Journal: Nature Communications

Article Title: Osteoclast-derived exosomal miR-214-3p inhibits osteoblastic bone formation

doi: 10.1038/ncomms10872

Figure Lengend Snippet: ( a ) A schematic diagram illustrating the experimental design. Serum and bone samples were collected from elderly woman patients with or without fractures, and divided into 60–69, 70–79 and 80–89 groups, respectively, according to age. ( b ) Real-time PCR analysis of the age-related changes in miR-214-3p levels in whole serum (left), serum exosomes (middle) and bone specimens (right) from elderly patients with or without fractures, respectively. The relative miR-214-3p level in each group was normalized to the mean value of the 60–69 group. Human RUN6B was used as the internal control. All data are the mean±s.d. * P <0.05, ** P <0.01. Two-way analysis of variance (ANOVA) with a Turkey's multiple comparisons test was performed. Both the time effect (age), the group effect (with or without fractures) and the time-by-group interaction effect were all statistically significant for all the examined parameters. ( c ) Real-time PCR analysis showing the age-related changes in BGLAP mRNA levels in bone specimens from elderly patients with fractures, respectively. The relative BGLAP mRNA level in each group was normalized to the mean value of the 60–69 group. Human GAPDH mRNA was used as the internal control. Data are the mean±s.d. ** P <0.01. One-way ANOVA with a post-hoc test was performed. ( d ) Western blot analysis of the osteoclast marker proteins (CTSK, TRAcP5 and Sema4D) in the lysates of serum exosomes isolated from elderly patients with or without fractures. OCs, the lysates of osteoclasts differentiated from human peripheral blood mononuclear cells. Exo 1, the lysates of serum exosomes from elderly patients without fractures. Exo 2, the lysates of serum exosomes from elderly patients with fracture. ( e ) Correlation analysis between exosomal and intra-osseous miR-214-3p levels (left), between exosomal miR-214-3p level and intra-osseous BGLAP mRNA level (middle) and between intra-osseous miR-214-3p level and intra-osseous BGLAP mRNA level (right), respectively, in elderly patients with fractures. The n value for each group is indicated at the bottom of each histogram.

Article Snippet: The blotting membrane was blocked with bovine serum albumin and incubated with goat polyclonal anti-Sema4D antibody (1:100; Santa Cruz, sc-16632), rabbit polyclonal anti-CTSK antibody (1:100; Santa Cruz, sc-30056), goat polyclonal anti-tartrate-resistant acid phosphatase antibody (1:100; Santa Cruz, sc-30833), rabbit polyclonal anti-CD9 antibody (1:100; Santa Cruz, sc-9148), rabbit polyclonal anti-CD63 antibody (1:100; Santa Cruz, sc-15363), rabbit polyclonal anti-Flotillin-1 antibody (1:100; Santa Cruz, sc-25506), goat polyclonal anti-TSG101 antibody (1:100; Santa Cruz, sc-6037) and rabbit polyclonal anti-calnexin antibody (1:100; Santa Cruz, sc-11397) followed by incubation with horseradish peroxidase (HRP)-coupled goat anti-rabbit IgG H&L (1:5,000, Abcam, ab6721) or HRP-coupled rabbit anti-goat IgG H&L (1:5,000, Abcam, ab6741), respectively.

Techniques: Real-time Polymerase Chain Reaction, Control, Western Blot, Marker, Isolation

Journal: Nature Communications

Article Title: Osteoclast-derived exosomal miR-214-3p inhibits osteoblastic bone formation

doi: 10.1038/ncomms10872

Figure Lengend Snippet: ( a ) Representative biophotonic images of the tissue distribution of fluorescence signal in mice at 4 and 8 h after intravenous injection of purified PKH67-labelled exosomes isolated from the supernatant of OC-miR-214-3p osteoclasts (OC-Exo). ( b ) Representative biophotonic images of the tissue distribution of fluorescence signal in mice at 8 h after intravenous injection of purified PKH67 exosomes isolated from the supernatant of either OC-miR-214-3p osteoclast (OC-Exo) or HEK 293T cells (HEK-Exo). ( c ) Western blot analysis of the Sema4D protein expression in pellets of sucrose gradient fractions from the osteoclast-derived exosome preparations. The density of each fraction was determined by refraction index measurements. ( d , e ) Flow cytometry analysis of PKH67 + osteoblasts after incubation with PKH67-labelled exosomes derived from OC-miR-214-3p osteoclasts. The PKH67-labelled exosomes were pre-treated with either anti-Sema4D (20 μg ml −1 ) or isotype-matched control (Sema4D iso) and then added to osteoblasts for 4 h incubation. ( f ) Real-time PCR analysis of the levels of pri-miR-214, pre-miR-214 and mature miR-214 in osteoblasts after incubation with either anti-Sema4D-treated exosomes (OC Exo+Anti-Sema4D) or Sema4D isotype-treated exosomes (OC Exo+Sema4D iso), respectively. All data are the mean±s.d. of four independent experiments. * P <0.05. One-way ANOVA with a post-hoc test was performed.

Article Snippet: The blotting membrane was blocked with bovine serum albumin and incubated with goat polyclonal anti-Sema4D antibody (1:100; Santa Cruz, sc-16632), rabbit polyclonal anti-CTSK antibody (1:100; Santa Cruz, sc-30056), goat polyclonal anti-tartrate-resistant acid phosphatase antibody (1:100; Santa Cruz, sc-30833), rabbit polyclonal anti-CD9 antibody (1:100; Santa Cruz, sc-9148), rabbit polyclonal anti-CD63 antibody (1:100; Santa Cruz, sc-15363), rabbit polyclonal anti-Flotillin-1 antibody (1:100; Santa Cruz, sc-25506), goat polyclonal anti-TSG101 antibody (1:100; Santa Cruz, sc-6037) and rabbit polyclonal anti-calnexin antibody (1:100; Santa Cruz, sc-11397) followed by incubation with horseradish peroxidase (HRP)-coupled goat anti-rabbit IgG H&L (1:5,000, Abcam, ab6721) or HRP-coupled rabbit anti-goat IgG H&L (1:5,000, Abcam, ab6741), respectively.

Techniques: Fluorescence, Injection, Purification, Isolation, Western Blot, Expressing, Derivative Assay, Refractive Index, Flow Cytometry, Incubation, Control, Real-time Polymerase Chain Reaction

Journal: EBioMedicine

Article Title: Transcriptome Analysis Uncovers a Growth-Promoting Activity of Orosomucoid-1 on Hepatocytes

doi: 10.1016/j.ebiom.2017.09.008

Figure Lengend Snippet: Orm1 is induced in humans and mics after PH. (a)–(c) Human data. (a) Postoperative serum Orm1 levels were examined in 10 patients who had undergone liver resection for HCC. (b) Correlation between changes in serum Orm1 at 24 h post-liver resection with the rates of liver resection (the resection volume counts per total liver volume counts). POD, postoperative days. (c) Representative photographs of IHC staining of Orm1 in commercially obtained liver sections of HCC and normal adjacent tissues in HCC patients. As a negative control (Ctl), human Orm1 antibody that had been incubated with a 5-fold (by weight) excess of blocking peptide. (d)–(g) Mouse data. (d) Time course of Orm1 gene expression in whole-liver tissues after PH (n = 3). (e) Orm1 transcript levels in whole-liver tissues and (f) serum protein levels at 48 h after PH (n = 4). (g) IHC staining of Orm1 in mouse liver tissues at 48 h after PH. P -value was assessed using Student's t -test or Mann-Whitney U test. * P < 0.05 indicates statistical significance. Scale bars, 100 μm.

Article Snippet: The sections were then incubated with goat anti-human Orm1 antibody (0.5 μg/mL; sc-51018; Santa Cruz Biotechnology) and goat IgG overnight at 4 °C.

Techniques: Immunohistochemistry, Negative Control, Incubation, Blocking Assay, Gene Expression, MANN-WHITNEY

Journal: EBioMedicine

Article Title: Transcriptome Analysis Uncovers a Growth-Promoting Activity of Orosomucoid-1 on Hepatocytes

doi: 10.1016/j.ebiom.2017.09.008

Figure Lengend Snippet: Transcriptional profiling of LSECs and HPCs during liver regeneration. (a) Schematic overview of transcriptome experimental procedures (n = 3). (b) Hierarchical clustering with Ward's method of 16,499 genes measured by CAGE technology in LSECs and HPCs. The mean average from three biological replicates at each time point was shown. Low expression tags were deleted and the tags that were expressed at least at one time point in either LSECs or HPCs were kept with the expression cutoff at 1 tags per million (TPM). PCA score plots of differentially expressed genes in the process of liver regeneration for (c) LSECs and (d) HPCs. Numbers of significantly differentially expressed genes compared with the sham control at each time point in (e) LSECs and (f) HPCs. Top canonical pathways associated with significantly differentially expressed genes during liver regeneration in (g) LSECs and (h) HPCs in IPA program. Functional annotations identified using DAVID software with top (i) LSEC and (j) HPC genes selected by evaluating the relative contribution of each gene to differential gene expression during the process of liver regeneration in PLS-DA modeling. (k) Expression of Orm1 and Orm2 in LSECs and HPCs during liver regeneration measured by CAGE analysis.

Article Snippet: The sections were then incubated with goat anti-human Orm1 antibody (0.5 μg/mL; sc-51018; Santa Cruz Biotechnology) and goat IgG overnight at 4 °C.

Techniques: Expressing, Control, Functional Assay, Software, Gene Expression

Journal: EBioMedicine

Article Title: Transcriptome Analysis Uncovers a Growth-Promoting Activity of Orosomucoid-1 on Hepatocytes

doi: 10.1016/j.ebiom.2017.09.008

Figure Lengend Snippet: Regulatory role of Orm1 on the proliferation of mouse regenerating HPCs and human hepatic cells. (a) Schematic overview of loss-of-function experimental procedures. Orm1 expression at transcript levels in (b) whole-liver tissue and (c) serum protein levels. (d) IHC staining and (e) gene expression of cell proliferation marker Ki-67 in whole-liver tissue at 48 h post-PH. The average percentage of Ki-67 positive cells presented in (d) was quantified from five randomly selected areas in three slides from each mouse (n = 2–4). Scale bar, 50 μm. siCtl, control siRNA; siOrm1, Orm1 siRNA. The quantitative data were presented as the mean plus standard error (n = 4). (f) Data mining of Orm1 expression in human tissues obtained from the freely available FANTOM5 database. Reference RNA, commercially obtained universal human reference total RNA. Others, other human hepatic non-parenchymal cells, primary cells, tissues and cancer cell lines. (g) The gene expression of Orm1 was examined in human liver cancer cell lines FLC4, FLC7, and HepG2, human hepatic stellate cell line LX2, and immortalized human liver endothelial cell line M1 using real-time RT-PCR. Gene expression was normalized to that of GAPDH. Effect of Orm1 knockdown on (h) gene expression of Orm1 and cyclin D1 and (i) cell proliferation of human hepatic cell line FLC4 in the in the absence [ORM1(−)] and presence [ORM1(+)] of 25 ng/mL recombinant human Orm1. The quantitative data were presented as the mean plus the standard deviation of three replicates. * P < 0.05 assessed using two-tailed Student's t -test, Mann-Whitney U test or ANOVA with post-hoc Tukey HSD Calculator for multiple comparison.

Article Snippet: The sections were then incubated with goat anti-human Orm1 antibody (0.5 μg/mL; sc-51018; Santa Cruz Biotechnology) and goat IgG overnight at 4 °C.

Techniques: Expressing, Immunohistochemistry, Gene Expression, Marker, Control, Quantitative RT-PCR, Knockdown, Recombinant, Standard Deviation, Two Tailed Test, MANN-WHITNEY, Comparison

Journal: EBioMedicine

Article Title: Transcriptome Analysis Uncovers a Growth-Promoting Activity of Orosomucoid-1 on Hepatocytes

doi: 10.1016/j.ebiom.2017.09.008

Figure Lengend Snippet: Molecular targets of Orm1 in mouse regenerating livers. Microarray analysis ( GSE83733 ) was performed in control siRNA (siCtl) and Orm1 siRNA (siOrm1)-injected mouse livers at 48 h post-PH (n = 2). (a) Heatmap visualization of 188 differentially expressed genes with a fold change of > 2 in the livers between groups of mice receiving siOrm1 or siCtl. (b) Top five associated signaling pathways performed using SEA analysis in GeneSpring GX13. (c) The top diseases or functions annotation and (d) top canonical pathway analysis performed in IPA platform. The pathways were ranked according to their-log10 of P values. The ratio indicates the number of enriched genes of interest relative to the total number of genes associated with that pathway in the IPA database. (e) Gene expression levels of MCM2 , MCM4 and MCM6 involved in the enriched “cell cycle control of chromosomal replication” singling pathway in whole-liver tissues before (PH 0 h) and at 48 h post-PH (PH 48 h) were verified using RT-PCR and presented as fold change compared to PH 0 h (n = 4). The data were presented in a Box-and-Whisker plot. * P < 0.05 assessed using the Mann-Whitney U test or two-tailed Student's t -test.

Article Snippet: The sections were then incubated with goat anti-human Orm1 antibody (0.5 μg/mL; sc-51018; Santa Cruz Biotechnology) and goat IgG overnight at 4 °C.

Techniques: Microarray, Control, Injection, Protein-Protein interactions, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Whisker Assay, MANN-WHITNEY, Two Tailed Test

Journal: EBioMedicine

Article Title: Transcriptome Analysis Uncovers a Growth-Promoting Activity of Orosomucoid-1 on Hepatocytes

doi: 10.1016/j.ebiom.2017.09.008

Figure Lengend Snippet: Schematic diagram of a regulatory role of Orm1 on regenerating HPC proliferation. In this study, beginning with the transcriptome profiling of LSECs and HPCs during mouse liver regeneration, early transcriptional changes in injury response pathways in LSECs was observed, followed by activation of cell cycle control pathways in HPCs, and Orm1, predominantly expressed in HPCs, was found and characterized as a promising factor responsible for that by regulating HPC proliferation during liver regeneration.

Article Snippet: The sections were then incubated with goat anti-human Orm1 antibody (0.5 μg/mL; sc-51018; Santa Cruz Biotechnology) and goat IgG overnight at 4 °C.

Techniques: Activation Assay, Control