided into 4 major groups which have high similarity. The highlighted nucleotides were used to derive the 4 consensus sequences (as light blue). An asterisk indicates the sequence identity among all miRNAs within the group.

Techniques Used: Sequencing

Expression profiles of microRNAs (miRNAs) between control and arsenic exposure in differentiating P19 cells. P19 cells were induced to differentiate with or without 0.5 µM of arsenic for 9 days. MicroRNA expression was detected via a miRNA microarray and plotted as a heat map using CIM Miner (NIH). Darker shading indicates increased expression. Only statistically different miRNAs are listed in the map (Student’s t test; P value < .05).
Figure Legend Snippet: Expression profiles of microRNAs (miRNAs) between control and arsenic exposure in differentiating P19 cells. P19 cells were induced to differentiate with or without 0.5 µM of arsenic for 9 days. MicroRNA expression was detected via a miRNA microarray and plotted as a heat map using CIM Miner (NIH). Darker shading indicates increased expression. Only statistically different miRNAs are listed in the map (Student’s t test; P value < .05).

Techniques Used: Expressing, Control, Microarray

Validation of miRNA transcript levels by qPCR. Day 9 differentiated P19 cells were used to confirm miRNA expression by qPCR (n = 3 per treatment). Significantly changed miRNAs with known roles in development were examined, including miR-92a (A), miR-291a (B), miR-709 (C), miR-199a (D), and miR-9 (E). Expression values were normalized with U6 snRNA and fold differences calculated from control cells using the delta Ct method. Values are expressed as mean ± SD and statistical differences were determined by Student’s t test (*P < .05).
Figure Legend Snippet: Validation of miRNA transcript levels by qPCR. Day 9 differentiated P19 cells were used to confirm miRNA expression by qPCR (n = 3 per treatment). Significantly changed miRNAs with known roles in development were examined, including miR-92a (A), miR-291a (B), miR-709 (C), miR-199a (D), and miR-9 (E). Expression values were normalized with U6 snRNA and fold differences calculated from control cells using the delta Ct method. Values are expressed as mean ± SD and statistical differences were determined by Student’s t test (*P < .05).

Techniques Used: Biomarker Discovery, Expressing, Control

Arsenic exposure induces members of the miR-466-669 cluster along with its host gene, Sfmbt2. P19 cells were differentiated and RNA was extracted from cells exposed to 0 or 0.5 μM arsenite on days 0, 2, 5, and 9 (n = 3 per treatment per day). MicroRNA or mRNA expression was determined by qPCR. Day 9 samples were used to determine the expression of miRNA-466-669 cluster genes (A) and the host gene Sfmbt2 (B). Samples from days 0, 2, 5, to 9 were used to determine the expression of miR-467d (C), miR-669p (D), and Sfmbt2 (E). Expression values were normalized with U6 snRNA for the miRNAs, and Gapdh for Sfmbt2. Fold changes were compared with unexposed cells, and time-dependent qPCR expression fold changes were compared with day 0 unexposed cells. Data are shown as mean ± SD. Statistical differences were determined by ANOVA followed by Tukey’s test or by Student’s t test (*P < .05).
Figure Legend Snippet: Arsenic exposure induces members of the miR-466-669 cluster along with its host gene, Sfmbt2. P19 cells were differentiated and RNA was extracted from cells exposed to 0 or 0.5 μM arsenite on days 0, 2, 5, and 9 (n = 3 per treatment per day). MicroRNA or mRNA expression was determined by qPCR. Day 9 samples were used to determine the expression of miRNA-466-669 cluster genes (A) and the host gene Sfmbt2 (B). Samples from days 0, 2, 5, to 9 were used to determine the expression of miR-467d (C), miR-669p (D), and Sfmbt2 (E). Expression values were normalized with U6 snRNA for the miRNAs, and Gapdh for Sfmbt2. Fold changes were compared with unexposed cells, and time-dependent qPCR expression fold changes were compared with day 0 unexposed cells. Data are shown as mean ± SD. Statistical differences were determined by ANOVA followed by Tukey’s test or by Student’s t test (*P < .05).

Techniques Used: Expressing

Inhibiting the miR-466-669 cluster during differentiation rescues the morphological loss of neurons following arsenic exposure. P19 cells were transfected with 100 nM anti-miRNA oligonucleotides which target 4 consensus sequences of the miRNA-466-467-669 cluster. Cells were coexposed to 0 or 0.5 μM arsenic for the 5 days of embryoid body formation. Only the arsenic exposure was maintained for the entire 9 days of differentiation, after which cell morphology was observed. Transfections include oligonucleotides sequences that do not target any miRNAs, designated as negative control, (N.C.), and a mixed transfection that combined all consensus anti-miRNAs. Arrows indicate neuronal cells (A). The distance of cells differentiating away from the embryoid body was quantitated using ImageJ and is expressed in mm (n = 6 replicate embryoid bodies per group) (B). mRNA levels of the neuronal cell marker NeuroD1 on day 9 was assessed by qPCR (C). mRNA expression levels were normalized with Gapdh using the comparative delta Ct method. Fold changes were compared with N.C. anti-miRNA. Data are shown as mean ± SD. Two-way ANOVA followed by Bonferroni (P < .05) was run to determine interactions and statistical differences between arsenic concentrations (*) and between the N.C. anti-miRNA and consensus anti-miRNA transfections (#).
Figure Legend Snippet: Inhibiting the miR-466-669 cluster during differentiation rescues the morphological loss of neurons following arsenic exposure. P19 cells were transfected with 100 nM anti-miRNA oligonucleotides which target 4 consensus sequences of the miRNA-466-467-669 cluster. Cells were coexposed to 0 or 0.5 μM arsenic for the 5 days of embryoid body formation. Only the arsenic exposure was maintained for the entire 9 days of differentiation, after which cell morphology was observed. Transfections include oligonucleotides sequences that do not target any miRNAs, designated as negative control, (N.C.), and a mixed transfection that combined all consensus anti-miRNAs. Arrows indicate neuronal cells (A). The distance of cells differentiating away from the embryoid body was quantitated using ImageJ and is expressed in mm (n = 6 replicate embryoid bodies per group) (B). mRNA levels of the neuronal cell marker NeuroD1 on day 9 was assessed by qPCR (C). mRNA expression levels were normalized with Gapdh using the comparative delta Ct method. Fold changes were compared with N.C. anti-miRNA. Data are shown as mean ± SD. Two-way ANOVA followed by Bonferroni (P < .05) was run to determine interactions and statistical differences between arsenic concentrations (*) and between the N.C. anti-miRNA and consensus anti-miRNA transfections (#).

Techniques Used: Transfection, Negative Control, Marker, Expressing

Consensus anti-miRNAs are active and functional and can rescue the expression of miR-466-467-669 target genes. P19 cells were transfected with all 4 miRNA inhibitors, with or without 0.5 μM arsenic (n = 3 replicates), allowed to form embryoid bodies for 5 days, and examined for mRNA expression of each of the 4 consensus sequences (A–D), 1 individual miRNA, miR-669a-3p (E), the host gene Sfmbt2 (F), and a known target gene for the consensus 1 cluster, Lats2 (G). mRNA expression levels were normalized with Gapdh, and miRNA expression levels were normalized with shRNA U6, using the comparative delta Ct method. Fold changes were compared with N.C. anti-miRNA. Data are shown as mean ± SD. Two-way ANOVA followed by Bonferroni (P < .05) was run to determine interactions and statistical differences between arsenic concentrations (*) and between the N.C. anti-miRNA and consensus anti-miRNA transfections (#) in (A–F). A 1-way ANOVA followed by Tukey’s test (P < .05) was run to determine significance (#) in (G).
Figure Legend Snippet: Consensus anti-miRNAs are active and functional and can rescue the expression of miR-466-467-669 target genes. P19 cells were transfected with all 4 miRNA inhibitors, with or without 0.5 μM arsenic (n = 3 replicates), allowed to form embryoid bodies for 5 days, and examined for mRNA expression of each of the 4 consensus sequences (A–D), 1 individual miRNA, miR-669a-3p (E), the host gene Sfmbt2 (F), and a known target gene for the consensus 1 cluster, Lats2 (G). mRNA expression levels were normalized with Gapdh, and miRNA expression levels were normalized with shRNA U6, using the comparative delta Ct method. Fold changes were compared with N.C. anti-miRNA. Data are shown as mean ± SD. Two-way ANOVA followed by Bonferroni (P < .05) was run to determine interactions and statistical differences between arsenic concentrations (*) and between the N.C. anti-miRNA and consensus anti-miRNA transfections (#) in (A–F). A 1-way ANOVA followed by Tukey’s test (P < .05) was run to determine significance (#) in (G).

Techniques Used: Functional Assay, Expressing, Transfection, shRNA

Mixed consensus miRNA inhibitors rescue arsenic’s inhibitory effects on NeuroD1 expression. P19 cells were transfected with a combined mixture of the 4 anti-miRNAs, with or without 0.5 μM arsenic (n = 3 replicates per anti-miRNA and per exposure group), and allowed to form embryoid bodies for 5 days. Immunohistochemistry was used to examine expression of NeuroD1 protein (red) in the embryoid bodies. Cells were counterstained with DAPI (blue) to indicate the nuclei (A). High magnification images of cells are shown in the (A) inserts. For 10 representative cells (examples are shown in the blue boxes), expression of NeuroD1 in the whole cell, cytoplasm, and nuclei were quantified using ImageJ (B). NeuroD1 transcript levels were quantified by qPCR, and normalized with Gapdh using the comparative delta Ct method with fold changes compared with N.C. (B). Data are shown as mean ± SD. Two-way ANOVA followed by Bonferroni (P < .05) was run to determine interactions and statistical differences between arsenic concentrations (*) and between the N.C. anti-miRNA and consensus anti-miRNA transfections (#).
Figure Legend Snippet: Mixed consensus miRNA inhibitors rescue arsenic’s inhibitory effects on NeuroD1 expression. P19 cells were transfected with a combined mixture of the 4 anti-miRNAs, with or without 0.5 μM arsenic (n = 3 replicates per anti-miRNA and per exposure group), and allowed to form embryoid bodies for 5 days. Immunohistochemistry was used to examine expression of NeuroD1 protein (red) in the embryoid bodies. Cells were counterstained with DAPI (blue) to indicate the nuclei (A). High magnification images of cells are shown in the (A) inserts. For 10 representative cells (examples are shown in the blue boxes), expression of NeuroD1 in the whole cell, cytoplasm, and nuclei were quantified using ImageJ (B). NeuroD1 transcript levels were quantified by qPCR, and normalized with Gapdh using the comparative delta Ct method with fold changes compared with N.C. (B). Data are shown as mean ± SD. Two-way ANOVA followed by Bonferroni (P < .05) was run to determine interactions and statistical differences between arsenic concentrations (*) and between the N.C. anti-miRNA and consensus anti-miRNA transfections (#).

Techniques Used: Expressing, Transfection, Immunohistochemistry



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Differentially expressed miRNA in exosomes from tuberculous pleural effusion and transudative pleural effusion (as control) by miRNAs sequencing. A Heatmap of differential exosomes miRNAs expression in pleural effusion between control individuals and tuberculous pleurisy patients; n = 9. RPKM values are represented by gradient colors and shown for each sample. Red represents a higher RPKM; blue represents a lower RPKM. Results are based on nine RNA sequencing samples. B Venn diagram showing the overlap between differentially expressed miRNAs in TPE exosomes and transudate exosomes. C Volcanic map of differential exosomes miRNAs expression in pleural effusion between control individuals and tuberculous pleurisy patients. Adjusted P value < 0.05 and fold change > 1 was set as restrictive conditions to identify the differentially expressed genes. D Pathway enrichment analysis showed the significant target genes of differentially expressed miRNAs associated with various KEGG pathways. E The expression of three differentially expressed miRNAs in TPE exosomes and transudate exosomes was verified by RT-qPCR and normalized by the U6. Data are expressed as mean ± SEM. n = 6, **** P < 0.0001 (Paired student’s t-test)

Journal: Respiratory Research

Article Title: Exosomal microRNAs of tuberculous pleural effusion orchestrating TGF-β signaling mediate pleural fibrosis

doi: 10.1186/s12931-026-03541-5

Figure Lengend Snippet: Differentially expressed miRNA in exosomes from tuberculous pleural effusion and transudative pleural effusion (as control) by miRNAs sequencing. A Heatmap of differential exosomes miRNAs expression in pleural effusion between control individuals and tuberculous pleurisy patients; n = 9. RPKM values are represented by gradient colors and shown for each sample. Red represents a higher RPKM; blue represents a lower RPKM. Results are based on nine RNA sequencing samples. B Venn diagram showing the overlap between differentially expressed miRNAs in TPE exosomes and transudate exosomes. C Volcanic map of differential exosomes miRNAs expression in pleural effusion between control individuals and tuberculous pleurisy patients. Adjusted P value < 0.05 and fold change > 1 was set as restrictive conditions to identify the differentially expressed genes. D Pathway enrichment analysis showed the significant target genes of differentially expressed miRNAs associated with various KEGG pathways. E The expression of three differentially expressed miRNAs in TPE exosomes and transudate exosomes was verified by RT-qPCR and normalized by the U6. Data are expressed as mean ± SEM. n = 6, **** P < 0.0001 (Paired student’s t-test)

Article Snippet: Exosomes from pleural effusion were obtained by ultracentrifugation of samples isolated from nine control individuals and nine patients with tuberculous pleurisy patients and processed by Novogene Co., Ltd (Beijing, China) for miRNA microarray analysis (lllumina SE50).

Techniques: Control, Sequencing, Expressing, RNA Sequencing, Quantitative RT-PCR

miR-503-5p regulated Smurf1/Smad7 signaling pathway. A , B Venn diagram showing overlap between differentially up-regulated miRNAs in TPE exosomes and miRNAs targeting Smurf1 or Smad7 predicted by the miRTarBase. The online analysis database “miRTarBase” ( https://miRTarBase.cuhk.edu.cn/ ) was used. C Human PMCs were transfected with miR-503-5p mimics (50 nmol/ml) or negative control (NC) for 24 h, after which miR-503-5p expression levels in cells were determined by qRT-PCR and normalized by the U6. D - E Human PMCs were transfected with miR-25-3p mimics or miR-503-5p mimics or miR-92a-3p mimics (50 nmol/ml) or negative control (NC) for 24 h, after which intracellular mRNA levels of Smurf1 were measured by RT-qPCR and normalized to GAPDH ( D ). The protein expression of Smurf1 and TGF-β receptor (TGFBR) were detected by western blotting. Bar graphs revealed changes in relative ratio of Smurf1 and TGFBR to GAPDH. F Human PMCs were transfected with miR-503-5p mimics (50 nmol/ml) or negative control (NC) for 24 h, after which mRNA expression of Smurf1, TGFBR and COL1A1 were detected by qRT-PCR. G Human PMCs were incubated with miR-503-5p mimics. After 24 h, Smurf1 protein was detected by immunofluorescence staining and nuclei with DAPI staining. Bar scale: 50 μm. H - K Human PMCs were transfected with miR-503-5p mimics (50 nmol/ml) or negative control (NC) for 24 h. miR-503-5p expression levels in cells were determined by qRT-PCR and normalized by the U6 ( H ). The protein expression of S Smurf1, TGFBR and COL1A1 were detected by western blotting ( I ). Bar graphs revealed changes in the relative ratio to GAPDH ( J ). mRNA levels of S Smurf1, TGFBR and COL1A1 were measured by RT-qPCR and normalized to GAPDH ( K ). Data are mean ± SEM. n = 3. * P < 0.05 (student’s t-test)

Journal: Respiratory Research

Article Title: Exosomal microRNAs of tuberculous pleural effusion orchestrating TGF-β signaling mediate pleural fibrosis

doi: 10.1186/s12931-026-03541-5

Figure Lengend Snippet: miR-503-5p regulated Smurf1/Smad7 signaling pathway. A , B Venn diagram showing overlap between differentially up-regulated miRNAs in TPE exosomes and miRNAs targeting Smurf1 or Smad7 predicted by the miRTarBase. The online analysis database “miRTarBase” ( https://miRTarBase.cuhk.edu.cn/ ) was used. C Human PMCs were transfected with miR-503-5p mimics (50 nmol/ml) or negative control (NC) for 24 h, after which miR-503-5p expression levels in cells were determined by qRT-PCR and normalized by the U6. D - E Human PMCs were transfected with miR-25-3p mimics or miR-503-5p mimics or miR-92a-3p mimics (50 nmol/ml) or negative control (NC) for 24 h, after which intracellular mRNA levels of Smurf1 were measured by RT-qPCR and normalized to GAPDH ( D ). The protein expression of Smurf1 and TGF-β receptor (TGFBR) were detected by western blotting. Bar graphs revealed changes in relative ratio of Smurf1 and TGFBR to GAPDH. F Human PMCs were transfected with miR-503-5p mimics (50 nmol/ml) or negative control (NC) for 24 h, after which mRNA expression of Smurf1, TGFBR and COL1A1 were detected by qRT-PCR. G Human PMCs were incubated with miR-503-5p mimics. After 24 h, Smurf1 protein was detected by immunofluorescence staining and nuclei with DAPI staining. Bar scale: 50 μm. H - K Human PMCs were transfected with miR-503-5p mimics (50 nmol/ml) or negative control (NC) for 24 h. miR-503-5p expression levels in cells were determined by qRT-PCR and normalized by the U6 ( H ). The protein expression of S Smurf1, TGFBR and COL1A1 were detected by western blotting ( I ). Bar graphs revealed changes in the relative ratio to GAPDH ( J ). mRNA levels of S Smurf1, TGFBR and COL1A1 were measured by RT-qPCR and normalized to GAPDH ( K ). Data are mean ± SEM. n = 3. * P < 0.05 (student’s t-test)

Article Snippet: Exosomes from pleural effusion were obtained by ultracentrifugation of samples isolated from nine control individuals and nine patients with tuberculous pleurisy patients and processed by Novogene Co., Ltd (Beijing, China) for miRNA microarray analysis (lllumina SE50).

Techniques: Transfection, Negative Control, Expressing, Quantitative RT-PCR, Western Blot, Incubation, Immunofluorescence, Staining

Triple miRNAs inhibitor attenuated TPE-Exo induced pleural fibrosis. C57BL/6 mice were intra-pleural injected by using PBS (100 µl/mouse), TPE-Exo (100 µl/mouse), TPE-Exo plus control inhibitor, or TPE-Exo plus triple miRNAs inhibitor with carbon particles (0.1 mg/mouse) as descriptions in the Methods. TPE-Exo from 50 ml TPE was administered at days 1, 5, 9. In TPE-Exo plus triple miRNAs inhibitor group, TPE-Exo was co-incubated with triple miRNAs inhibitor which restrained expressions of miR-150-3p, miR-424-3p and miR-503-5p. All mice were euthanized at day 21, and tissues were taken for analysis. A Representative Masson’s trichrome staining images of visceral pleura from lung sections, parietal pleura from chest wall and diaphragm sections. Original magnification, ×400. B Changes in pleural thickness. C Changes in collagen percentages of visceral and parietal pleura. Data are expressed as mean ± SEM. n = 6 mice. *** P < 0.001 (One-way ANOVA followed by the Bonferroni’s test)

Journal: Respiratory Research

Article Title: Exosomal microRNAs of tuberculous pleural effusion orchestrating TGF-β signaling mediate pleural fibrosis

doi: 10.1186/s12931-026-03541-5

Figure Lengend Snippet: Triple miRNAs inhibitor attenuated TPE-Exo induced pleural fibrosis. C57BL/6 mice were intra-pleural injected by using PBS (100 µl/mouse), TPE-Exo (100 µl/mouse), TPE-Exo plus control inhibitor, or TPE-Exo plus triple miRNAs inhibitor with carbon particles (0.1 mg/mouse) as descriptions in the Methods. TPE-Exo from 50 ml TPE was administered at days 1, 5, 9. In TPE-Exo plus triple miRNAs inhibitor group, TPE-Exo was co-incubated with triple miRNAs inhibitor which restrained expressions of miR-150-3p, miR-424-3p and miR-503-5p. All mice were euthanized at day 21, and tissues were taken for analysis. A Representative Masson’s trichrome staining images of visceral pleura from lung sections, parietal pleura from chest wall and diaphragm sections. Original magnification, ×400. B Changes in pleural thickness. C Changes in collagen percentages of visceral and parietal pleura. Data are expressed as mean ± SEM. n = 6 mice. *** P < 0.001 (One-way ANOVA followed by the Bonferroni’s test)

Article Snippet: Exosomes from pleural effusion were obtained by ultracentrifugation of samples isolated from nine control individuals and nine patients with tuberculous pleurisy patients and processed by Novogene Co., Ltd (Beijing, China) for miRNA microarray analysis (lllumina SE50).

Techniques: Injection, Control, Incubation, Staining

Results of a microarray analysis. Eight miRNAs were significantly downregulated in the patients with functional dyspepsia (FD) compared to those in healthy controls using method 1. However, six miRNAs were downregulated using method 2. Notably, five miRNAs were consistently downregulated between the two methods. Furthermore, five miRNAs and seven miRNAs were significantly upregulated using methods 1 and 2, respectively. Therefore, four miRNAs were consistently upregulated in patients with FD.

Journal: Internal Medicine

Article Title: Exosomal hsa-miR-3649 and hsa-miR-202-3p in Gastric Juice as Potential Biomarkers for Functional Dyspepsia with a Previous Helicobacter pylori Infection

doi: 10.2169/internalmedicine.6047-25

Figure Lengend Snippet: Results of a microarray analysis. Eight miRNAs were significantly downregulated in the patients with functional dyspepsia (FD) compared to those in healthy controls using method 1. However, six miRNAs were downregulated using method 2. Notably, five miRNAs were consistently downregulated between the two methods. Furthermore, five miRNAs and seven miRNAs were significantly upregulated using methods 1 and 2, respectively. Therefore, four miRNAs were consistently upregulated in patients with FD.

Article Snippet: An miRNA microarray analysis of the discovery cohort was performed using 3D-Gene R (Toray Industries, Tokyo, Japan), as described in our previous report ( ).

Techniques: Microarray, Functional Assay