Journal: Scientific Reports

Article Title: Knockdown of LncRNA H19 inhibits vascularization and endochondral ossification via the MiRNA-21a-5p-Smad7/p-Smad2/3 pathway in fracture repair

doi: 10.1038/s41598-025-11300-7

Figure Lengend Snippet: Expression of H19 during postnatal skeletal development. ( A ) H19 exhibited expression within the spongiosa of developing endochondral bones and cortical bone. The expression of H19 gene (red) in the forelimb metaphyseal to diaphyseal region was detected by fluorescence in situ hybridization at postnatal day 0. H19 detected in hypertrophic chondrocytes that do not express COL1α1 (green) (shown by red arrow). ( B ) H19 expression localization in cells, H19 (red) expression is mainly located in the cytoplasm. DAPI, 4′, 6-diaminyl-2-phenylindole, COL1α1, Collagen 1α1.

Article Snippet: This involved combining the hybridization buffer included in the kit with Cy3-tagged mouse lncRNA H19 probes that were made by Ribobio (Guangzhou, China).

Techniques: Expressing, Fluorescence, In Situ Hybridization

Journal: Scientific Reports

Article Title: Knockdown of LncRNA H19 inhibits vascularization and endochondral ossification via the MiRNA-21a-5p-Smad7/p-Smad2/3 pathway in fracture repair

doi: 10.1038/s41598-025-11300-7

Figure Lengend Snippet: Effect of H19 loss on the process of fracture repair. ( A ) Expression of red fluorescent protein (RFP) derived from adenovirus simH19 in tibial fracture of AdsimH19 mice. Immunohistochemical staining of fracture callus revealed RFP-positive cells (brown) in periosteum, external cartilage callus and internal cartilage callus. ( B ) Quantitative analysis of H19 in the tissues around the fracture site. Quantitative PCR analysis of the tissues around the fracture site showed that the expression of H19 in the AdsimH19 group was significantly lower than that in the AdGFP group, * p < 0.05. ( C ) The effect of H19 on the process of fracture repair. X-ray images of fractures showed initial signs of external callus formation in the AdGFP group at the 2nd week post operation (PO-2 W), and cortical space disappeared by bridging internal and external callus at the 4th week post operation (PO-4 W). In the AdsimH19 group, obvious callus was formed and extended outward from the fracture line at week 2 (PO-2 W) and week 3 (PO-3 W), and fracture healing was delayed at week 4 (PO-4 W). ( D ) Micro-CT (µCT) analysis showed that the callus of fractures in the AdGFP group was almost completely bridged with PO-2 W and completely bridged with PO-4 W. The AdsimH19 group showed a large amount of radiative space between the cortices of the PO-2 W fissure and continued to PO-4 W. ( E ) Quantitative analysis by micro-CT (µCT).BV, bone tissue volume; BV/TV, bone volume fraction; SMI, structure model index; Tb.N, trabecular number; Tb.Th, trabecular thickness; Conn.Dn, Connectivity Density; * p < 0.05, ** p < 0.01, compared with AdGFP group.

Article Snippet: This involved combining the hybridization buffer included in the kit with Cy3-tagged mouse lncRNA H19 probes that were made by Ribobio (Guangzhou, China).

Techniques: Expressing, Derivative Assay, Immunohistochemical staining, Staining, Real-time Polymerase Chain Reaction, Micro-CT

Journal: Scientific Reports

Article Title: Knockdown of LncRNA H19 inhibits vascularization and endochondral ossification via the MiRNA-21a-5p-Smad7/p-Smad2/3 pathway in fracture repair

doi: 10.1038/s41598-025-11300-7

Figure Lengend Snippet: Silencing H19 delays endochondral osteogenesis and remodeling during fracture repair. ( A ) Histological evaluation of fracture repair was performed with alcian blue/hematoxylin/Orange-g (ABH/OG) staining. External calluses in PO-1 W, AdsimH19 and AdGFP groups showed obvious signs of early recruitment of mesenchymal cells and formation of cartilage (blue). Cartilage callus (blue) in the AdsimH19 group was more than that in the AdGFP group at PO-2 W, and the AdGFP group showed a transition from cartilage (blue) to bone (red) callus formation. The fractures in the PO-4 W and AdGFP group showed cortical unity through the bridge callus, accompanied by bone healing and active new bone remodeling. Partial fractures of periosteal and intramedullary region were not uniform in AdsimH19 group. ( B ) quantitative analysis of the tissue composition of fracture callus. In the process of fracture repair, the ratio of cartilage area to bone area in AdsimH19 group was significantly increased compared with AdGFP group, * p < 0.05, ** p < 0.01. ( C ) Tartrate-tolerant acid phosphatase (TRAP) staining was used to detect osteoclasts. The total number of trap positive cells (purplish red) in AdsimH19 group was less than that in AdGFP control group. ( D ) TRAP staining was used for quantitative analysis of osteoclasts, * p < 0.05, compared with AdGFP group.

Article Snippet: This involved combining the hybridization buffer included in the kit with Cy3-tagged mouse lncRNA H19 probes that were made by Ribobio (Guangzhou, China).

Techniques: Staining, Control

Journal: Scientific Reports

Article Title: Knockdown of LncRNA H19 inhibits vascularization and endochondral ossification via the MiRNA-21a-5p-Smad7/p-Smad2/3 pathway in fracture repair

doi: 10.1038/s41598-025-11300-7

Figure Lengend Snippet: Silencing H19 obstructs endochondral osteogenesis leading to delayed bone remodeling. ( A ) The same proportion of AdBMP2, AdBMP2+simH19, AdGFP, AdBMP2+H19-infected cartilage fragments were subcutaneously implanted into the lateral abdomen of nude mice without thymus (nu/nu) for 3 weeks. In appearance, the tumor size of AdGFP group was the smallest. The masses in AdGFP and AdBMP2+simH19 groups showed a more distinct cartilage phenotype than those in AdBMP2 and AdBMP2+H19 groups. ( B ) Micro-CT quantitative analysis showed that compared with AdGFP, bone mass and trabecular mass parameters in AdBMP2 group were higher, while bone volume fraction and trabecular mass parameters in AdBMP2+simH19 group were lower than AdBMP2 group. “*” means p < 0.01 as compared with AdBMP2 group; “#” means p < 0.01 as compared with AdBMP2+simH19 group. BV, bone tissue volume; BV/TV, bone volume fraction; SMI, structure model index; Tb.N, trabecular number; Tb.Th, trabecular thickness; Conn.Dn, Connectivity Density. ( C ) The proportion analysis of cartilage hypertrophy, calcification and mineral deposition determined by saffranine O staining of cartilage mass showed that compared with AdGFP, chondrocytes in AdBMP2 group were significantly hypertrophic; Compared with AdBMP2, hypertrophic chondrocytes decreased in AdBMP2+simH19 group. Compared with AdBMP2+simH19, hypertrophic chondrocytes increased in AdBMP2+H19 group. ( D ) Immunofluorescence (IF) detection of CD31 in cartilage mass showed that compared with AdBMP2, the expression of CD31 (red) in cartilage ossification area in AdBMP2+simH19 group was significantly reduced. ( E ) Immunofluorescence (IF) detection of CD31 in mouse fracture sections showed that in the PO-2 W and PO-4 W, the expression of CD31 (red) of AdsimH19 groups was significantly lower than that in the AdGFP group in the cartilage ossification region, and the silence of H19 hindered vascularization, resulting in delayed endochondral osteogenesis.

Article Snippet: This involved combining the hybridization buffer included in the kit with Cy3-tagged mouse lncRNA H19 probes that were made by Ribobio (Guangzhou, China).

Techniques: Infection, Micro-CT, Staining, Immunofluorescence, Expressing

Journal: Scientific Reports

Article Title: Knockdown of LncRNA H19 inhibits vascularization and endochondral ossification via the MiRNA-21a-5p-Smad7/p-Smad2/3 pathway in fracture repair

doi: 10.1038/s41598-025-11300-7

Figure Lengend Snippet: LncRNA H19 binds miR-21a-5p to regulate the expression of samd7. ( A ) BMP2, BMP2 + simH19 induced changes in miRNA expression. C3H10T1/2 cells were infected with AdBMP2 or AdBMP2+AdsimH19 and micro cultured. The changes of miRNA expression in C3H10T1/2 cells were detected by microarray at 12 days after infection, and the results showed significant changes in miRNA expression. ( B ) microarray results showed that eight miRNAs were significantly up-regulated in the AdBMP2+simH19 group, among which miR-21a-5p was increased threefold compared with the control group. ( C ) AdsimH19 increased the expression of miR-21a-5p in chondrocytes. qPCR was used to detect the expression of miR-21a-5p in cultured mouse chondrocytes infected with AdBMP2 or AdBMP2+AdsimH19 in vitro. Expression of miR-21a-5p at day 3, day 7 and day12 showed increased in the AdBMP2+AdsimH19 group. D, H19 wild type, miR-21a-5p simulator and H19 mutant double luciferase gene sequence. ( E ) H19 and miR-21a-5p have binding sites. Luciferase activity of HEK293 cells transfected with NC mimics + H19 wt, mmu-miR-21a-5p + H19 wt, NC mimics + H19 mu and mmu-miR-21a-5p + H19 mu was detected. The results showed that compared with the NC mimics + H19 wt group, the luciferase activity of the mmu-miR-21a-5p + H19 wt group was decreased, and compared with the mmu-miR-21a-5p + H19 wt group, the luciferase activity of the mmu-miR-21a-5p + H19 mu group was increased. “*” means p < 0.05, “**” means p < 0.01. ( F ) BMP2-induced mRNA expression of H19 and smad7. Stimulated by AdBMP2, the relative expression levels of H19 and smad7 were measured by reverse transcription and quantitative real-time PCR (RT-qPCR) at a series of times (days 1, 2, 3, 5, 7, 9, and 12). AdGFP was used as a control. Values for AdBMP2 / AdGFP are shown (each assay was triplicate and/or performed in at least three separate experiments).

Article Snippet: This involved combining the hybridization buffer included in the kit with Cy3-tagged mouse lncRNA H19 probes that were made by Ribobio (Guangzhou, China).

Techniques: Expressing, Infection, Cell Culture, Microarray, Control, In Vitro, Mutagenesis, Luciferase, Sequencing, Binding Assay, Activity Assay, Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Knockdown of LncRNA H19 inhibits vascularization and endochondral ossification via the MiRNA-21a-5p-Smad7/p-Smad2/3 pathway in fracture repair

doi: 10.1038/s41598-025-11300-7

Figure Lengend Snippet: Silencing of H19 resulted in increased miR-21a-5p, decreased smad7 expression, and increased smad2/3 phosphorylation. ( A ) BMP2 induced increased expression of smad7 protein. Blot of smad7 expression was performed on day 7 after transfection of AdBMP2 into mouse chondrocytes. Using beta-actin as a control, the relative protein expression was analyzed by Image Lab software. Compared with the AdGFP group, “*” indicates p < 0.05. Each assay was triplicate and/or performed in at three separate experiments; Representative results are shown. Full-length/ uncropped gels and blots were included in a Supplementary Information file. ( B ) Decreased miR-21a-5p increases the phosphorylation of smad2/3. On the 7th day after transfection of AdBMP2+simH19 + miRNC, AdBMP2+simH19+AntmiR-21a-5p, the expression of smad7, p-smad2/3 and samd2/3 were imprinted. Using beta-actin as a control, the relative protein expression was analyzed by Image Lab software. Compared with AdBMP2+simH19 + miRNC group, “*” indicates p < 0.05. Each assay was triplicate and/or performed in at three separate experiments; Representative results are shown. Full-length/ uncropped gels and blots were included in a Supplementary Information file. ( C ) silencing H19 leads to decreased smad7 expression. The expression of smad7 in the callus transfected with AdGFP and AdsimH19 was detected by immunohistochemistry. Compared with the AdGFP group, the expression of smad7 (brown) in the AdsimH19 group was significantly reduced. ( D ) silencing H19 promotes smad2/3 phosphorylation. The expression of p-smad2/3 in callus transfected with AdGFP and AdsimH19 was detected by immunofluorescence. Compared with AdGFP group, the expression of p-smad2/3 (red) in AdsimH19 group was significantly increased.

Article Snippet: This involved combining the hybridization buffer included in the kit with Cy3-tagged mouse lncRNA H19 probes that were made by Ribobio (Guangzhou, China).

Techniques: Expressing, Phospho-proteomics, Transfection, Control, Software, Immunohistochemistry, Immunofluorescence

Journal: Scientific Reports

Article Title: Knockdown of LncRNA H19 inhibits vascularization and endochondral ossification via the MiRNA-21a-5p-Smad7/p-Smad2/3 pathway in fracture repair

doi: 10.1038/s41598-025-11300-7

Figure Lengend Snippet: Effect of miR-21a-5p inhibitors on silencing H19-induced delayed fracture healing. ( A ) The effect of Ant-miR-21a-5p on the process of silent-H19-induced fracture repair, fracture radiographs showed that obvious callus was formed in the AdsimH19 + miRNC group at week 2 (PO-2 W) and week 3 (PO-3 W) after surgery, and fracture healing was delayed at week 4 (PO-4 W). AdsimH19 + Ant miR-21a-5p group restored the normal formation of periosteum along the periosteum and extended outward from the fracture line at PO-2 W and PO-3 W. The cortical space disappeared through internal and external callus bridging at the 4th week after surgery (PO-4 W), and the fracture was successfully unified. ( B ) micro-CT (µCT) analysis showed that the AdsimH19 + miRNC group showed a large amount of radiation-permeable space between the crack cortex of PO-2 W and continued to PO-4 W, and the callus of the fracture of AdsimH19 + Ant-miR-21a-5p group was almost completely bridged with PO-4 W. ( C ) Histological evaluation of fracture repair was performed by alcian blue/hematoxylin/Orange g (ABH/OG) staining. At PO-1 W, the external calluses of AdsimH19 + miRNC group and AdsimH19 + Ant miR-21a-5p group showed signs of early recruitment of mesenchymal cells and formation of cartilage (blue). At PO-2 W, Cartilage callus(blue) in AdsimH19 + miRNC group was more than that in AdsimH19 + Ant-miR-21a-5p group, and the transition from cartilage (blue) to bone (red) callus formation in AdsimH19 + Ant-miR-21a-5p group was observed. At PO-4 W, partial fractures in periosteum and intramedullary region in AdsimH19 + miRNC group were not uniform, and fractures in AdsimH19 + Ant miR-21a-5p group showed cortical unity through the bridge callus, accompanied by bone healing and active new bone remodeling.

Article Snippet: This involved combining the hybridization buffer included in the kit with Cy3-tagged mouse lncRNA H19 probes that were made by Ribobio (Guangzhou, China).

Techniques: Micro-CT, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Dexamethasone induces transgenerational inheritance of fetal-derived glomerulosclerosis phenotype in offspring through GR/DNMT3a mediated alterations of the lncRNA- Meg3 /Notch signaling pathway

doi: 10.1186/s12964-025-02346-1

Figure Lengend Snippet: The effect of PDE on Meg3 in kidney tissue and oocytes of female rats. A : Volcano map showing the differentially expressed lncRNAs and mRNAs in the kidney of PDE female fetal rats. B : The expression of Meg3 in F1-F3 fetal kidneys. n = 8–12. C : The expression of Meg3 in F1 and F2 oocytes. n = 8–12. D : CpG islands of DNA strand of Meg3 promoter (range: chr6:128489808–128524209). E , G , I : Methylation status of the 8 CpG sites in CpG island3 at Meg3 DMR in DNA strand. F , H , J : The average methylation ratio of CpG island3 in F1-F3 fetal kidneys. n = 3. * P < 0.05, ** P < 0.01 vs. control

Article Snippet: LncRNA Meg3 Smart Silencer was acquired from Guangzhou RiboBio Co., Ltd. (China).

Techniques: Expressing, Methylation, Control

Journal: Cell Communication and Signaling : CCS

Article Title: Dexamethasone induces transgenerational inheritance of fetal-derived glomerulosclerosis phenotype in offspring through GR/DNMT3a mediated alterations of the lncRNA- Meg3 /Notch signaling pathway

doi: 10.1186/s12964-025-02346-1

Figure Lengend Snippet: Meg3 mediated PDE-induced renal developmental toxicity in F1-F3 female offspring by inhibiting Notch signaling pathway. A , B , C : The mRNA expression of Notch signal channel in F1-F3 fetal kidneys. n = 8–12. D , E : The protein expression of NICD in F1-F3 fetal kidneys. n = 3. F : The expression of Meg3 in the primary MMSCs by different concentrations of dexamethasone. n = 6. G - I : The mRNA expressions of Notch1 , Notch2 and Hes1 in the MMSCs by different concentrations of dexamethasone. n = 6. J , K : The protein expression of NICD in the MMSCs by different concentrations of dexamethasone. n = 3. L : The expression of Meg3 in MMSCs by dexamethasone in combination with Meg3 Smart Silencer. n = 6. M - O : The mRNA expressions of Notch1 , Notch2 and Hes1 in MMSCs by dexamethasone in combination with Meg3 Smart Silencer. n = 6. P , Q : The protein expression of NICD in MMSCs by dexamethasone in combination with Meg3 Smart Silencer. n = 3. * P < 0.05, ** P < 0.01 vs. control. # P < 0.05, ## P < 0.01 vs. DEX. & P < 0.05, && P < 0.01 vs. control

Article Snippet: LncRNA Meg3 Smart Silencer was acquired from Guangzhou RiboBio Co., Ltd. (China).

Techniques: Expressing, Control

Journal: Cell Communication and Signaling : CCS

Article Title: Dexamethasone induces transgenerational inheritance of fetal-derived glomerulosclerosis phenotype in offspring through GR/DNMT3a mediated alterations of the lncRNA- Meg3 /Notch signaling pathway

doi: 10.1186/s12964-025-02346-1

Figure Lengend Snippet: DNMT3a mediated PDE-induced upregulation of renal Meg3 in F1 female offspring rats. A : The mRNA expressions of Dnmt1 , Dnmt3a , and Dnmt3b in the F1 fetal kidney. n = 8–12. B : The mRNA expression of DNMTs in MMSCs treated with dexamethasone. n = 6. C , D : The protein expression of DNMT3a in MMSCs treated with dexamethasone. n = 3. E : The mRNA expression of Dnmt3a in MMSCs treated with dexamethasone in combination with DNMT3a plasmids. n = 6. F , G : The protein expression of DNMT3a in MMSCs treated with dexamethasone in combination with DNMT3a plasmids. n = 3. H : The expression of Meg3 in MMSCs treated with dexamethasone in combination with DNMT3a plasmids, n = 6. I , J : The mRNA expressions of Notch1 and Hes1 in MMSCs treated with dexamethasone in combination with DNMT3a plasmids, n = 6. * P < 0.05, ** P < 0.01 vs. control. # P < 0.05, ## P < 0.01 vs. DEX. & P < 0.05, && P < 0.01 vs. control

Article Snippet: LncRNA Meg3 Smart Silencer was acquired from Guangzhou RiboBio Co., Ltd. (China).

Techniques: Expressing, Control

Journal: Cell Communication and Signaling : CCS

Article Title: Dexamethasone induces transgenerational inheritance of fetal-derived glomerulosclerosis phenotype in offspring through GR/DNMT3a mediated alterations of the lncRNA- Meg3 /Notch signaling pathway

doi: 10.1186/s12964-025-02346-1

Figure Lengend Snippet: GR mediated PDE-induced inhibition of renal DNMT3a in F1 female offspring rats. A-D : The protein expression of GR in the F1 fetal kidney (magnification: ×400). n = 3. E , F : The protein expression of GR in MMSCs treated with dexamethasone. n = 3. G : The mRNA expression of GR in MMSCs treated with dexamethasone in combination with si-GR. n = 6. H-J : The protein expressions of GR and DNMT3a in MMSCs treated with dexamethasone in combination with si-GR. n = 3. K-N : The mRNA expressions of Dnmt3a , Meg3 , Notch1 , and Hes1 in MMSCs treated with dexamethasone in combination with si-GR. n = 6. * P < 0.05, ** P < 0.01 vs. control. # P < 0.05, ## P < 0.01 vs. DEX. & P < 0.05, && P < 0.01 vs. control.

Article Snippet: LncRNA Meg3 Smart Silencer was acquired from Guangzhou RiboBio Co., Ltd. (China).

Techniques: Inhibition, Expressing, Control