miR-196b-5p inhibits proliferation of Wharton's jelly umbilical cord stem cells.

Human umbilical cord mesenchymal stem cells can be obtained from different parts of the umbilical cord, including Wharton's jelly. Transplantation of Wharton's jelly umbilical cord stem cells (WJCMSCs) is a promising strategy for the treatment of various diseases. However, the molecular mechanisms underlying the proliferation of WJCMSCs are incompletely understood. Here, we report that overexpression of miR-196b-5p in WJCMSCs suppresses proliferation and arrests the cell cycle in G0/G1 phase, whereas knockdown of miR-196b-5p promotes WJCMSC proliferation and cell-cycle progression. Moreover, miR-196b-5p overexpression resulted in decreased levels of Cyclin A, Cyclin D, Cyclin E and cyclin-dependent kinases 2 and increased levels of p15INK4b , whereas miR-196b-5p knockdown had the opposite effects. In conclusion, our data suggests that miR-196b-5p inhibits WJCMSC proliferation by enhancing G0/G1-phase arrest.

Depletion of SNRNP200 inhibits the osteo-/dentinogenic differentiation and cell proliferation potential of stem cells from the apical papilla.

BACKGROUND: Tissue regeneration mediated by mesenchymal stem cells (MSCs) is deemed a desirable way to repair teeth and craniomaxillofacial tissue defects. Nevertheless, the molecular mechanisms about cell proliferation and committed differentiation of MSCs remain obscure. Previous researches have proved that lysine demethylase 2A (KDM2A) performed significant function in the regulation of MSC proliferation and differentiation. SNRNP200, as a co-binding factor of KDM2A, its potential effect in regulating MSCs' function is still unclear. Therefore, stem cells from the apical papilla (SCAPs) were used to investigate the function of SNRNP200 in this research. METHODS: The alkaline phosphatase (ALP) activity assay, Alizarin Red staining, and osteogenesis-related gene expressions were used to examine osteo-/dentinogenic differentiation potential. Carboxyfluorescein diacetate, succinimidyl ester (CFSE) and cell cycle analysis were applied to detect the cell proliferation. Western blot analysis was used to evaluate the expressions of cell cycle-related proteins. RESULTS: Depletion of SNRNP200 caused an obvious decrease of ALP activity, mineralization formation and the expressions of osteo-/dentinogenic genes including RUNX2, DSPP, DMP1 and BSP. Meanwhile, CFSE and cell cycle assays revealed that knock-down of SNRNP200 inhibited the cell proliferation and blocked cell cycle at the G2/M and S phase in SCAPs. In addition, it was found that depletion of SNRNP200 up-regulated p21 and p53, and down-regulated the CDK1, CyclinB, CyclinE and CDK2. CONCLUSIONS: Depletion of SNRNP200 repressed osteo-/dentinogenic differentiation potentials and restrained cell proliferation through blocking cell cycle progression at the G2/M and S phase, further revealing that SNRNP200 has crucial effects on preserving the proliferation and differentiation potentials of dental tissue-derived MSCs.

miR-4651 inhibits cell proliferation of gingival mesenchymal stem cells by inhibiting HMGA2 under nifedipine treatment.

Drug-induced gingival overgrowth (DIGO) is recognized as a side effect of nifedipine (NIF); however, the underlying molecular mechanisms remain unknown. In this study, we found that overexpressed miR-4651 inhibits cell proliferation and induces G0/G1-phase arrest in gingival mesenchymal stem cells (GMSCs) with or without NIF treatment. Furthermore, sequential window acquisition of all theoretical mass spectra (SWATH-MS) analysis, bioinformatics analysis, and dual-luciferase report assay results confirmed that high-mobility group AT-hook 2 (HMGA2) is the downstream target gene of miR-4651. Overexpression of HMGA2 enhanced GMSC proliferation and accelerated the cell cycle with or without NIF treatment. The present study demonstrates that miR-4651 inhibits the proliferation of GMSCs and arrests the cell cycle at the G0/G1 phase by upregulating cyclin D and CDK2 while downregulating cyclin E through inhibition of HMGA2 under NIF stimulation. These findings reveal a novel mechanism regulating DIGO progression and suggest the potential of miR-4651 and HMGA2 as therapeutic targets.

Epiregulin promotes osteogenic differentiation and inhibits neurogenic trans-differentiation of adipose-derived mesenchymal stem cells via MAPKs pathway.

Mesenchymal stem cells (MSCs) exists low efficiency to trans-differentiate into other germinal layer cell types. One key issue is to discover the effect of important factor on MSCs differentiation abiltiy. In this study, we investigated the role and mechanism of epiregulin (EREG) on the osteogenic differentiation and neurogenic trans-differentiation in adipose-derived stem cells (ADSCs). We discovered that the depletion of EREG inhibited the osteogenic differentiation in vitro. And 25 ng/mL recombinant human epiregulin protein (rhEREG) effectively improved the osteogenic differentiation of EREG-depleted-ADSCs. Depletion of EREG promoted the formation of neural spheres, and increased the expressions of nestin, ßIII-tubulin, NeuroD, NCAM, TH, and NEF in ADSCs. Then, 25 ng/mL rhEREG significantly inhibited these neurogenic differentiation indicators. Inhibition of p38 MAPK, JNK, or Erk1/2 signaling pathway separately, blocked the rhEREG-enhanced osteogenic differentiation ability and the rhEREG-inhibited neurogenic trans-differentiation ability of ADSCs. In conclusions, EREG promoted the osteogenic differentiation and inhibited the neurogenic trans-differentiation potentials of ADSCs via MAPK signaling pathways.

SFRP2 promotes stem cells from apical papilla-mediated periodontal tissue regeneration in miniature pig.

Mesenchymal stem cell-based therapy is a reliable treatment for periodontal tissue regeneration, while ideal regeneration rate is still a facing problem. In previous study, we found SFRP2 a promising gene in modulating mesenchymal stem cells potential. We further investigated its role on periodontal tissue regeneration. We created periodontitis model in miniature pigs and locally injected with stem cells from apical papilla (SCAP). The periodontitis models were classed into three groups, SFRP2-SCAP group (injected with SCAP overexpressing with SFRP2), SCAP group (injected with SCAP transduced with vector backbone) and saline group (vehicle group injected with saline). Clinical assignment, CT scanning, histopathological assessment and quantitative analysis were applied to evaluate the regeneration effect. Twelve weeks after the injection, we found healthier gingival status in SFRP2-SCAP group than the other two groups. Clinical assignment results showed values of probing depth, gingival recession and attachment loss were improved in SFRP2-SCAP group than that of SCAP group and saline group. The volume of newborn bone was also enhanced in SFRP2-SCAP group than SCAP group and saline group. The difference of clinical assignments and newborn bone between each group was significant relevant. HE staining demonstrated increased tissue regeneration in SFRP2-SCAP group than SCAP group and saline group. Our findings revealed that SFRP2 could enhance SCAP-mediated periodontal tissue regeneration and provide a potential target for improving the regeneration of periodontal tissue.

Analysis of the characteristics and expression profiles of coding and noncoding RNAs of human dental pulp stem cells in hypoxic conditions.

BACKGROUND: Human dental pulp stem cell (DPSC)-mediated regenerative endodontics is a promising therapy for damaged teeth; however, the hypoxic environment in root canals can affect tissue regeneration. In this study, we investigate the characteristics and possible regulatory mechanisms of DPSC function under hypoxic conditions. METHODS: Human DPSCs were cultured under normoxia (20% O2) and hypoxia (3% O2). DPSC proliferation and osteo/odontogenic differentiation potential were assessed by Cell Counting Kit-8 (CCK8) assay, carboxyfluorescein succinimidyl ester (CFSE) assay, alkaline phosphatase (ALP) activity, Alizarin red staining, real-time RT-PCR assays, and western blot analysis. Microarray and bioinformatic analyses were performed to investigate the differences in the mRNA, lncRNA, and miRNA expression profiles of DPSCs. RESULTS: DPSCs exhibited a more powerful proliferation ability and lower osteo/odontogenic differentiation potential in hypoxic conditions. A total of 60 mRNAs (25 upregulated and 35 downregulated), 47 lncRNAs (20 upregulated and 27 downregulated), and 14 miRNAs (7 upregulated and 7 downregulated) in DPSCs were differentially expressed in the hypoxia group compared with the normoxia group. Bioinformatic analysis identified that 7 mRNAs (GRPR, ERO1L, ANPEP, EPHX1, PGD, ANGPT1, and NQO1) and 5 lncRNAs (AF085958, AX750575, uc002czn.2, RP3-413H6.2, and six-twelve leukemia (STL)) may be associated with DPSCs during hypoxia according to CNC network analysis, while 28 mRNAs (including GYS1, PRKACB, and NQO1) and 13 miRNAs (including hsa-miR-3916 and hsa-miR-192-5p) may be involved according to miRNA target gene network analysis. The depletion of one candidate lncRNA, STL, inhibited the osteo/odontogenic differentiation potentials of DPSCs. CONCLUSIONS: Our results revealed that hypoxia could enhance the proliferation ability and impair the osteo/odontogenic differentiation potential of DPSCs in vitro. Furthermore, our results identified candidate coding and noncoding RNAs that could be potential targets for improving DPSC function in regenerative endodontics and lead to a better understanding of the mechanisms of hypoxia's effects on DPSCs.