Journal: Cellular & Molecular Biology Letters

Article Title: LncRNA DNM3OS regulates GREM2 via miR-127-5p to suppress early chondrogenic differentiation of rat mesenchymal stem cells under hypoxic conditions

doi: 10.1186/s11658-021-00269-6

Figure Lengend Snippet: Primer sequences used for RT-qPCR

Article Snippet: After finishing blocking in 0.1% BSA, the rMSCs were incubated with the primary antibody against GREM2 overnight at 4 °C, followed by incubation with CY3-conjugated goat anti-rabbit IgG (1:100 dilution; Boster Biological Technology) for 1 h at room temperature.

Techniques:

Journal: Cellular & Molecular Biology Letters

Article Title: LncRNA DNM3OS regulates GREM2 via miR-127-5p to suppress early chondrogenic differentiation of rat mesenchymal stem cells under hypoxic conditions

doi: 10.1186/s11658-021-00269-6

Figure Lengend Snippet: GREM2 was a target of miR-127-5p. A Nucleotide sequences of the predicted target site for miR-127-5p in the GREM2 3ʹ-UTR. B Luciferase reporter assays were performed to identify direct interaction between miR-127-5p and the GREM2 3ʹ-UTR. Wild type and mutant miR-127-5p target binding sequences in the GREM2 3ʹ-UTR were cloned into a reporter luciferase vector and co-transfected with the synthetized miR-127-5p (or NC) into rMSCs. *** p < 0.001, compared with NC; C GREM2 protein expression was measured in rMSCs from the control, induction, hypoxia, and hypoxia/induction groups. D GREM2 protein expression was measured in rMSCs from the control, induction, and si-SHP/hypoxia groups. E GREM2 protein expression was measured in rMSCs from the control, induction, and miR-127-5p inhibitor/induction groups. F GREM2 protein expression in rMSCs was measured by immunofluorescence. * p < 0.05, *** p < 0.001, compared with control; # p < 0.05, ### p < 0.001, compared with hypoxia or induction group

Article Snippet: After finishing blocking in 0.1% BSA, the rMSCs were incubated with the primary antibody against GREM2 overnight at 4 °C, followed by incubation with CY3-conjugated goat anti-rabbit IgG (1:100 dilution; Boster Biological Technology) for 1 h at room temperature.

Techniques: Luciferase, Mutagenesis, Binding Assay, Clone Assay, Plasmid Preparation, Transfection, Expressing, Control, Immunofluorescence

Journal: Cellular & Molecular Biology Letters

Article Title: LncRNA DNM3OS regulates GREM2 via miR-127-5p to suppress early chondrogenic differentiation of rat mesenchymal stem cells under hypoxic conditions

doi: 10.1186/s11658-021-00269-6

Figure Lengend Snippet: Overexpression of miR-127-5p promoted chondrogenic differentiation of rMSCs by regulating GREM2-mediated SMAD-dependent BMP signaling. The rMSCs were transfected with miR-127-5p mimics, followed by hypoxia intervention (1% O 2 ) and induction of chondrogenic differentiation for 14 days. A MiR-127-5p and GREM2 expression levels were determined by quantitative real-time PCR analysis. Results are expressed as mean ± standard deviation of data obtained from three independent experiments. *** p < 0.001, compared with control; ### p < 0.001, compared with induction group; p < 0.001, compared with hypoxia/induction; B Images from Alcian blue staining assays of rMSCs. C Levels of COL1A1, COL2A1, SOX9, and ACAN protein expression were detected in rMSCs. D Levels of GREM2, BMP2, p-SMAD1, and p-SMAD2 protein expression in rMSCs were detected by western blotting. E GREM2 protein expression in rMSCs was detected by immunofluorescence

Article Snippet: After finishing blocking in 0.1% BSA, the rMSCs were incubated with the primary antibody against GREM2 overnight at 4 °C, followed by incubation with CY3-conjugated goat anti-rabbit IgG (1:100 dilution; Boster Biological Technology) for 1 h at room temperature.

Techniques: Over Expression, Transfection, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation, Control, Staining, Western Blot, Immunofluorescence

Journal: Cellular & Molecular Biology Letters

Article Title: LncRNA DNM3OS regulates GREM2 via miR-127-5p to suppress early chondrogenic differentiation of rat mesenchymal stem cells under hypoxic conditions

doi: 10.1186/s11658-021-00269-6

Figure Lengend Snippet: DNM3OS inhibited chondrogenic differentiation of rMSCs via miR-127-5p regulation of GREM2. The rMSCs were transfected with a DNM3OS overexpression plasmid, followed by induction of chondrogenic differentiation for 14 days. Accordingly, the rMSCs were classified into control, induction, hypoxia, hypoxia/induction, and DNM3OS/induction groups. A The putative binding site for miR-127-5p in the DNM3OS 3′-UTR is shown. B DNM3OS expression was determined by quantitative real-time PCR analysis. C A luciferase reporter assay was performed to identify direct action between DNM3OS and the GREM2 3ʹ-UTR. D Levels of DNM3OS, miR-127-5p, and GREM2 expression were determined by quantitative real-time PCR analysis. Results are expressed as mean ± standard deviation of data obtained from three independent experiments. ** p < 0.01, *** p < 0.001, compared with control; # p < 0.05, ### p < 0.001, compared with induction group; E Images from Alcian blue staining assays of rMSCs. (F) Levels of COL1A1, COL2A1, SOX9, and ACAN protein expression were detected in rMSCs. G Levels of GREM2, BMP2, p-SMAD1, and p-SMAD2 in rMSCs were detected by western blotting. H GREM2 protein expression in rMSCs was measured by immunofluorescence

Article Snippet: After finishing blocking in 0.1% BSA, the rMSCs were incubated with the primary antibody against GREM2 overnight at 4 °C, followed by incubation with CY3-conjugated goat anti-rabbit IgG (1:100 dilution; Boster Biological Technology) for 1 h at room temperature.

Techniques: Transfection, Over Expression, Plasmid Preparation, Control, Binding Assay, Expressing, Real-time Polymerase Chain Reaction, Luciferase, Reporter Assay, Standard Deviation, Staining, Western Blot, Immunofluorescence

Journal: Cellular & Molecular Biology Letters

Article Title: LncRNA DNM3OS regulates GREM2 via miR-127-5p to suppress early chondrogenic differentiation of rat mesenchymal stem cells under hypoxic conditions

doi: 10.1186/s11658-021-00269-6

Figure Lengend Snippet: DNM3OS knockdown weakened the inhibitory effect of hypoxia on chondrogenic induction. The rMSCs were transfected with si-DNM3OS, followed by induction of chondrogenic differentiation under hypoxic conditions (1% O 2 ) for 14 days. A Levels of miR-127-5p, DNM3OS, and GREM2 expression were determined by quantitative real-time PCR analysis. *** p < 0.001, compared with si-NC + hypoxia/induction group. B Levels of GREM2, BMP2, p-SMAD1, and p-SMAD2 in rMSCs were detected by western blotting. C Images from Alcian blue staining assays of rMSCs. (D) Levels of COL1A1, COL2A1, SOX9, and ACAN protein expression were detected in rMSCs. si-NC: negative control

Article Snippet: After finishing blocking in 0.1% BSA, the rMSCs were incubated with the primary antibody against GREM2 overnight at 4 °C, followed by incubation with CY3-conjugated goat anti-rabbit IgG (1:100 dilution; Boster Biological Technology) for 1 h at room temperature.

Techniques: Knockdown, Transfection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Staining, Negative Control

Journal: Regenerative Therapy

Article Title: Gremlin 2 suppresses differentiation of stem/progenitor cells in the human skin

doi: 10.1016/j.reth.2021.06.007

Figure Lengend Snippet: The expressions of GREM2 and differentiation markers in the epidermis. To examine GREM2 expression in the epidermis and epidermal differentiation, immunohistochemistry against GREM2 and epidermal differentiation markers, Keratin 10 (KRT10) and Keratin 14 (KRT14), was performed (n = 14; 65–89 years of age, average 80.9 years of age). (A) Representative images of a low and a high GREM2 expression in the epidermis. The skin samples were from the donors No. 6 (Low, 83 years of age) and No. 4 (High, 75 years of age) in Table 1 . Red, GREM2; blue, nuclei. (B) Double staining with KRT10 and KRT14 in the epidermis of the same samples as (A). Red, KRT14; green, KRT10; blue nuclei. (C, D) Fluorescence intensities of GREM2, KRT10 and KRT14 per unit area in the epidermis were measured, and the average value of each of the three proteins for each donor was calculated. The average values for the donor No. 1 ( Table 1 , 65 years of age) were set as 1, and the relative values of (other) donors were calculated. Then the residuals of the relative values against age were plotted. (C) Partial correlation analysis revealed that the expression level of KRT10 was negatively correlated with that of GREM2 (∗ p < 0.05). (D) Partial correlation analysis did not reveal any significant correlation between the expression levels of GREM2 and KRT14.

Article Snippet: After blocking with 3% BSA, 1% Triton-X in PBS at room temperature for 1 h, immunohistochemistry was performed in a conventional way against GREM2 (GeneTex, CA, USA), Integrin beta 1 (Abcam, Cambridge, UK), p75 NGF receptor/CD271 (ORIGENE, MD, USA), Endoglin/CD105 (Santa Cruz Biotechnology, TX, USA), Keratin 10 (Neomaekers, CA, USA), Keratin 14 (BioLegend, CA, USA) and Pro-COL3A1 (Santa Cruz Biotechnology) in the conditions shown in .

Techniques: Expressing, Immunohistochemistry, Double Staining, Fluorescence

Journal: Regenerative Therapy

Article Title: Gremlin 2 suppresses differentiation of stem/progenitor cells in the human skin

doi: 10.1016/j.reth.2021.06.007

Figure Lengend Snippet: The expressions of stem cell markers in the epidermis with different levels of GREM2. Immunostaining against epidermal stem cell markers, ITGB1 and CD271, was performed. (A) Immunohistological images against ITGB1 (red in the upper panels) and CD271 (green in the lower panels) of representative samples with a low and a high GREM2 expression (Low, No. 6, 83 years of age; High, No. 4, 75 years of age), which were from the same samples shown in Fig. 1 . Blue, nuclei. (B) The percentage of ITGB1-positive cells per basal cells was calculated. The residuals of GREM2 expression level and the ratio of ITGB1-positive cells (%) against age were plotted. Partial correlation analysis revealed no significant correlation. (C) The percentage of CD271-positive cells per basal cells was calculated. The residuals of GREM2 expression level and the ratio of CD271-positive cells (%) against age were plotted. Partial correlation analysis revealed no significant correlation.

Article Snippet: After blocking with 3% BSA, 1% Triton-X in PBS at room temperature for 1 h, immunohistochemistry was performed in a conventional way against GREM2 (GeneTex, CA, USA), Integrin beta 1 (Abcam, Cambridge, UK), p75 NGF receptor/CD271 (ORIGENE, MD, USA), Endoglin/CD105 (Santa Cruz Biotechnology, TX, USA), Keratin 10 (Neomaekers, CA, USA), Keratin 14 (BioLegend, CA, USA) and Pro-COL3A1 (Santa Cruz Biotechnology) in the conditions shown in .

Techniques: Immunostaining, Expressing

Journal: Regenerative Therapy

Article Title: Gremlin 2 suppresses differentiation of stem/progenitor cells in the human skin

doi: 10.1016/j.reth.2021.06.007

Figure Lengend Snippet: The expressions of GREM2 and type 3 procollagen in the dermis. To examine GREM2 expression and dermal differentiation, immunohistochemistry against GREM2 and a dermal differentiation marker, type 3 procollagen (Pro-COL3A1), was performed in the dermis (n = 14; 65–89 years of age, average 80.9 years of age). (A) Representative images of a low and a high GREM2 expression in the dermis. The boxed areas in the left panels are magnified and shown in the right. The samples were from the donors No. 9 (Low, 86 years of age) and No. 11 (High, 86 years of age) in Table 1 . Red, GREM2; blue, nuclei. (B) Immunohistochemistry against type 3 procollagen (green) in the dermis of the same samples as (A). Blue, nuclei. (C) Fluorescence intensities of GREM2 and type 3 procollagen per unit area in the dermal papillary layer were measured, and the average value of each of the two proteins for each donor was calculated. The average values for the donor No. 1 ( Table 1 and 65 years of age) were set as 1, and the relative values of (other) donors were calculated. Then the residuals of the relative values against age were plotted. Partial correlation analysis revealed that the expression level of type 3 procollagen was negatively correlated with that of GREM2 (∗ p < 0.05).

Article Snippet: After blocking with 3% BSA, 1% Triton-X in PBS at room temperature for 1 h, immunohistochemistry was performed in a conventional way against GREM2 (GeneTex, CA, USA), Integrin beta 1 (Abcam, Cambridge, UK), p75 NGF receptor/CD271 (ORIGENE, MD, USA), Endoglin/CD105 (Santa Cruz Biotechnology, TX, USA), Keratin 10 (Neomaekers, CA, USA), Keratin 14 (BioLegend, CA, USA) and Pro-COL3A1 (Santa Cruz Biotechnology) in the conditions shown in .

Techniques: Expressing, Immunohistochemistry, Marker, Fluorescence

Journal: Regenerative Therapy

Article Title: Gremlin 2 suppresses differentiation of stem/progenitor cells in the human skin

doi: 10.1016/j.reth.2021.06.007

Figure Lengend Snippet: The expressions of stem cell markers in the dermis with different levels of GREM2. Immunostaining against dermal stem cell markers, CD105 and CD271, was performed. (A and B) Immunohistological images against CD105 (green in A) and CD271 (green in B) of representative samples with a low and a high GREM2 expression (Low, No. 9, 86 years of age; High, No. 11, 86 years of age), which were from the same samples shown in Fig. 3 . Blue, nuclei. The arrowheads indicate positive cells for CD105 in A or CD271 in B. (C) The percentage of CD105-positive cells per total number of cells in the dermal papillary layer was calculated. The residuals of GREM2 expression level and the ratio of CD105-positive cells (%) against age were plotted. Partial correlation analysis revealed no significant correlation. (D) The percentage of CD271-positive cells per total number of cells in the dermal papillary layer was calculated. The residuals of GREM2 expression level and the ratio of CD271-positive cells (%) against age were plotted. Partial correlation analysis revealed no significant correlation.

Article Snippet: After blocking with 3% BSA, 1% Triton-X in PBS at room temperature for 1 h, immunohistochemistry was performed in a conventional way against GREM2 (GeneTex, CA, USA), Integrin beta 1 (Abcam, Cambridge, UK), p75 NGF receptor/CD271 (ORIGENE, MD, USA), Endoglin/CD105 (Santa Cruz Biotechnology, TX, USA), Keratin 10 (Neomaekers, CA, USA), Keratin 14 (BioLegend, CA, USA) and Pro-COL3A1 (Santa Cruz Biotechnology) in the conditions shown in .

Techniques: Immunostaining, Expressing

Journal: Regenerative Therapy

Article Title: Gremlin 2 suppresses differentiation of stem/progenitor cells in the human skin

doi: 10.1016/j.reth.2021.06.007

Figure Lengend Snippet: The effect of GREM2 on the differentiation of HDK1cells. HDK1 cells were cultured for seven days to generate 3D epidermis in the differentiation medium with GREM2. (A, B) mRNA was collected from 3D reconstructed epidermis, and the gene expression levels of differentiation markers, Keratin 10 ( KRT10 in A) and Involucrin ( IVL in B), were examined. The expressions of both genes were suppressed by GREM2 in a dose-dependent manner and a significant suppression was observed when GREM2 was added at 25 ng/ml (n = 3, mean ± SD, ∗ p < 0.05). (C–F) Sections of 3D reconstructed epidermis (cultured with GREM2 at 25 ng/mL or without GREM2) were prepared, and HE staining (C) and immunohistochemistry against KRT10 and KRT14 (D) were performed. The 3D reconstructed epidermis cultured with GREM2 was thinner than GREM2-negative control. Fluorescence intensities of KRT10 (E) and KRT14 (F) in immunohistochemical images were measured and compared between when cultured with and without GREM2. The expression of KRT10, a differentiation marker, was significantly lower in the 3D epidermis cultured with GREM2 (n = 3, mean ± SD, ∗ p < 0.05). The addition of GREM2 did not change the expression level of KRT14, an undifferentiation marker, in the 3D reconstructed epidermis, especially in the lower layers.

Article Snippet: After blocking with 3% BSA, 1% Triton-X in PBS at room temperature for 1 h, immunohistochemistry was performed in a conventional way against GREM2 (GeneTex, CA, USA), Integrin beta 1 (Abcam, Cambridge, UK), p75 NGF receptor/CD271 (ORIGENE, MD, USA), Endoglin/CD105 (Santa Cruz Biotechnology, TX, USA), Keratin 10 (Neomaekers, CA, USA), Keratin 14 (BioLegend, CA, USA) and Pro-COL3A1 (Santa Cruz Biotechnology) in the conditions shown in .

Techniques: Cell Culture, Gene Expression, Staining, Immunohistochemistry, Negative Control, Fluorescence, Immunohistochemical staining, Expressing, Marker

Journal: Regenerative Therapy

Article Title: Gremlin 2 suppresses differentiation of stem/progenitor cells in the human skin

doi: 10.1016/j.reth.2021.06.007

Figure Lengend Snippet: The effect of GREM2 on the differentiation of SF8428 cells. SF8428 cells were cultured in the differentiation medium with GREM2 for 24 h. (A, B) mRNA was collected from cells, and the gene expressions of fibrogenesis markers, type 1 collagen ( COL1A1 in A) and type 3 collagen ( COL3A1 in B), were compared. The expression levels of both genes were significantly suppressed by GREM2 in a dose-dependent manner (n = 3, mean ± SD, ∗∗∗ p < 0.001, ∗ p < 0.05). (C–F) SF8428 cells were cultured in the differentiation medium with GREM2 for four days to examine its effect on collagen fiber formation. Then cells were fixed with 4% PFA, and immunohistochemistry against COL1A1 (red in C) and COL3A1 (red in D) was performed. Blue, nuclei. In the cells cultured with GREM2, collagen fiber formation and the expressions of collagen genes were suppressed. Fluorescence intensities of COL1A1 (E) and COL3A1 (F) in immunohistochemical images were measured and compared between when cultured with and without GREM2. The expressions of both proteins were significantly suppressed when GREM2 was added (n = 3, mean ± SD, ∗∗ p < 0.01, ∗ p < 0.05).

Article Snippet: After blocking with 3% BSA, 1% Triton-X in PBS at room temperature for 1 h, immunohistochemistry was performed in a conventional way against GREM2 (GeneTex, CA, USA), Integrin beta 1 (Abcam, Cambridge, UK), p75 NGF receptor/CD271 (ORIGENE, MD, USA), Endoglin/CD105 (Santa Cruz Biotechnology, TX, USA), Keratin 10 (Neomaekers, CA, USA), Keratin 14 (BioLegend, CA, USA) and Pro-COL3A1 (Santa Cruz Biotechnology) in the conditions shown in .

Techniques: Cell Culture, Expressing, Immunohistochemistry, Fluorescence, Immunohistochemical staining

Journal: Hereditas

Article Title: Overexpression of miR-671-3p alleviates postmenopausal osteoporosis by targeting GREM2 to activate BMP2/SMAD signaling pathway.

doi: 10.1186/s41065-025-00467-8

Figure Lengend Snippet: Fig. 3 WT/MT-GREM2 3’UTR sequences and miR-671-3p binding sites (A). Dual-luciferase reporter gene assay (B) and RNA immunoprecipitation (RIP) experiment (C) indicated the binding between miR-671-3p and GREM2. The expression of GREM2 was upregulated in PMOP patients’ serum (D) and negatively related to the levels of miR-671-3p (E). GREM2 was upregulated in MG, and overexpression of miR-671-3p inhibited its expression (F). GREM2 overexpression had no impact on miR-671-3p expression (G). WT-GREM2: Wild-type GREM2 3’UTR luciferase reporter; MT-GREM2: Mutant GREM2 3’UTR luciferase reporter; Ctrl: Normal culture conditions; MG: Cells treated with simulated MG for 72 h; MG + miR-NC: MG + miR- NC transfection; MG + miR- mimic: MG + miR-671-3p mimic transfection; MG + miR + GREM2-NC: MG + miR-mimic + empty vector transfection; MG + miR + oe-GREM2: MG + miR- mimic + GREM2 overexpression plasmid transfection. Bar graphs show mean ± SD (n = 5). two-tailed t-test for WT vs. MT; one-way ANOVA with Tukey’s test for other groups

Article Snippet: Proteins were detected using antibodies against GREM2 (1:1000, Proteintech), BMP2 (1:1000, CST), p-SMAD1/5 (1:1000, Abcam), and GAPDH (1:5000, Proteintech).

Techniques: Binding Assay, Luciferase, Reporter Gene Assay, RNA Immunoprecipitation, Expressing, Over Expression, Mutagenesis, Transfection, Plasmid Preparation, Two Tailed Test

Journal: Hereditas

Article Title: Overexpression of miR-671-3p alleviates postmenopausal osteoporosis by targeting GREM2 to activate BMP2/SMAD signaling pathway.

doi: 10.1186/s41065-025-00467-8

Figure Lengend Snippet: Fig. 4 miR-671-3p inhibits GREM2 protein expression (A). Elevated GREM2 exacerbates MG-induced inhibition of cell differentiation (B-C), disrupted vi ability (D) and apoptosis (E-F), counteracting the protective effect of miR-671-3p. Ctrl: Normal culture conditions; MG: Cells treated with simulated MG for 72 h; MG + miR-NC: MG + miR- NC transfection; MG + miR-mimic: MG + miR-671-3p mimic transfection; MG + miR + GREM2-NC: MG + miR-mimic + empty vector transfection; MG + miR + oe-GREM2: MG + miR-mimic + GREM2 overexpression plasmid transfection. Bar graphs show mean ± SD (n = 5). one-way ANOVA with Tukey’s post hoc test

Article Snippet: Proteins were detected using antibodies against GREM2 (1:1000, Proteintech), BMP2 (1:1000, CST), p-SMAD1/5 (1:1000, Abcam), and GAPDH (1:5000, Proteintech).

Techniques: Expressing, Inhibition, Cell Differentiation, Transfection, Plasmid Preparation, Over Expression

Journal: Hereditas

Article Title: Overexpression of miR-671-3p alleviates postmenopausal osteoporosis by targeting GREM2 to activate BMP2/SMAD signaling pathway.

doi: 10.1186/s41065-025-00467-8

Figure Lengend Snippet: Fig. 5 Reduced expression of BMP2 (A) and P-SMAD1/5 (B-C) proteins under MG is promoted by miR-671-3p upregulation and inhibited by GREM2 overexpression. Ctrl: Normal culture conditions; MG: Cells treated with simulated MG for 72 h; MG + miR-NC: MG + miR- NC transfection; MG + miR-mimic: MG + miR-671-3p mimic transfection; MG + miR + GREM2-NC: MG + miR-mimic + empty vector transfection; MG + miR + oe-GREM2: MG + miR-mim ic + GREM2 overexpression plasmid transfection. Bar graphs show mean ± SD (n = 5). one-way ANOVA with Tukey’s post hoc test

Article Snippet: Proteins were detected using antibodies against GREM2 (1:1000, Proteintech), BMP2 (1:1000, CST), p-SMAD1/5 (1:1000, Abcam), and GAPDH (1:5000, Proteintech).

Techniques: Expressing, Over Expression, Transfection, Plasmid Preparation