OBJECTIVE: To investigate the effects of melatonin (MT) on bone mass and serum inflammatory factors in rats received ovariectomy (OVX) and to investigate the effects of MT on the levels of inflammatory factors in culture medium and osteogenic ability of bone marrow mesenchymal stem cells (BMSCs) stimulated by lipopolysaccharide. METHODS: Fifteen 12-week-old Sprague Dawley (SD) rats were randomly divided into 3 groups. The rats in Sham group only received bilateral lateral abdominal incision and suture, the rats in OVX group received bilateral OVX, and the rats in OVX+MT group received 100 mg/(kg·d) MT oral intervention after bilateral OVX. After 8 weeks, the levels of serum inflammatory factors [interleukin-1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α)] were detected using ELISA assay. Besides, the distal femurs were detected by Micro-CT to observe changes in bone mass and microstructure, and quantitatively measured bone volume fraction, trabecular thickness, and trabecular number. The BMSCs were extracted from the femurs of three 3-week-old SD rats using whole bone marrow culture method and passaged. The 3rd-5th passage BMSCs were cultured with different concentrations of MT (0, 1, 10, 100, 1 000 µmol/L), and the cell viability was then detected using cell counting kit 8 (CCK-8) to select the optimal concentration of MT for subsequent experiments. Cells were devided into osteogenic induction group (group A) and osteogenic induction+1/5/10 μg/mL lipopolysaccharide group (group B-D). The levels of inflammatory factors (IL-1β, IL-6 and TNF-α) in cell culture medium were detected using ELISA assay after corresponding intervention. According to the results of CCK-8 method and ELISA detection, the cells were intervened with the most significant concentration of lipopolysaccharide for stimulating inflammation and the optimal concentration of MT with osteogenic induction, defining as group E, and the cell culture medium was collected to detect the levels of inflammatory factors by ELISA assay. After that, alkaline phosphatase (ALP) staining and alizarin red staining were performed respectively in groups A, D, and E, and the expression levels of osteogenic related genes [collagen type Ⅰ alpha 1 chain (Col1a1) and RUNX family transcription factor 2 (Runx2)] were also detected by real time fluorescence quantitative PCR (RT-qPCR). RESULTS: ELISA and Micro-CT assays showed that compared with Sham group, the bone mass of the rats in the OVX group significantly decreased, and the expression levels of serum inflammatory factors (IL-1β, IL-6, and TNF-α) in OVX group significantly increased (P<0.05). Significantly, the above indicators in OVX+MT group were all improved (P<0.05). Rat BMSCs were successfully extracted, and CCK-8 assay showed that 100 µmol/L was the maximum concentration of MT that did not cause a decrease in cell viability, and it was used in subsequent experiments. ELISA assays showed that compared with group A, the expression levels of inflammatory factors (IL-1β, IL-6, and TNF-α) in the cell culture medium of groups B-D were significantly increased after lipopolysaccharide stimulation (P<0.05), and in a concentration-dependent manner. Moreover, the expression levels of inflammatory factors in group D were significantly higher than those in groups B and C (P<0.05). After MT intervention, the expression levels of inflammatory factors in group E were significantly lower than those in group D (P<0.05). ALP staining, alizarin red staining, and RT-qPCR assays showed that compared with group A, the percentage of positive area of ALP and alizarin red and the relative mRNA expressions of Col1a1 and Runx2 in group D significantly decreased, while the above indicators in group E significantly improved after MT intervention (P<0.05). CONCLUSION MT may affect the bone mass of postmenopausal osteoporosis by reducing inflammation in rats; MT can reduce the inflammation of BMSCs stimulated by lipopolysaccharide and weaken its inhibition of osteogenic differentiation of BMSCs.
Female ; Rats ; Animals ; Tumor Necrosis Factor-alpha ; Osteogenesis ; Rats, Sprague-Dawley ; Core Binding Factor Alpha 1 Subunit ; Melatonin/pharmacology* ; Interleukin-6/genetics* ; Lipopolysaccharides/pharmacology* ; Coloring Agents ; Inflammation

Objective: To analyze the clinical features, efficacy and prognosis factors of core binding factor (CBF) acute myeloid leukemia (AML) children in South China. Methods: This was a retrospective cohort study. Clinical data of 584 AML patients from 9 hospitals between January 2015 to December 2020 was collected. According to fusion gene results, all patients were divided into two groups: CBF-AML group (189 cases) and non-CBF-AML group (395 cases). CBF-AML group were divided into AML1-ETO subgroup (154 cases) and CBFβ-MYH11 subgroup (35 cases). Patients in CBF-AML group chosen different induction scheme were divided into group A (fludarabine, cytarabine, granulocyte colony stimulating factor and idarubicin (FLAG-IDA) scheme, 134 cases) and group B (daunorubicin, cytarabine and etoposide (DAE) scheme, 55 cases). Age, gender, response rate, recurrence rate, mortality, molecular genetic characteristics and other clinical data were compared between groups. Kaplan-Meier method was used for survival analysis and survival curve was drawn. Cox regression model was used to analyze prognostic factors. Results: A total of 584 AML children were diagnosed, including 346 males and 238 females. And a total of 189 children with CBF-AML were included, including 117 males and 72 females. The age of diagnosis was 7.3 (4.5,10.0)years, and the white blood cell count at initial diagnosis was 21.4 (9.7, 47.7)×10⁹/L.The complete remission rate of the first course (CR1) of induction therapy, relapse rate, and mortality of children with CBF-AML were significantly different from those in the non-CBF-AML group (91.0% (172/189) vs. 78.0% (308/395); 10.1% (19/189) vs. 18.7% (74/395); 13.2% (25/189) vs. 25.6% (101/395), all P<0.05). In children with CBF-AML, the CBFβ-MYH11 subgroup had higher initial white blood cells and lower proportion of extramedullary invasion than the AML1-ETO subgroup, with statistical significance (65.7% (23/35) vs. 14.9% (23/154), 2.9% (1/35) vs. 16.9% (26/154), both P<0.05). AML1-ETO subgroup had more additional chromosome abnormalities (75/154), especially sex chromosome loss (53/154). Compared with group B, group A had more additional chromosome abnormalities and a higher proportion of tumor reduction regimen, with statistical significance (50.0% (67/134) vs. 29.1% (16/55), 34.3% (46/134) vs. 18.2% (10/55), both P<0.05). Significant differences were found in 5-years event free survival (EFS) rate and 5-year overall survival (OS) rate between CBF-AML group and non-CBF-AML group ((77.0±6.4)%vs. (61.9±6.7)%,(83.7±9.0)%vs. (67.3±7.2)%, both P<0.05).EFS and OS rates of AML1-ETO subgroup and CBFβ-MYH11 subgroup in children with CBF-AML were not significantly different (both P>0.05). Multivariate analysis showed in the AML1-ETO subgroup, CR1 rate and high white blood cell count (≥50×10⁹/L) were independent risk factors for EFS (HR=0.24, 95%CI 0.07-0.85,HR=1.01, 95%CI 1.00-1.02, both P<0.05) and OS (HR=0.24, 95%CI 0.06-0.87; HR=1.01, 95%CI 1.00-1.02; both P<0.05). Conclusions: In CBF-AML, AML1-ETO is more common which has a higher extramedullary involvement and additional chromosome abnormalities, especially sex chromosome loss. The prognosis of AML1-ETO was similar to that of CBFβ-MYH11. The selection of induction regimen group FLAG-IDA for high white blood cell count and additional chromosome abnormality can improve the prognosis.
Male ; Female ; Humans ; Child ; Retrospective Studies ; RUNX1 Translocation Partner 1 Protein/genetics* ; Core Binding Factor Alpha 2 Subunit/therapeutic use* ; Prognosis ; Leukemia, Myeloid, Acute/genetics* ; Cytarabine/therapeutic use* ; Oncogene Proteins, Fusion/genetics* ; Chromosome Aberrations

Objective: To investigate the effect of the AML1-ETO (AE) fusion gene on the biological function of U937 leukemia cells by establishing a leukemia cell model that induces AE fusion gene expression. Methods: The doxycycline (Dox) -dependent expression of the AE fusion gene in the U937 cell line (U937-AE) were established using a lentivirus vector system. The Cell Counting Kit 8 methods, including the PI and sidanilide induction, were used to detect cell proliferation, cell cycle-induced differentiation assays, respectively. The effect of the AE fusion gene on the biological function of U937-AE cells was preliminarily explored using transcriptome sequencing and metabonomic sequencing. Results: ①The Dox-dependent Tet-on regulatory system was successfully constructed to regulate the stable AE fusion gene expression in U937-AE cells. ②Cell proliferation slowed down and the cell proliferation rate with AE expression (3.47±0.07) was lower than AE non-expression (3.86 ± 0.05) after inducing the AE fusion gene expression for 24 h (P<0.05). The proportion of cells in the G(0)/G(1) phase in the cell cycle increased, with AE expression [ (63.45±3.10) %) ] was higher than AE non-expression [ (41.36± 9.56) %] (P<0.05). The proportion of cells expressing CD13 and CD14 decreased with the expression of AE. The AE negative group is significantly higher than the AE positive group (P<0.05). ③The enrichment analysis of the transcriptome sequencing gene set revealed significantly enriched quiescence, nuclear factor kappa-light-chain-enhancer of activated B cells, interferon-α/γ, and other inflammatory response and immune regulation signals after AE expression. ④Disorder of fatty acid metabolism of U937-AE cells occurred under the influence of AE. The concentration of the medium and short-chain fatty acid acylcarnitine metabolites decreased in cells with AE expressing, propionyl L-carnitine, wherein those with AE expression (0.46±0.13) were lower than those with AE non-expression (1.00±0.27) (P<0.05). The metabolite concentration of some long-chain fatty acid acylcarnitine increased in cells with AE expressing tetradecanoyl carnitine, wherein those with AE expression (1.26±0.01) were higher than those with AE non-expression (1.00±0.05) (P<0.05) . Conclusion: This study successfully established a leukemia cell model that can induce AE expression. The AE expression blocked the cell cycle and inhibited cell differentiation. The gene sets related to the inflammatory reactions was significantly enriched in U937-AE cells that express AE, and fatty acid metabolism was disordered.
Humans ; U937 Cells ; RUNX1 Translocation Partner 1 Protein ; Leukemia/genetics* ; Core Binding Factor Alpha 2 Subunit/genetics* ; Oncogene Proteins, Fusion/genetics* ; Leukemia, Myeloid, Acute/genetics*

OBJECTIVE: To explore the clinical phenotypes and genetic diagnosis of 2 sporadic cases for cleidocranial dysplasia. METHODS: The clinical data of two cases of CCD admitted to the Third Affiliated Hospital of Zhengzhou University on December 16, 2021 and December 9, 2021 were analyzed retrospectively, and the whole exome sequencing (WES), chromosome microarray analysis and copy number variation sequencing were performed. RESULTS: The main ultrasonographic findings of the fetus had included poorly calcified skull bones, budging of parieto-occipital area, compression and deformation of skull, and loss of nasal bone. The infant's clinical phenotypes included delayed closure of anterior fontanelle, recurrent respiratory tract infection, growth retardation, and clavicular hypoplasia. By WES analysis, the fetus was found to harbor a heterozygous c.911_914delinsTTT variant of the RUNX2 gene, whilst the infant was found to harbor a heterozygous c.1008delT variant of the RUNX2 gene. Both variants were verified by Sanger sequencing to have occurred de novo. CONCLUSION For sporadic cases featuring cleidocranial dysplasia, prenatal ultrasonography is particularly important. Hypoplastic clavicle, skull calcification and nasal bone absence are the main features. Diagnosis should also be suspected for infants featuring growth retardation, recurrent respiratory tract infections and clavicular dysplasia. The identification of the c.911_914delinsTTT and c.1008delT variants of the RUNX2 gene has facilitated genetic counseling and prenatal diagnosis, and also expanded the mutational spectrum of the RUNX2 gene.
Female ; Humans ; Pregnancy ; Cleidocranial Dysplasia/genetics* ; Core Binding Factor Alpha 1 Subunit ; DNA Copy Number Variations ; Growth Disorders ; Retrospective Studies

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*

Humans ; Core Binding Factor Alpha 2 Subunit/genetics* ; Translocation, Genetic ; Leukemia, Myeloid, Acute/genetics* ; Prognosis ; High-Throughput Nucleotide Sequencing ; RUNX1 Translocation Partner 1 Protein/genetics* ; Oncogene Proteins, Fusion/genetics*

OBJECTIVE: To investigate the effect of RUNX2 gene overexpression vector modified exosomes derived from bone marrow mesenchymal stem cells (BMSCs) combined with calcium carbonate scaffold system in bone defect. METHODS: Rabbit BMSCs were used as the research object, and BMSCs were identified by flow cytometry. Construct RUNX2 gene overexpression vector, transfect BMSCs with lentivirus, and collect exosomes by ultracentrifugation. The morphology of exosomes was observed by transmission electron microscope, the expression of exosome marker CD63 was detected by Western blot, and the calcium carbonate scaffold was constructed by three chamber parallel automatic temperature control reaction system. According to whether the RUNX2 gene overexpression vector was transfected or not, the complex of BMSCs and calcium carbonate scaffold was divided into three groups, namely BMSCs group, RUNX2 overexpression group and exosome group. The osteogenic differentiation of BMSCs was detected by oil red O staining and RT-PCR. There were 9 clean adult healthy male New Zealand white rabbits, aged (12.97±1.21) months, with a body weight of (19.3±3.6) kg, with 3 rabbits in each group. The animal model of skull defect was constructed by surgical method, and the repair of bone defect was evaluated by imaging, he staining and Masson staining. RESULTS: The results of flow cytometry showed that the expression of CD29 protein, CD44 protein, CD11b protein and CD45 protein on the surface of BMSCs were 99.5%, 100%, 0.1% and 0.1%, respectively. Transmission electron microscopy showed that the exosomes were bilayer vesicles with a diameter of 50 to 150 nm. Western blot showed that the molecular marker CD63 of exosomes was positive. Oil red O staining showed that the osteogenic differentiation of BMSCs in exosome group was significantly higher than that in RUNX2 overexpression group and BMSCs group. The results of RT-PCR showed that the relative expressions of RUNX2, BMP-2 and ALP mRNA in BMSCs in exosome group were significantly higher than those in RUNX2 overexpression group and BMSCs group (P<0.05). The imaging results showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group. HE staining and Masson staining showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group (P<0.05). MSCs in exosome group was significantly higher than that in RUNX2 overexpression group and BMSCs group. The results of RT-PCR showed that the relative expressions of RUNX2, BMP-2 and ALP mRNA in BMSCs in exosome group were significantly higher than those in RUNX2 overexpression group and BMSCs group(P<0.05). The imaging results showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group. HE staining and Masson staining showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group(P<0.05). CONCLUSION Compared with RUNX2 gene overexpression vector transfection, extraction of exosomes directly can promote the differentiation of BMSCs into osteoblasts more efficiently, and the combination with calcium carbonate scaffold can better promote the healing of bone defects. So as to provide new ideas and methods for the clinical treatment of bone defects.
Animals ; Calcium Carbonate/metabolism* ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Exosomes/metabolism* ; Humans ; Male ; Osteogenesis/genetics* ; RNA, Messenger/metabolism* ; Rabbits

OBJECTIVE: To detect the genetic variant of a child with cleidocranial dysplasia (CCD) and to find out the causation of the illness. METHODS: Gene variant was identified by the second generation targeted sequencing and Sanger sequencing. RESULTS: The gene sequencing revealed that the RUNX2 gene had c.196C>T(p.Glu66*) nonsense variant, which was predicted to be a pathogenic variant according to the ACMG guidelines(PVS1+PS2). CONCLUSION The variant of c.196C > T in the RUNX2 gene may be the cause of the child with CCD, and the novel variant enriches the RUNX2 gene variant spectrum.
Asians/genetics* ; Child ; China ; Cleidocranial Dysplasia/genetics* ; Core Binding Factor Alpha 1 Subunit/genetics* ; Humans ; Mutation

OBJECTIVE: To analyze the clinical features and pathogenic variant in a Chinese pedigree affected with cleidocranial dysplasia (CCD). METHODS: Clinical data of 8 patients from the pedigree was collected, including physical examination and X-ray images of head, face, spine, limbs, and mouth. Peripheral blood samples were collected from 6 affected members for the extraction of genomic DNA. The proband and other 3 patients were subjected to trio-whole exome sequencing. Candidate variant was verified by Sanger sequencing of the other 2 affected members from the pedigree. RESULTS: This pedigree has included 22 members (8 affected) from four generations. Genetic testing revealed that the proband has harbored a novel pathogenic variant of the RUNX2 gene [NM_001024630: c.1268_1277del (p.P425Afs*56)], which was inherited from her mother and carried by all affected members in the pedigree. The same variant was not detected among the unaffected members, suggesting co-segregation with the phenotype. CONCLUSION The c.1268_1277del (p.P425Afs*56) variant of the RUNX2 gene probably underlay the pathogenesis of CCD in this pedigree. Genetic testing has facilitated the definite diagnosis and enabled prenatal diagnosis.
Humans ; Pregnancy ; Female ; Cleidocranial Dysplasia/genetics* ; Pedigree ; Core Binding Factor Alpha 1 Subunit/genetics* ; Phenotype ; China ; Mutation

OBJECTIVE: To construct a luciferase reporter gene vector carrying human nuclear factor of activated T cells 2 (NFATc2) gene promoter and examine the effects of metformin and lipopolysaccharide (LPS) on the transcriptional activity of NFATc2 gene. METHODS: The promoter sequence of human NFATc2 gene was acquired from UCSC website for PCR amplification. NFATc2 promoter fragment was inserted into pGL3-basic plasmid double cleaved with Kpn Ⅰ and Hind Ⅲ. The resultant recombinant plasmid pGL3-NFATC2-promoter was co-transfected with the internal reference plasmid pRL-TK in 293F cells, and luciferase activity in the cells was detected. Reporter gene vectors of human NFATc2 gene promoter with different fragment lengths were also constructed and assayed for luciferase activity. The changes in transcription activity of NFATc2 gene were assessed after treatment with different concentrations of metformin and LPS for 24 h. We also examined the effect of mutation in RUNX2-binding site in NFATC2 gene promoter on the regulatory effects of metformin and LPS on NFATc2 transcription. RESULTS: We successfully constructed pGL3-NFATc2-promoter plasmids carrying different lengths (2170 bp, 2077 bp, 1802 bp, 1651 bp, 1083 bp, 323 bp) of NFATc2 promoter sequences as verified by enzymatic digestion and sequencing. Transfection of 293F cells with the plasmid carrying a 1651 bp NFATc2 promoter (pGL3-1651 bp) resulted in the highest transcriptional activity of NFATc2 gene, and the luciferase activity was approximately 3.3 times that of pGL3-2170 bp (1.843 ± 0.146 vs 0.547 ± 0.085). Moderate (5 mmol/L) and high (10 mmol/L) concentrations of metformin significantly upregulated the transcriptional activity of pGL3-1651 bp by up to 2.5 and 3 folds, respectively. LPS at different doses also upregulated the transcriptional activity of pGL3-1651 bp by at least 1.6 folds. The mutation in the RUNX2 binding site on pGL3-1651 bp obviously reduced metformin- and LPS-induced enhancement of pGL3-1651bp transcription by 1.7 and 2 folds, respectively. CONCLUSION pGL3-NFATc2-promoter can be transcribed and activated in 293F cells, and LPS and metformin can activate the transcription of pGL3- NFATc2-promoter in a RUNX2-dependent manner.
Core Binding Factor Alpha 1 Subunit/genetics* ; Humans ; Lipopolysaccharides/pharmacology* ; Luciferases/genetics* ; Metformin/pharmacology* ; NFATC Transcription Factors/genetics* ; Promoter Regions, Genetic ; T-Lymphocytes ; Transcription, Genetic/drug effects* ; Transfection