OBJECTIVE: To investigate the regulatory effects of miR-26a-5p on the osteogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) by regulating cAMP response element binding protein 1 (CREB1). METHODS: The adipose tissues of four 3-4 weeks old female C57BL/6 mice were collected and the cells were isolated and cultured by digestion separation method. After morphological observation and identification by flow cytometry, the 3rd-generation cells were subjected to osteogenic differentiation induction. At 0, 3, 7, and 14 days after osteogenic differentiation induction, the calcium deposition was observed by alizarin red staining, ALP activity was detected, miR- 26a-5p and CREB1 mRNA expressions were examined by real-time fluorescence quantitative PCR, and CREB1 protein and its phosphorylation (phospho-CREB1, p-CREB1) level were measured by Western blot. After the binding sites between miR-26a-5p and CREB1 was predicted by the starBase database, HEK-293T cells were used to conduct a dual-luciferase reporter gene experiment to verify the targeting relationship (represented as luciferase activity after 48 hours of culture). Finally, miR-26a-p inhibitor (experimental group) and the corresponding negative control (control group) were transfected into ADSCs. Alizarin red staining, ALP activity, real-time fluorescent quantitative PCR (miR-26a-5p) and Western blot [CREB1, p-CREB1, Runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN)] were performed at 7 and 14 days after osteogenic induction culture. RESULTS: The cultured cells were identified as ADSCs. With the prolongation of osteogenic induction culture, the number of calcified nodules and ALP activity significantly increased ( P<0.05). The relative expression of miR-26a-5p in the cells gradually decreased, while the relative expressions of CREB1 mRNA and protein, as well as the relative expression of p-CREB1 protein were increased. The differences were significant between 7, 14 days and 0 day ( P<0.05). There was no significant difference in p-CREB1/CREB1 between different time points ( P>0.05). The starBase database predicted that miR-26a-5p and CREB1 had targeted binding sequences, and the dual-luciferase reporter gene experiment revealed that overexpression of miR-26a-5p significantly suppressed CREB1 wild-type luciferase activity ( P<0.05). After 7 and 14 days of osteogenic induction, compared with the control group, the number of calcified nodules, ALP activity, and relative expressions of CREB1, p-CREB1, OCN, and RUNX2 proteins in the experimental group significantly increased ( P<0.05). There was no significant difference in p-CREB1/CREB1 between the two groups ( P>0.05). CONCLUSION Knocking down miR-26a-5p promoted the osteogenic differentiation of ADSCs by up-regulating CREB1 and its phosphorylation.
Animals ; Female ; Mice ; Cell Differentiation ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Cyclic AMP Response Element-Binding Protein/metabolism* ; Mesenchymal Stem Cells ; Mice, Inbred C57BL ; MicroRNAs/metabolism* ; Osteocalcin/metabolism* ; Osteogenesis/genetics* ; RNA, Messenger/genetics*

OBJECTIVE: To compare the mRNA level of cell proliferation-related genes Twist1, SIRT1, FGF2 and TGF-β3 in placenta mesenchymal stem cells (PA-MSCs), umbilical cord mensenchymals (UC-MSCs) and dental pulp mesenchymal stem cells (DP-MSCs). METHODS: The morphology of various passages of PA-MSCs, UC-MSCs and DP-MSCs were observed by microscopy. Proliferation and promoting ability of the three cell lines were detected with the MTT method. Real-time PCR (RT-PCR) was used to determine the mRNA levels of Twist1, SIRT1, FGF2, TGF-β3. RESULTS: The morphology of UC-MSCs and DP-MSCs was different from that of PA-MSCs. Proliferation ability and promoting ability of the PA-MSCs was superior to that of UC-MSCs and DP-MSCs. In PA-MSCs, expression level of Twist1 and TGF-β3 was the highest and FGF2 was the lowest. SIRT1 was highly expressed in UC-MSCs. With the cell subcultured, different expression levels of Twist1, SIRT1, FGF2, TGF-β3 was observed in PA-MSCs, UC-MSCs and DP-MSCs. CONCLUSION Up-regulated expression of the Twist1, SIRT1 and TGF-β3 genes can promote proliferation of PA-MSCs, UC-MSCs and DP-MSCs, whilst TGF-β3 may inhibit these. The regulatory effect of Twist1, SIRT1, FGF2 and TGF-β3 genes on PA-MSCs, UC-MSCs and DP-MSCs are different.
Cell Differentiation ; Cell Proliferation/genetics* ; Cells, Cultured ; Dental Pulp/cytology* ; Female ; Fibroblast Growth Factor 2/genetics* ; Humans ; Mesenchymal Stem Cells/cytology* ; Nuclear Proteins/genetics* ; Placenta/cytology* ; Pregnancy ; Sirtuin 1/genetics* ; Transforming Growth Factor beta3/genetics* ; Twist-Related Protein 1/genetics* ; Umbilical Cord/cytology*

OBJECTIVE: To explore the role of small nuclear noncoding RNA 7SK in embryonic stem cell (ESCs) proliferation and the value of 7SK as a target for early diagnosis and treatment for primordial dwarfism (PD). METHODS: ESC line R1 was transfected with the CRISPR/Cas9 system, and sequencing of the PCR product and glycerol gradient analysis were performed to identify novel 7SK deletion mutations. A lentivirus system was used to knock down cyclin-dependent kinase 9 (CDK9) in clones with 7SK deletion mutations, and the effect of CDK9 knockdown on the protein level of cell division cycle 6 (CDC6) was analyzed with Western blotting. RESULTS: We identified a novel deletion mutation of 7SK at 128-179 nt in the ESCs, which resulted in deficiency of cell proliferation. 7SK truncation at 128-179 nt significantly reduced the protein expressions of La-related protein 7 (LARP7) and CDC6. CONCLUSIONS 7SK truncation at 128-179 nt can significantly impair proliferation of ESCs by downregulating CDC6. 7SK is a key regulator of proliferation and mediates the growth of ESCs through a mechanism dependent on CDK9 activity, suggesting the value of 7SK truncation at 128-179 nt as a potential target for early diagnosis and treatment of PD.
Cell Cycle Proteins ; Cell Proliferation ; Embryonic Stem Cells/metabolism* ; HeLa Cells ; Humans ; Nuclear Proteins ; Positive Transcriptional Elongation Factor B/metabolism* ; RNA, Long Noncoding/genetics* ; RNA-Binding Proteins ; Ribonucleoproteins ; Transcription Factors

Combined radiation-wound injury (CRWI) is characterized by blood vessel damage and pro-inflammatory cytokine deficiency. Studies have identified that the direct application of leptin plays a significant role in angiogenesis and inflammation. We established a sustained and stable leptin expression system to study the mechanism. A lentivirus method was employed to explore the angiogenic potential and peripheral inflammation of irradiated human umbilical vein endothelial cells (HUVECs). Leptin was transfected into human placenta-derived mesenchymal stem cells (HPMSCs) with lentiviral vectors. HUVECs were irradiated by X-ray at a single dose of 20 Gy. Transwell migration assay was performed to assess the migration of irradiated HUVECs. Based on the Transwell systems, co-culture systems of HPMSCs and irradiated HUVECs were established. Cell proliferation was measured by cell counting kit-8 (CCK-8) assay. The secretion of pro-inflammatory cytokines (human granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-1α, IL-6, and IL-8) was detected by enzyme-linked immunosorbent assay (ELISA). The expression of pro-angiogenic factors (vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF)) mRNA was detected by real-time quantitative polymerase chain reaction (RT-qPCR) assay. Relevant molecules of the nuclear factor-κB (NF-κB) and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways were detected by western blot assay. Results showed that leptin-modified HPMSCs (HPMSCs/ leptin) exhibited better cell proliferation, migration, and angiogenic potential (expressed more VEGF and bFGF). In both the single HPMSCs/leptin and the co-culture systems of HPMSCs/leptin and irradiated HUVECs, the increased secretion of pro-inflammatory cytokines (human GM-CSF, IL-1α, and IL-6) was associated with the interaction of the NF-κB and JAK/STAT signaling pathways. We conclude that HPMSCs/leptin could promote angiogenic potential and peripheral inflammation of HUVECs after X-ray radiation.
Cell Proliferation ; Cells, Cultured ; Cytokines/biosynthesis* ; Female ; Human Umbilical Vein Endothelial Cells/radiation effects* ; Humans ; Inflammation/etiology* ; Leptin/pharmacology* ; Mesenchymal Stem Cells/physiology* ; Neovascularization, Physiologic/physiology* ; Placenta/cytology* ; Pregnancy ; STAT3 Transcription Factor/genetics* ; Transcription Factor RelA/genetics* ; X-Rays

Objective: To clarify the prevalence, clinical features and molecular characteristics of germline GATA2 mutations in pediatric primary myelodysplastic syndromes (MDS) . Methods: Next-generation sequencing technology was used to detect mutations in GATA2 and other myeloid malignancy genes in 129 children with primary MDS from Jan. 2007 to Jan. 2018. The relationship between genotypes and phenotypes was analyzed. Results: Germline GATA2 mutations accounted for 8.5% (11/129) of all primary MDS cases, and 14.0% (11/50) of MDS with excess blasts (MDS-EB) and acute myeloid leukaemia with myelodysplasia-related changes (AML-MRC) . Compared with GATA2 wild-type patients, GATA2 mutated patients were older at diagnosis[8 (1-16) years old vs 6 years old (range: 1 month old-18 years old) , P=0.035]and higher risk of monosomy 7 (72.7%vs 5.2%, P<0.001) and classified into MDS-EB and AML-MRC compared with refractory cytopenia of childhood (RCC) (63.6%vs 36.4%, P=0.111) . The multivariate analysis showed SETBP1 mutation (P=0.041, OR=9.003, 95%CI 1.098-73.787) and isolated monosomy 7 (P=0.002, OR=24.835, 95%CI 3.305-186.620) were significantly associated with germline mutated GATA2. Overall survival (OS) and outcomes of hematopoietic stem cell transplantation (HSCT) were not influenced by GATA2 mutational status. Conclusions: Our data identify germline GATA2 mutations have a high prevalence in older pediatric patients with monosomy 7, and high risk of progression into advanced MDS subtypes. GATA2 mutation status does not affect OS in pediatric primary MDS.
Adolescent ; Child ; Child, Preschool ; GATA2 Transcription Factor/genetics* ; Germ-Line Mutation ; Hematopoietic Stem Cell Transplantation ; Humans ; Infant ; Leukemia, Myeloid, Acute ; Myelodysplastic Syndromes/genetics*

To study the molecular mechanism for DNA hypomethylation of STAT3 promoter in CD4+ T cells from acute graft-versus-host disease (aGVHD) patients.
 Methods: We collected CD4+ T cells from peripheral blood of 42 patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) from HLA-identical sibling donors. GADD45A expression level in CD4+ T cells was measured by real-time PCR and Western blot. The binding level between HMGB1 and GADD45A in CD4+ T cells was analyzed by co-immunoprecipitation, while the binding levels of HMGB1/GADD45A with STAT3 promoter were detected by chromatin immunoprecipitation-quantitative real-time PCR (ChIP-qPCR). After overexpression of HMGB1 and knockdown of GADD45A in normal CD4+ T cells, STAT3 expression and DNA methylation were measured by Western blot and bisulfite sequencing PCR, respectively.
 Results: GADD45A expression was significantly up-regulated in patients with aGVHD compared with that in the patients without aGVHD. More HMGB1-GADD45A complexes were found in CD4+ T cells from patients with aGVHD compared with that in patients without aGVHD. The bindings of HMGB1/GADD45A with STAT3 promoter were significantly increased, and the binding levels of HMGB1/GADD45A were negatively correlated with STAT3 promoter DNA methylation. The expression of STAT3 was significantly reduced and the DNA methylation of STAT3 promoter was significantly increased in CD4+ T cells with overexpression of HMGB1 and knockdown of GADD45A compared with CD4+ T cells only with overexpression of HMGB1.
 Conclusion: The increased expression of HMGB1/GADD45A plays an importent role in STAT3 promoter DNA hypomethylation, thereby promoting STAT3 expression in CD4+ T cells from aGVHD patients.
CD4-Positive T-Lymphocytes ; Cell Cycle Proteins ; metabolism ; DNA Demethylation ; Gene Expression Regulation ; genetics ; Graft vs Host Disease ; genetics ; HMGB1 Protein ; metabolism ; Hematopoietic Stem Cell Transplantation ; Humans ; Nuclear Proteins ; metabolism ; Promoter Regions, Genetic ; genetics ; STAT3 Transcription Factor ; genetics ; metabolism

Objective: To investigate the survival and prognostic factors of allogeneic hematopoietic stem-cell transplantation (allo-HSCT) for patients with myeloid neoplasms and RUNX1 mutations. Methods: From July 2014 to April 2018, the clinical data of forty-two AML/MDS patients with RUNX1 mutations in the First Affiliated Hospital of Soochow University were retrospectively analyzed. The clinical characteristic features and distribution of the mutations frequently observed with RUNX1 mutations were summarized, the prognosis of allo-HSCT for these patients was also analyzed. Results: Among 42 AML/MDS patients with RUNX1 mutations, 27 were male, 15 were female. The median age was 43.5 (16-68) years old. There are 31 patients in allo-HSCT group and 11 patients in chemotherapy group. RUNX1 mutations co-occurred with many other gene mutations, the most frequent mutations were FLT3 (26.2%, 11/42) . Interestingly, FLT3 mutations only occurred in AML patients compared with MDS patients (P=0.014) . ASXL1 (25%, 3/12) mutations were observed as the most frequent co-mutations in MDS patients. One-year overall survival (OS) , disease-free survival (DFS) of allo-HSCT and chemotherapy patients were (70.6±9.0) %, (61.0±9.4) % and (34.4±16.7) %, (22.4±15.3) %, respectively. When OS and DFS between allo-HSCT and chemotherapy patients were compared, significant differences (χ(2)=4.843, 4.320, P<0.05) were showed. In univariate analysis, transplant age >45 years was a negative effect for OS [HR=4.819 (95% CI 1.145-20.283) , P=0.032] and DFS [HR=5.945 (95% CI 1.715-20.604) , P=0.005]. Also, complex chromosome karyotype abnormality was a negative effect for OS [HR=5.572 (95%CI 1.104-28.113) , P=0.038]. Conclusion: Transplant age (>45 years) and complex chromosome karyotype abnormality were negative prognostic factors in allo-HSCT for myeloid neoplasms patients with RUNX1 mutations.
Adult ; Aged ; Core Binding Factor Alpha 2 Subunit/genetics* ; Female ; Hematopoietic Stem Cell Transplantation ; Humans ; Leukemia, Myeloid, Acute/genetics* ; Male ; Middle Aged ; Mutation ; Prognosis ; Retrospective Studies ; Transplantation, Homologous

OBJECTIVESTo investigate the hair growth-promoting effect of Miscanthus sinensis var. purpurascens (MSP) flower extracton on in vitro and in vivo models.

METHODSMSP flower extract was extracted in 99.9% methanol and applied to examine the proliferation of human dermal papilla cells (hDPCs) in vitro at the dose of 3.92-62.50 μg/mL and hair growth of C57BL/6 mice in vivo at the dose of 1000 μg/mL. The expression of transforming growth factor β1 (TGF-β1), hepatocyte growth factor (HGF), β-catenin, substance P was measured by relative quantitative realtime polymerase chain reaction. Histopathological and immunohistochemical analysis were performed.

RESULTSMSP (7.81 μg/mL) down-regulated TGF-β1 and up-regulated HGF and β-catenin in hDPCs (P<0.01). MSP (1000 μg/mL)-treated mice showed the earlier transition of hair follicles from the telogen to the anagen phase. The number of mast cells was lower in the MSP-treated mice than in other groups (P<0.05 vs. NCS group). Substance P and TGF-β1 were expressed in hair follicles and skin of the MSP group lower than that in negative control. Stem cell factor in hair follicles was up-regulated in the MSP-treated mice (P<0.01).

CONCLUSIONSThe MSP flower extract may have hair growth-promotion activities.

Animals ; Antioxidants ; pharmacology ; Cell Count ; Cell Proliferation ; drug effects ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Female ; Flowers ; chemistry ; Hair Follicle ; cytology ; drug effects ; growth & development ; Hepatocyte Growth Factor ; metabolism ; Humans ; Mast Cells ; cytology ; Mice, Inbred C57BL ; Phosphorylation ; drug effects ; Plant Extracts ; pharmacology ; Poaceae ; chemistry ; RNA, Messenger ; genetics ; metabolism ; Skin ; metabolism ; Stem Cell Factor ; metabolism ; Stress, Psychological ; pathology ; Substance P ; metabolism ; Transforming Growth Factor beta ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; beta Catenin ; metabolism

Irreversible destruction of bronchi and alveoli can lead to multiple incurable lung diseases. Identifying lung stem/progenitor cells with regenerative capacity and utilizing them to reconstruct functional tissue is one of the biggest hopes to reverse the damage and cure such diseases. Here we showed that a rare population of SOX9 basal cells (BCs) located at airway epithelium rugae can regenerate adult human lung. Human SOX9 BCs can be readily isolated by bronchoscopic brushing and indefinitely expanded in feeder-free condition. Expanded human SOX9 BCs can give rise to alveolar and bronchiolar epithelium after being transplanted into injured mouse lung, with air-blood exchange system reconstructed and recipient's lung function improved. Manipulation of lung microenvironment with Pirfenidone to suppress TGF-β signaling could further boost the transplantation efficiency. Moreover, we conducted the first autologous SOX9 BCs transplantation clinical trial in two bronchiectasis patients. Lung tissue repair and pulmonary function enhancement was observed in patients 3-12 months after cell transplantation. Altogether our current work indicated that functional adult human lung structure can be reconstituted by orthotopic transplantation of tissue-specific stem/progenitor cells, which could be translated into a mature regenerative therapeutic strategy in near future.
Bronchiectasis ; genetics ; metabolism ; Humans ; Pulmonary Alveoli ; cytology ; metabolism ; SOX9 Transcription Factor ; genetics ; metabolism ; Stem Cell Transplantation ; methods ; Stem Cells ; cytology ; metabolism

PURPOSE: Angiopoietin-1 (Ang1) is a critical factor for vascular stabilization and endothelial survival via inhibition of endothelial permeability and leukocyte- endothelium interactions. Hence, we hypothesized that treatment with umbilical cord mesenchymal stem cells (UCMSCs) carrying the Ang1 gene (UCMSCs-Ang1) might be a potential approach for acute lung injury (ALI) induced by lipopolysaccharide (LPS). MATERIALS AND METHODS: UCMSCs with or without transfection with the human Ang1 gene were delivered intravenously into rats one hour after intra-abdominal instillation of LPS to induce ALI. After the rats were sacrificed at 6 hours, 24 hours, 48 hours, 8 days, and 15 days post-injection of LPS, the serum, the lung tissues, and bronchoalveolar lavage fluid (BALF) were harvested for analysis, respectively. RESULTS: Administration of fluorescence microscope confirmed the increased presence of UCMSCs in the injured lungs. The evaluation of UCMSCs and UCMSCs-Ang1 actions revealed that Ang1 overexpression further decreased the levels of the pro-inflammatory cytokines TNF-α, TGF-β1, and IL-6 and increased the expression of the anti-inflammatory cytokine IL-10 in the injured lungs. This synergy caused a substantial decrease in lung airspace inflammation and vascular leakage, characterized by significant reductions in wet/dry ratio, differential neutrophil counts, myeloperoxidase activity, and BALF. The rats treated by UCMSCs-Ang1 showed improved survival and lower ALI scores. CONCLUSION: UCMSCs-Ang1 could improve both systemic inflammation and alveolar permeability in ALI. UC-derived MSCs-based Ang1 gene therapy may be developed as a potential novel strategy for the treatment of ALI.
Acute Lung Injury/chemically induced/*therapy ; Angiopoietin-1/*genetics ; Animals ; Bronchoalveolar Lavage Fluid ; Cytokines/metabolism ; Endotoxins ; Genetic Therapy ; Interleukin-10/metabolism ; Interleukin-6/metabolism ; Leukocyte Count ; Lipopolysaccharides ; Lung/metabolism ; Male ; *Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells/metabolism ; Neutrophils/metabolism ; Rats ; Transforming Growth Factor beta1/metabolism ; Tumor Necrosis Factor-alpha/metabolism ; Umbilical Cord/*cytology