Journal: Cell metabolism

Article Title: Nondigestible stachyose binds membranous HSP90β on small intestinal epithelium to regulate the exosomal miRNAs: A new function and mechanism.

doi: 10.1016/j.cmet.2024.10.012

Figure Lengend Snippet: Figure 2. Knockout of HSP90b in MODE-K cells eliminates the accumulation of stachyose on the cell membrane and the regulatory effects of stachyose on the exosomal miRNA expression (A–C) Identification of exosome-sized extracellular vesicles secreted by MODE-K cells. The data were representative of three independent experiments. See also Figure S2B. (A) Morphology of exosomes secreted by MODE-K cells treated with 2 mM of stachyose was visualized by electron microscopy on the 100-mesh copper mesh (with carbon film). Representative from three experiments. Scale bar, 100 nm. (B) Nanoparticle tracking analysis measured by NanoSight. (C) The protein levels of Tsg 101 and CD 63 in exosomes were measured by western blotting. (D–F) Profiling of exosomal miRNAs released by MODE-K cells treated with 0 (Sta0), 2 mM (Sta2), or 4 mM (Sta4) stachyose based on small RNA sequencing results (n = 4). (D) PCA was performed using the OmicStudio tools. p value and R value were calculated by vegan package of R. PCA was performed by stats package. Figures were drawn by ggplot2 package. (E) Volcano plots of miRNA levels in stachyose-treated versus non-treated MODE-K cells. (F) Heatmap of highly expressed miRNAs selected from all detected 327 mouse miRNAs. Highly expressed miRNAs were identified as relative expression > 203.3 (mean relative expression of all miRNAs). (G) The subcellular localization of stachyose on the HSP90b-WT and HSP90b-KO MODE-K cells. Scale bar, 10 mm. (H) The relative expression levels of 12 miRNAs in the exosomes derived from HSP90b-WT and HSP90b-KO MODE-K cells after treated with 0, 2, and 4 mM stachyose. *p < 0.05.

Article Snippet: 100 mg of mouse feces were input for All-in-One miRNA qRT-PCR detection kits (Gene Copoeia, USA) and tested on CFX 96 Touch Real-time PCR System (BIO-RED, USA) following the manufacturer’s protocol.

Techniques: Knock-Out, Membrane, Expressing, Electron Microscopy, Western Blot, RNA Sequencing, Derivative Assay

Journal: Cell metabolism

Article Title: Nondigestible stachyose binds membranous HSP90β on small intestinal epithelium to regulate the exosomal miRNAs: A new function and mechanism.

doi: 10.1016/j.cmet.2024.10.012

Figure Lengend Snippet: Figure 3. Stachyose reconstitutes fecal miRNA profiles in mice and humans (A and B) Fecal miRNA profile of mice receiving 0 (NC), 200 (S-L), 400 (S-M), and 800 (S-H) mg/kg$bw stachyose tested by small RNA sequencing analysis (n = 4). (A) Heatmap of highly expressed miRNAs selected from all detected 107 mouse miRNAs. Highly expressed miRNAs were identified as relative expres- sion > 156.34 (mean relative expression of all miRNAs). (B) Differentially expressed miRNAs filtered by Log2FC of relative expression and expression variation (| Log2FC|>1, miRNA expression variation > 50, the expression variation was defined as the absolute differences in the relative expression levels between two groups). The significant ones (p < 0.05) were emphasized in yellow. (C) Relative expression of miR-191-5p, miR-30a-5p, and miR-21a-5p. Data were represented as mean ± SEM (n = 3). *p < 0.05. (D–F) Fecal miRNA profile of healthy human subjects (six males and six females). Feces from 12 human subjects were collected pre and post stachyose (5 g/kg$bw, recommended daily intake) administration. (D) Clinical trial protocol. All subjects received stachyose supplementation for 8 weeks. (E) PCAs of miRNAs in human feces based on small RNA sequencing results. The method for PCA analysis was same as Figure 2. (F) Log2FC values of 28 highly expressed miRNAs (miRNA expression > 50) among all detected 138 human miRNAs. (G) UpSetR and Venn analyses of all downregulated highly expressed miRNAs detected both in mice and human feces. UpSetR analysis was performed by UpSetR package of R. Venn analysis was performed by yyplot package.

Article Snippet: 100 mg of mouse feces were input for All-in-One miRNA qRT-PCR detection kits (Gene Copoeia, USA) and tested on CFX 96 Touch Real-time PCR System (BIO-RED, USA) following the manufacturer’s protocol.

Techniques: RNA Sequencing, Expressing

Journal: Cell metabolism

Article Title: Nondigestible stachyose binds membranous HSP90β on small intestinal epithelium to regulate the exosomal miRNAs: A new function and mechanism.

doi: 10.1016/j.cmet.2024.10.012

Figure Lengend Snippet: Figure 5. The axis of stachyose-fecal miRNA-gut microbiota: Stachyose directly regulates fecal miRNAs, which, in turn, shape the gut microbiota (A) Relative expression of 16S rRNA gene in fecal samples from normal and antibiotic-treated pseudo-germ-free (PGF) mice. The relative 16S rRNA level was calculated based on Ct values tested by qPCR analysis (n = 3). *p < 0.05, **p < 0.01. (B–D) Fecal miRNA profile of stachyose-treated (Ab-S) and -untreated (Ab) PGF mice (n = 4). (B) PCA analysis of fecal miRNA levels based on small RNA sequencing results. (C) Differentially expressed miRNAs filtered by Log2FC of relative expression and expression variation (|Log2FC|>1, miRNA expression variation > 10) in PGF mice. The significant ones (p < 0.05) were emphasized in yellow. (D) Venn analysis of all downregulated highly expressed miRNAs detected both in PGF mice (Ab-S vs. Ab) and normal mice (S-L S-M S-H vs. NC) feces. The highly expressed miRNAs are shown in Figure S4H. (E–G) Gut microbiome structure of antibiotics-pretreated mice receiving fecal miRNAs harvested from mice in Ab-S or Ab group (n = 5). (E) After 4 weeks of pre- treatment with antibiotics, feces collected from donors (stachyose-treated [Ab-S] and -untreated [Ab] PGF mice) were heat inactivated, and the isolated fecal miRNAs were transplanted to recipients (miR-Ab/miR-Ab-S) for 8 weeks. (F) PCA with unweighted UniFrac distance of all detected genera in feces from miR-Ab and miR-Ab-S mice. (G) Log2FC values of significantly altered five genera with mean relative abundance > 0.5. The color represents the phylum that the genus belonged to.

Article Snippet: 100 mg of mouse feces were input for All-in-One miRNA qRT-PCR detection kits (Gene Copoeia, USA) and tested on CFX 96 Touch Real-time PCR System (BIO-RED, USA) following the manufacturer’s protocol.

Techniques: Expressing, RNA Sequencing, Isolation

Journal: Cell metabolism

Article Title: Nondigestible stachyose binds membranous HSP90β on small intestinal epithelium to regulate the exosomal miRNAs: A new function and mechanism.

doi: 10.1016/j.cmet.2024.10.012

Figure Lengend Snippet: Figure 6. miR-30a-5p restrains the proliferation of Lactobacillus reuteri (A) Linear regression analysis on the relative expression levels of stachyose-downregulated fecal miRNAs in both normal and PGF mice (related to Figure 5D) and relative abundance of Lactobacillus genus. The data of Lactobacillus genus were based on 16S rRNA sequencing data for normal and PGF mice. Linear regression analysis was performed by GraphPad Prism with 95% confidence interval. (B) Venn analysis of all downregulated highly expressed miRNAs detected in normal mice feces, PGF mice feces, human feces, and MODE-K-cell-secreted exosomes. (C) FITC-labeled (green) miR-30a-5p and miR-191-5p were co-cultured with Lactobacillus gasseri (Lg) and Lactobacillus reuteri (Lr). Fluorescence signal was captured by fluorescence microscope (ZEISS, DMi8 automated, left). Representative from three experiments. The relative expression of miR-30a-5p and miR- 191-5p in Lg (blue fill) and Lr (yellow fill) was detected by qPCR analysis (n = 3, right). See also Figures S6D and S6E. (D) Target site sequence alignment of miR-191-5p and miR-30a-5p against Lr mRNA sequence (matching sites highlighted). The interaction energy was measured by Targetscan score and Miranda energy. (E) Lg and Lr were grown in culture media with 2-mM miRNA mimics and their scrambled controls for 24 h. Bacterial growth was monitored as absorbance at 600 nm (OD600). Representative growth curves of three independent experiments with duplicates were presented. See Figure S6F for additional growth curves. *p < 0.05 compared with vehicle, #p < 0.05 compared with scramble control. Data were represented as mean ± SEM.

Article Snippet: 100 mg of mouse feces were input for All-in-One miRNA qRT-PCR detection kits (Gene Copoeia, USA) and tested on CFX 96 Touch Real-time PCR System (BIO-RED, USA) following the manufacturer’s protocol.

Techniques: Expressing, Sequencing, Labeling, Cell Culture, Fluorescence, Microscopy, Control

Journal: Cell metabolism

Article Title: Nondigestible stachyose binds membranous HSP90β on small intestinal epithelium to regulate the exosomal miRNAs: A new function and mechanism.

doi: 10.1016/j.cmet.2024.10.012

Figure Lengend Snippet: Figure 7. The crosstalk between gut microbiota and fecal miRNA (A) After 4 weeks of pre-treatment with antibiotics, fecal materials containing both miRNAs and bacteria collected from donors (mice receiving 0 [NC] and 400 [S-M] mg/kg$bw stachyose, same as Figure 4) were transplanted to recipients (Naive-NC/Naive-S) for 8 weeks. (B) Relative expression of 16S rRNA gene in feces samples from normal and antibiotics-pretreated recipient mice. The relative 16S rRNA expression was calculated by Ct value tested by qPCR analysis (n = 3). (C–E) PCA with unweighted UniFrac distance (C), genera distribution (D), and the change ratio of Lactobacillus (E) for antibiotics-pretreated miR-Ab, miR-Ab-S, Naive-NC, and Naive-S mice (n = 5).*p < 0.05, **p < 0.01. (F) Lg and Lr were grown in culture media with bacterial metabolites from miR-Ab and miR-Ab-S. Growth was monitored as absorbance at 600 nm (OD600) for 24 h. Representative growth curves of three independent experiments with duplicates were presented. *p < 0.05, **p < 0.01. Data were represented as mean ± SEM. (G and H) PCA (G) and the change ratio of miR-30a-5p (H) for antibiotics-pretreated miR-Ab, miR-Ab-S, Naive-NC, and Naive-S mice (n = 4).

Article Snippet: 100 mg of mouse feces were input for All-in-One miRNA qRT-PCR detection kits (Gene Copoeia, USA) and tested on CFX 96 Touch Real-time PCR System (BIO-RED, USA) following the manufacturer’s protocol.

Techniques: Bacteria, Expressing

Journal: BMC cancer

Article Title: Suppressing Dazl modulates tumorigenicity and stemness in human glioblastoma cells.

doi: 10.1186/s12885-020-07155-y

Figure Lengend Snippet: Fig. 2 Dazl knockdown inhibited the proliferation and germline traits of glioblastoma cells in vitro. a Western blot analysis detected whether Cas9 protein was transfected into GBM cells successfully and whether Dazl protein was deleted. b A CCK-8 cell proliferation assay was performed after Dazl deletion in A172, U251, and LN229 cells. c An alkaline phosphatase stain assay was performed between the WT GBM cell lines and the Dazl deletion cells. Images were taken from the inverted microscope (magnification × 200). All experiments were carried out in triplicate. Data are shown as the mean ± SE (*P < 0.05, **P < 0.01)

Article Snippet: GBM cells were transfected with CRISPR plasmids and the lenti-cas9 pSpCas9(BB)-2A-GFP (PX458) plasmid (Addgene plasmid #48138) using XtremeGENE 9 DNA Transfection Reagent (6,365,787, 001, Sigma-Aldrich, USA).

Techniques: Knockdown, In Vitro, Western Blot, Transfection, CCK-8 Assay, Proliferation Assay, Staining, Inverted Microscopy