Journal: Research
Article Title: Novel lncRNA LncMSTRG.11341.25 Promotes Osteogenic Differentiation of Human Bone Marrow Stem Cells via the miR-939-5p/PAX8 Axis
doi: 10.34133/research.0601
Figure Lengend Snippet: LncMSTRG25–PAX8–miR-939-5p regulatory axis regulates hBMSC osteogenesis. (A) Potential target-binding sites of PAX8 and miR-939-5p were predicted using TargetScan and StarBase, and then validated through a dual-luciferase reporter assay. (B) The results from the dual-luciferase reporter gene assay indicated that cotransfection with the miR-939-5p plasmid and PAX8-WT1 and PAX8-WT2 plasmids caused a significant decrease in luciferase activity. (C to E) RNA RIP experiments were performed on hBMSCs using PAX8 antibody to evaluate the enrichment effect of anti-PAX8 on LncMSTRG25 and miR-939-5p. qPCR was used to detect RNA levels in the immunoprecipitates. The miR-939-5p level in the anti-PAX8 group was higher compared with that in the anti-IgG group, and the level of LncMSTRG25 was significantly increased in the anti-PAX8 group compared with that in the anti-IgG group. The LncMSTRG25 PCR product was subjected to agarose electrophoresis, and in the anti-PAX8 group, the expression level was markedly increased. (F and G) Silver staining and Western blotting results of RNA pull-down samples showed that the biotin-labeled LncMSTRG25-specific probe group significantly enriched PAX8. (H and I) Using Lv-LncMSTRG25, Lv-NC, siPAX8, or an siRNA negative control, osteogenic differentiation was induced for 7 d. Protein expression levels of PAX8, ALP, BMP2, RUNX2, COLL1, OPN, and OCN were analyzed by Western blotting. (J) To detect the relative expression levels of PAX8, ALP, BMP2, RUNX2, COLL1, OPN, and OCN, PCR was performed. Osteogenic differentiation continued for 14 d. (K) ARS and ALP staining were used to monitor osteogenic differentiation. (L) Specific antibody immunofluorescence staining was used to detect the expression of PAX8, BMP2, and RUNX2 after 7 d of osteogenic differentiation. Results are presented as the mean ± SD of 3 independent trials, with significance levels of * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.
Article Snippet: For 14 d, the third-generation hBMSCs were cultured in osteogenic differentiation induction medium provided by Cyagen (USA); medium changes were performed every 3 d. After induction, the hBMSCs were fixed with 4% paraformaldehyde.
Techniques: Binding Assay, Luciferase, Reporter Assay, Reporter Gene Assay, Cotransfection, Plasmid Preparation, Activity Assay, Electrophoresis, Expressing, Silver Staining, Western Blot, Labeling, Negative Control, Staining, Immunofluorescence
![( A ) Experimental flowchart of IFITM5 + cells isolation and characterization in vitro. ( B ) Flow cytometry gating strategies for sorting IFITM5 + cells ( n = 3 embryos). ( C ) Flow cytometry plots showing serial colony formation from a single IFITM5 + cell ( n = 3 clones). ( D ) Representative crystal violet staining of fibroblast colony-forming unit (CFU-F) colonies from IFITM5 − and IFITM5 + cells. ( E ) Numbers and mean diameters of CFU-F colonies ( n = 3 embryos). ( F , H , and J ) Representative alizarin red, alcian blue, and oil red O stainings after in vitro differentiation of clonally expanded IFITM5 − and IFITM5 + cells. ( G , I , and K ) qPCR analyses of osteogenic, chondrogenic, and adipogenic marker genes and quantification of alizarin red in clonally expanded IFITM5 − and IFITM5 + cells after in vitro differentiation ( n = 3 embryos). ( L ) Workflow of IFITM5 + cell characterization in vivo. ( M ) Bright-field images and H&E staining of subcapsular xenografts of IFITM5 − and IFITM5 + cells ( n = 5). ( N , P , and Q ) Immunofluorescence staining of COLII, COLI, OPN, and Stem101. ( O ) Micro–computed tomography (CT) three-dimensional (3D) reconstruction images (top) and coronal images (bottom) of mandibular defect repair after GelMA, IFITM5 − , and IFITM5 + cell transplantation. ( R ) Quantification of bone formation parameters at defect region ( n = 5). ( S ) H&E staining (bone defect edge demarcated by black dashed lines) and Masson staining in coronal sections of GelMA, IFITM5 − , and IFITM5 + cell groups. ( T ) Immunohistochemical staining for human nucleoli, RUNX2, and OPN in areas marked by white dashed lines in Masson staining images. Scale bars, 100 μm in all figures. [(E), (G), (I), and (K)] ** P < 0.01 and *** P < 0.001 determined by an unpaired two-tailed Student’s t test. (R) * P < 0.05, ** P < 0.01, and *** P < 0.001 versus GelMA; ### P < 0.001 versus IFITM5 − cells determined by one-way analysis of variance (ANOVA) with Tukey’s post hoc test. SSC-A, side scatter-area; OD, optical density.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1644/pmc11691644/pmc11691644__sciadv.ado7852-f5.jpg)
![( A ) Flow cytometry gating strategies for sorting CADM1 + cells ( n = 2 embryos). ( B ) Representative crystal violet staining of CFU-F colonies from CADM1 + and IFITM5 + cells. Scale bars, 100 μm. ( C ) Numbers and mean diameters of the CFU-F colonies ( n = 2 embryos). ( D ) Representative alizarin red staining after osteogenic differentiation of CADM1 + and IFITM5 + cells. Scale bars, 100 μm. ( E ) Quantification of alizarin red staining in CADM1 + and IFITM5 + cells after in vitro differentiation ( n = 2 embryos). ( F ) qPCR analyses of osteogenic marker genes in vitro differentiation ( n = 2 embryos). ( G ) Micro-CT coronal images (top) and 3D reconstruction images (bottom) of mouse mandibular defect repair after transplantation of CADM1 + and IFITM5 + cells. ( H ) Quantification of bone formation parameters at defect region ( n = 5). ( I ) Representative images of H&E staining (bone defect edge demarcated by black dashed lines) in coronal sections of GelMA, CADM1 + , and IFITM5 + cell groups. Scale bar, 100 μm. [(C), (E), and (F)] * P < 0.05, ** P < 0.01, and *** P < 0.001 determined by an unpaired two-tailed Student’s t test. (H) ** P < 0.01 and *** P < 0.001 versus GelMA; # P < 0.05 and ### P < 0.001 versus CADM1 + cells determined by one-way ANOVA with Tukey’s post hoc test.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1644/pmc11691644/pmc11691644__sciadv.ado7852-f6.jpg)
![( A ) Schematic diagram of characterization IFITM5 + cells in vivo and in vitro. ( B ) H&E and immunofluorescence staining of IFITM5 and RUNX2 in mandible fragments aged 19, 32, and 49 (white boxes indicating magnified regions). y, years. ( C ) Flow analysis with MSC-specific surface markers and morphology of BMSCs ( n = 3). ( D ) Gating scheme for sorting Zombie − IFITM5 + BMSCs ( n = 3 patients). ( E ) Crystal violet staining of CFU-F colonies from IFITM5 − and IFITM5 + BMSCs. ( F ) Numbers and mean diameters of CFU-F colonies ( n = 3 patients). ( G , J , and L ) Alizarin red, alcian blue, and oil red O stainings after in vitro differentiation. ( H and M ) Quantification of alizarin red and oil red O stainings. ( I , K , and N ) qPCR analyses of osteogenic, chondrogenic, and adipogenic marker genes ( n = 3 patients). ( O ) Bright-field images and H&E staining of subcapsular xenografts of IFITM5 − and IFITM5 + BMSCs ( n = 5). ( P , Q , and R ) Immunofluorescence staining images of COLII, COLI, OPN, and Stem101. ( S ) Micro-CT 3D reconstruction images and coronal images of mandibular defect after GelMA, IFITM5 − , and IFITM5 + BMSC transplantation. ( T ) Quantification of bone formation parameters at defect region ( n = 5). ( U ) H&E staining (bone defect edge demarcated by black dashed lines) and Masson staining in coronal sections of GelMA, IFITM5 − , and IFITM5 + BMSC groups. ( V ) Immunohistochemical staining for Human nucleoli, RUNX2, and OPN in areas marked by white dashed lines in Masson staining images. Scale bars, 100 μm in all figures. [(F), (H), (I), (K), (M), and (N)] ** P < 0.01 and *** P < 0.001 determined by an unpaired two-tailed Student’s t test. (T) ** P < 0.01 and *** P < 0.001 versus GelMA; ## P < 0.01 and ### P < 0.001 versus IFITM5 − cells determined by one-way ANOVA with Tukey’s post hoc test.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1644/pmc11691644/pmc11691644__sciadv.ado7852-f7.jpg)
