Journal: Frontiers in Immunology

Article Title: Inhibition of miRNA associated with a disease-specific signature and secreted via extracellular vesicles of systemic lupus erythematosus patients suppresses target organ inflammation in a humanized mouse model

doi: 10.3389/fimmu.2023.1090177

Figure Lengend Snippet: RNA expression profiles from plasma-derived extracellular vesicles (EVs) in human and murine samples. (A) EVs were isolated from healthy human urine (N = 10) and plasma (N = 25) sources. (B) Plasma-derived EVs were isolated from lupus nephritis (LN) patients (N= 5) and healthy controls (N = 25). (A, B) RNA was isolated from EVs for comprehensive, global RNA-sequencing analysis for detectable small RNA sequences. Reads were aligned and principal component analysis was performed to evaluate grouping of the datasets. (C) A volcano plot was made from the data generated in (B) . (D) Plasma was isolated from NZM2410 mice with high and low blood urea nitrogen (BUN) levels (N = 15 per group). EVs were isolated and RNA was purified for sequencing to detect miRNAs. Data from all detected miRNAs (approximately 800) is shown as a heat map and expressed as log-transformed RNAseq reads. (E) Fold changes of all log-transformed miRNA expression levels detected in RNA-sequencing of isolated EVs from human (LN/healthy) and murine (high BUN/low BUN) samples were analyzed by pathway analysis software focused on miRNAs associated with LN pathology. Relative fold changes in expression level are shown in a heat map organized by hierarchy.

Article Snippet: Murine EVs isolated from plasma were submitted directly to System Biosciences (SBI) for RNA isolation and miRNA profiling according to the Exo-NGS service, exosomal RNA sequencing protocol.

Techniques: RNA Expression, Clinical Proteomics, Derivative Assay, Isolation, RNA Sequencing, Generated, Purification, Sequencing, Transformation Assay, Expressing, Software

Journal: Frontiers in Immunology

Article Title: Inhibition of miRNA associated with a disease-specific signature and secreted via extracellular vesicles of systemic lupus erythematosus patients suppresses target organ inflammation in a humanized mouse model

doi: 10.3389/fimmu.2023.1090177

Figure Lengend Snippet: Extracellular vesicles (EVs) and EV-encapsulated miRs that bind and activate TLR7 and TLR8 are upregulated in SLE patients. (A) Plasma derived EVs were isolated from healthy volunteers (N = 6) and SLE patients (N = 16; 14 with active disease and 2 inactive) by ultracentrifugation and quantified by an ELISA assay. (B) RNA was extracted from EVs isolated from healthy volunteers (N = 6) and active SLE patients (N = 6). Expression of miR-21, miR29a, miR-29b, and Let7a was measured by RT-PCR analysis. Data was normalized to RNU-44 internal control expression and shown as a relative fold-change. Values are the mean ± SEM with indicated p values calculated via paired, two-tailed, Student’s t tests. *p ≤ 0.05; **p ≤ 0.01.

Article Snippet: Murine EVs isolated from plasma were submitted directly to System Biosciences (SBI) for RNA isolation and miRNA profiling according to the Exo-NGS service, exosomal RNA sequencing protocol.

Techniques: Clinical Proteomics, Derivative Assay, Isolation, Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Inhibition of miRNA associated with a disease-specific signature and secreted via extracellular vesicles of systemic lupus erythematosus patients suppresses target organ inflammation in a humanized mouse model

doi: 10.3389/fimmu.2023.1090177

Figure Lengend Snippet: Schematic of the proposed mechanism of estrogen-mediated miR production and inflammation via extracellular vesicle signaling in SLE. Estrogen (E2) enters an immune cell, dimerizes with estrogen receptor (ER)α, translocates to the nucleus, and promotes the expression of miRNA (miR) processing machinery, including RNA polymerase III, Dicer1, AGO2, and Drosha. These miRs are packaged and secreted in EVs, which are taken up by recipient immune cells. When EV-derived miR cargo is taken up, it can function to regulate gene expression in the recipient cell via two pathways. The canonical pathway involves miRs binding to target mRNAs through the RNA-induced silencing complex (RISC). In the non-canonical pathway, EV-encapsulated miRs fuse with endosomes and bind to TRL7 or TLR8 to stimulate proinflammatory gene expression and additional EV secretion, which promotes SLE pathogenesis.

Article Snippet: Murine EVs isolated from plasma were submitted directly to System Biosciences (SBI) for RNA isolation and miRNA profiling according to the Exo-NGS service, exosomal RNA sequencing protocol.

Techniques: Expressing, Derivative Assay, Gene Expression, Binding Assay

Journal: Frontiers in Allergy

Article Title: miR-155 Is a Positive Regulator of FcεRI-Induced Cyclooxygenase-2 Expression and Cytokine Production in Mast Cells

doi: 10.3389/falgy.2022.835776

Figure Lengend Snippet: miRNA profiling in human SMCs. Heat map showing significant ( p < 0.01) changes in miRNA expression in human SMCs following FcεRI crosslinking. RNA was extracted from SMCs that were sensitized with chimeric human anti-NP IgE (1 μg/ml) and activated for 3 h at 37°C with the multivalent antigen NP-HSA (1 ng/ml). The heat map identifies 10 miRNAs that were significantly ( p < 0.01) upregulated (red) and 11 that were significantly downregulated (green) in Ag/IgE-activated SMCs ( n = 3 different cultures from different donors). All data can be found in the .

Article Snippet: miRNA profiling services were performed by LC Sciences (lcsciences.com, Houston TX, USA) using their μParaflo ® microfluidic biochip technology and optimized probes in a microfluidics microarray platform.

Techniques: Expressing

Journal: International Journal of Clinical and Experimental Pathology

Article Title: miR-1 and miR-145 act as tumor suppressor microRNAs in gallbladder cancer

doi:

Figure Lengend Snippet: Table 2

Article Snippet: The miRNA microarray experiments were performed by Thermo Scientific Dharmacon miRNA profiling service.

Techniques: Microarray, Expressing

Journal: Gene

Article Title: Comparative profiling of miRNA expression of lung adenocarcinoma cells in two-dimensional and three-dimensional cultures

doi: 10.1016/j.gene.2012.09.093

Figure Lengend Snippet: Distinct miRNA profiles of mK-ras-LE cells in 2-D and rBM 3-D cultures. A) mK-ras-LE cells were grown in 2-D and rBM 3-D culture. Morphology of the cells was visualized using fluorescent staining for F-actin. The images of rBM 3-D culture were captured at the central planes of cell clusters using a confocal fluorescent microscope on day 12 post-seeding. The images were captured at 600x magnification. Representative images of each culture condition were displayed. B) Total cell RNA was extracted from mK-ras-LE cells in 2-D and rBM 3-D cultures. miRNA expression profiles were compared between the two culture conditions using miRNA microarrays. A heatmap of the significantly differentially expressed miRNAs was generated using geWorkbench suite (n = 3, P < 0.01, FDR < 0.05). C) The expression of miR-17, miR-92b, and miR-21 was measured using qRT-PCR in total cell RNA collected from 2-D and rBM 3-D cultures of mK-ras-LE cells. A fold change of each miRNA of interest was obtained by setting the values of each miRNA in 2-D culture to one and normalization to the values of U6. D) Similar to part C except that the expression of miR-200a was compared between 2-D and rBM 3-D cultures. E) The culture conditions were similar to part A. The cell lysates were collected from the cell colonies extracted from the culture. The protein levels of ZEB2, PTEN, and -actin were determined using immunoblots. The data were presented in averages and standard deviations obtained from three independent experiments. and indicated a P value < 0.05 and 0.01, respectively.

Article Snippet: The miRNA expression profiling service was provided by LC Sciences (Houston, TX) using μParaflo® technology and proprietary probe hybridization (Cy3 and Cy5 dendrimer dyes) that enables highly sensitive and specific direct detection of mouse miRNAs.

Techniques: Staining, Microscopy, Expressing, Generated, Quantitative RT-PCR, Western Blot

Journal: Gene

Article Title: Comparative profiling of miRNA expression of lung adenocarcinoma cells in two-dimensional and three-dimensional cultures

doi: 10.1016/j.gene.2012.09.093

Figure Lengend Snippet: miRNA expression associated with aincar morphogenesis. A) A549 and A549LC cells were cultured in 2-D (A1 & A2) and rBM 3-D (A3 & A4) cultures. Morphology was recorded using an inverse phase contrast microscope. Polarized monolayer of the cells and the central lumen were pointed at by white arrows. B) The expression of miR-17, miR-92, and miR-21 was measured using qRT-PCR in total cell RNA collected from 2-D and rBM 3-D of A549 and A549LC cells. A fold change of each miRNA was obtained by setting the values of each miRNA from A549 cells in 2-D culture to one and normalization to the values of U6. C) Similar to part A except that the expression of miR-200a was compared across the groups. D) The culture conditions were similar to part A. The RNA levels of pri-miR-21 and VMP1 were measured in A549 cells using qRT-PCR. A fold change of each RNA was obtained by setting the values of RNA from A549 cells in 2-D culture to one and normalization to the values of 36B4. The data were presented in averages and standard deviations obtained from three independent experiments. , , and indicated a P value < 0.05, 0.01, and 0.001, respectively.

Article Snippet: The miRNA expression profiling service was provided by LC Sciences (Houston, TX) using μParaflo® technology and proprietary probe hybridization (Cy3 and Cy5 dendrimer dyes) that enables highly sensitive and specific direct detection of mouse miRNAs.

Techniques: Expressing, Cell Culture, Microscopy, Quantitative RT-PCR