Effect of Morus alba extract sanggenon C on growth and proliferation of glioblastoma cells / 中国中药杂志

Glioblastoma is the most common primary cranial malignancy, and chemotherapy remains an important tool for its treatment. Sanggenon C(San C), a class of natural flavonoids extracted from Morus plants, is a potential antitumor herbal monomer. In this study, the effect of San C on the growth and proliferation of glioblastoma cells was examined by methyl thiazolyl tetrazolium(MTT) assay and 5-bromodeoxyuridinc(BrdU) labeling assay. The effect of San C on the tumor cell cycle was examined by flow cytometry, and the effect of San C on clone formation and self-renewal ability of tumor cells was examined by soft agar assay. Western blot and bioinformatics analysis were used to investigate the mechanism of the antitumor activity of San C. In the presence of San C, the MTT assay showed that San C significantly inhibited the growth and proliferation of tumor cells in a dose and time-dependent manner. BrdU labeling assay showed that San C significantly attenuated the DNA replication activity in the nucleus of tumor cells. Flow cytometry confirmed that San C blocked the cell cycle of tumor cells in G_0/G_1 phase. The soft agar clone formation assay revealed that San C significantly attenuated the clone formation and self-renewal ability of tumor cells. The gene set enrichment analysis(GSEA) implied that San C inhibited the tumor cell division cycle by affecting the myelocytomatosis viral oncogene(MYC) signaling pathway. Western blot assay revealed that San C inhibited the expression of cyclin through the regulation of the MYC signaling pathway by lysine demethylase 4B(KDM4B), which ultimately inhibited the growth and proliferation of glioblastoma cells and self-renewal. In conclusion, San C exhibits the potential antitumor activity by targeting the KDM4B-MYC axis to inhibit glioblastoma cell growth, proliferation, and self-renewal.

Histone demethylase JMJD3 downregulation protects against aberrant force-induced osteoarthritis through epigenetic control of NR4A1 / 国际口腔科学杂志·英文版

Osteoarthritis (OA) is a prevalent joint disease with no effective treatment strategies. Aberrant mechanical stimuli was demonstrated to be an essential factor for OA pathogenesis. Although multiple studies have detected potential regulatory mechanisms underlying OA and have concentrated on developing novel treatment strategies, the epigenetic control of OA remains unclear. Histone demethylase JMJD3 has been reported to mediate multiple physiological and pathological processes, including cell differentiation, proliferation, autophagy, and apoptosis. However, the regulation of JMJD3 in aberrant force-related OA and its mediatory effect on disease progression are still unknown. In this work, we confirmed the upregulation of JMJD3 in aberrant force-induced cartilage injury in vitro and in vivo. Functionally, inhibition of JMJD3 by its inhibitor, GSK-J4, or downregulation of JMJD3 by adenovirus infection of sh-JMJD3 could alleviate the aberrant force-induced chondrocyte injury. Mechanistic investigation illustrated that aberrant force induces JMJD3 expression and then demethylates H3K27me3 at the NR4A1 promoter to promote its expression. Further experiments indicated that NR4A1 can regulate chondrocyte apoptosis, cartilage degeneration, extracellular matrix degradation, and inflammatory responses. In vivo, anterior cruciate ligament transection (ACLT) was performed to construct an OA model, and the therapeutic effect of GSK-J4 was validated. More importantly, we adopted a peptide-siRNA nanoplatform to deliver si-JMJD3 into articular cartilage, and the severity of joint degeneration was remarkably mitigated. Taken together, our findings demonstrated that JMJD3 is flow-responsive and epigenetically regulates OA progression. Our work provides evidences for JMJD3 inhibition as an innovative epigenetic therapy approach for joint diseases by utilizing p5RHH-siRNA nanocomplexes.

Loss of KDM4B impairs osteogenic differentiation of OMSCs and promotes oral bone aging / 国际口腔科学杂志·英文版

Aging of craniofacial skeleton significantly impairs the repair and regeneration of trauma-induced bony defects, and complicates dental treatment outcomes. Age-related alveolar bone loss could be attributed to decreased progenitor pool through senescence, imbalance in bone metabolism and bone-fat ratio. Mesenchymal stem cells isolated from oral bones (OMSCs) have distinct lineage propensities and characteristics compared to MSCs from long bones, and are more suited for craniofacial regeneration. However, the effect of epigenetic modifications regulating OMSC differentiation and senescence in aging has not yet been investigated. In this study, we found that the histone demethylase KDM4B plays an essential role in regulating the osteogenesis of OMSCs and oral bone aging. Loss of KDM4B in OMSCs leads to inhibition of osteogenesis. Moreover, KDM4B loss promoted adipogenesis and OMSC senescence which further impairs bone-fat balance in the mandible. Together, our data suggest that KDM4B may underpin the molecular mechanisms of OMSC fate determination and alveolar bone homeostasis in skeletal aging, and present as a promising therapeutic target for addressing craniofacial skeletal defects associated with age-related deteriorations.

Association of JMJD3, MMP-2 and VEGF expressions with clinicopathological features of invasive ductal breast carcinoma / 南方医科大学学报

OBJECTIVE@#To examine the expressions of JMJD3, matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) in invasive ductal breast carcinoma, their association with the clinicopathological features of the patients and the effect of JMJD3 overexpression on proliferation and MMP-2 and VEGF expressions in breast cancer cells.@*METHODS@#The protein and mRNA expressions of JMJD3, MMP-2, and VEGF in invasive ductal breast carcinoma and paired adjacent tissues were detected by immunohistochemistry and RT-PCR, respectively, and their correlation with the clinicopathological characteristics of the patients was analyzed. Kaplan-Meier survival analysis was used to evaluate the correlation of JMJD3, MMP-2 and VEGF expression levels with the survival of the patients. In breast cancer MDA-MB-231 cells transfected with a JMJD3-expression plasmid, the expression of Ki67 was examined immunohistochemically, the cell proliferation was assessed with CCK8 assay, and the mRNA expressions of MMP-2 and VEGF were detected with RT-PCR.@*RESULTS@#Breast cancer tissues had significantly lower JMJD3 expression and higher MMP-2 and VEGF expressions at both the mRNA and protein levels than the adjacent tissue (@*CONCLUSIONS@#The expressions of JMJD3, MMP-2 and VEGF in invasive ductal breast carcinoma are closely correlated to tumor proliferation, invasion, metastasis and prognosis and can be used for prognostic evaluation of breast cancer.

Expression Levels of JARID1B, Hes1 and MMP-9 Genes in CML Patients Treated with Imatinib Mesylate / 中国实验血液学杂志

OBJECTIVE@#To explore the relationship between the expression levels of JARID1B,Hes1 and MMP-9 genes and the stages of chronic myelogenous leukemia(CML) and the curative effect of imatinib mesylate (IM).@*METHODS@#Peripheral blood samples of 15 cases of CML in chronic phase and 10 cases of CML in progressive phase were collected from the Hematology Department of Taihe Hospital affiliated to Hubei University of Medicine and 15 cases of healthy people in the Physical Examination Center. CML patients were divided into effective group and ineffective group based on the efficacy after treatment with IM, then real-time PCR was used to detect the expression levels of JARID1B, Hes1 and MMP-9 mRNA, finally, the differences in the level of gene expression and their correlations with CML stages and IM curative efficacy were analysed.@*RESULTS@#The expression levels of Hes1 and MMP-9 in initially diagnosed patients in chronic and progressive phase without IM treatment were significantly higher than those of health people（P＜0.05）. There was no significant difference in the expression level of JARID1B between chronic phase patients and health people（P＞0.05）, but the expression level of JARID1B in the progressive phase patients was higher than that of health people （P＜0.05）. The expression levels of JARID1B and Hes1 in the IM-effective group were not significantly different from those in the IM-ineffective group (P=0.85,P=0.82）, while the expression level of MMP-9 in the IM-effective group [JP2]was significantly lower than that in the IM-ineffective group（P＜0.05）.@*CONCLUSION@#The expression levels of JARID1B Hes1 and MMP-9 relate with the different phase of CML; The expression levels of JARID1B and Hes1 have not significant relationship with IM curative efficacy, the MMP-9 gene expression level relates with IM curative efficacy.

Histone methyltransferases and demethylases: regulators in balancing osteogenic and adipogenic differentiation of mesenchymal stem cells / 国际口腔科学杂志·英文版

Mesenchymal stem cells (MSCs) are characterized by their self-renewing capacity and differentiation potential into multiple tissues. Thus, management of the differentiation capacities of MSCs is important for MSC-based regenerative medicine, such as craniofacial bone regeneration, and in new treatments for metabolic bone diseases, such as osteoporosis. In recent years, histone modification has been a growing topic in the field of MSC lineage specification, in which the Su(var)3-9, enhancer-of-zeste, trithorax (SET) domain-containing family and the Jumonji C (JmjC) domain-containing family represent the major histone lysine methyltransferases (KMTs) and histone lysine demethylases (KDMs), respectively. In this review, we summarize the current understanding of the epigenetic mechanisms by which SET domain-containing KMTs and JmjC domain-containing KDMs balance the osteogenic and adipogenic differentiation of MSCs.

Bivalent histone modifications during tooth development / 国际口腔科学杂志·英文版

Histone methylation is one of the most widely studied post-transcriptional modifications. It is thought to be an important epigenetic event that is closely associated with cell fate determination and differentiation. To explore the spatiotemporal expression of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3) epigenetic marks and methylation or demethylation transferases in tooth organ development, we measured the expression of SET7, EZH2, KDM5B and JMJD3 via immunohistochemistry and quantitative polymerase chain reaction (qPCR) analysis in the first molar of BALB/c mice embryos at E13.5, E15.5, E17.5, P0 and P3, respectively. We also measured the expression of H3K4me3 and H3K27me3 with immunofluorescence staining. During murine tooth germ development, methylation or demethylation transferases were expressed in a spatial-temporal manner. The bivalent modification characterized by H3K4me3 and H3K27me3 can be found during the tooth germ development, as shown by immunofluorescence. The expression of SET7, EZH2 as methylation transferases and KDM5B and JMJD3 as demethylation transferases indicated accordingly with the expression of H3K4me3 and H3K27me3 respectively to some extent. The bivalent histone may play a critical role in tooth organ development via the regulation of cell differentiation.

The PHD1 finger of KDM5B recognizes unmodified H3K4 during the demethylation of histone H3K4me2/3 by KDM5B

KDM5B is a histone H3K4me2/3 demethylase. The PHD1 domain of KDM5B is critical for demethylation, but the mechanism underlying the action of this domain is unclear. In this paper, we observed that PHD1KDM5B interacts with unmethylated H3K4me0. Our NMR structure of PHD1KDM5B in complex with H3K4me0 revealed that the binding mode is slightly different from that of other reported PHD fingers. The disruption of this interaction by double mutations on the residues in the interface (L325A/D328A) decreases the H3K4me2/3 demethylation activity of KDM5B in cells by approximately 50% and increases the transcriptional repression of tumor suppressor genes by approximately twofold. These findings imply that PHD1KDM5B may help maintain KDM5B at target genes to mediate the demethylation activities of KDM5B.

Expression of JMJD2A in infiltrating duct carcinoma was markedly higher than fibroadenoma, and associated with expression of ARHI, p53 and ER in infiltrating duct carcinoma.

Jumonji Domain Containing 2A (JMJD2A) may be a cancer-associated gene involved in human breast cancer. With a view to investigating expression of JMJD2A in human breast cancer and benign lesion tissues as well as relationship between JMJD2A and tumor related proteins, histological and immunohistochemical analysis, Western blot and quantitative real-time PCR in infiltrating duct carcinoma and fibroadenoma for JMJD2A and immunohistochemical analysis and quantitative real-time PCR in infiltrating duct carcinoma for tumor related proteins (ARHI, p53, ER, PR and CerbB-2) were performed. Histological examination validated the clinical diagnosis. The JMJD2A positive rate of infiltrating duct carcinoma was significantly higher than fibroadenoma by immunohistochemical analysis. The mean optical density of JMJD2A in infiltrating duct carcinoma was higher than fibroadenoma by western blot. JMJD2A mRNA level in infiltrating duct carcinoma was higher than fibroadenoma by quantitative real-time PCR. Spearman correlation analysis revealed that the expression of JMJD2A was associated with ARHI, p53 and ER from immunohistochemical results respectively. Pearson correlation analysis revealed that the expression of JMJD2A was associated with ARHI, p53 and ER from quantitative real-time PCR results respectively. Expression of JMJD2A in infiltrating duct carcinoma was higher, and associated with ARHI, p53 and ER. The results may take JMJD2A as a potential diagnostic and therapeutic target in human breast cancer.

ICSI causes abnormal H3K9 methylation in the male pronuclei and growth retardation of mouse embryos / 中华男科学杂志

<p><b>OBJECTIVE</b>To evaluate the safety of intracytoplasmic sperm injection (ICSI) in the mouse model.</p><p><b>METHODS</b>We simulated clinical ICSI technology and comprehensively evaluated it by parthenogenetic activation, immunofluorescence, embryo transplantation, examination of early implantation, and measurement of the crown-rump length (CRL).</p><p><b>RESULTS</b>ICSI significantly reduced the ability of preimplantation embryo development of the mouse, especially after the 8-cell stage (P < 0.01). The fluorescence of H3K9 dimethylation was abnormal at the male pronuclei of the embryos derived from ICSI. Further examination of the development of the transferred ICSI embryos indicated no significant difference in the rate of early implantation at E5. 5 days as compared with normal fertilization (P = 0.6), but the percentage of "normal embryos" was decreased significantly at E9.5 days (P < 0.01). Obvious growth retardation phenotype was observed even in the normal ICSI embryos at E9.5 days.</p><p><b>CONCLUSION</b>ICSI might result in growth retardation of embryos by affecting H3K9 dimethylation in the male pronuclei.</p>

KDM6B epigenetically regulates odontogenic differentiation of dental mesenchymal stem cells / 国际口腔科学杂志·英文版

Mesenchymal stem cells (MSCs) have been identified and isolated from dental tissues, including stem cells from apical papilla, which demonstrated the ability to differentiate into dentin-forming odontoblasts. The histone demethylase KDM6B (also known as JMJD3) was shown to play a key role in promoting osteogenic commitment by removing epigenetic marks H3K27me3 from the promoters of osteogenic genes. Whether KDM6B is involved in odontogenic differentiation of dental MSCs, however, is not known. Here, we explored the role of KDM6B in dental MSC fate determination into the odontogenic lineage. Using shRNA-expressing lentivirus, we performed KDM6B knockdown in dental MSCs and observed that KDM6B depletion leads to a significant reduction in alkaline phosphate (ALP) activity and in formation of mineralized nodules assessed by Alizarin Red staining. Additionally, mRNA expression of odontogenic marker gene SP7 (osterix, OSX), as well as extracellular matrix genes BGLAP (osteoclacin, OCN) and SPP1 (osteopontin, OPN), was suppressed by KDM6B depletion. When KDM6B was overexpressed in KDM6B-knockdown MSCs, odontogenic differentiation was restored, further confirming the facilitating role of KDM6B in odontogenic commitment. Mechanistically, KDM6B was recruited to bone morphogenic protein 2 (BMP2) promoters and the subsequent removal of silencing H3K27me3 marks led to the activation of this odontogenic master transcription gene. Taken together, our results demonstrated the critical role of a histone demethylase in the epigenetic regulation of odontogenic differentiation of dental MSCs. KDM6B may present as a potential therapeutic target in the regeneration of tooth structures and the repair of craniofacial defects.

The effects of JARID1B siRNA on proliferation and apoptosis in HL-60 cell / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the effect of small interfering RNA(siRNA) targeting JARID1B gene on the proliferation and apoptosis in HL-60 acute promyelocytic leukemia cell line, and to explore its mechanisms.</p><p><b>METHODS</b>The JARID1B siRNA was transfected into HL-60 cells using Lipofectamine(TM) 2000(Lipo) vector. The proliferation inhibition by siRNA targeting JARID1B was detected by MTT, cells apoptosis by flow cytometry, the mRNA expression of JARID1B by RT-PCR, the protein expression of JARID1B, Bcl-2, procaspase-9, procaspase-3, c-myc and P27 and histone methylated H3K4 by Western blot.</p><p><b>RESULTS</b>siRNA targeting JARID1B upregulated histone methylated H3K4 level, inhibited the proliferation of HL-60 cells, and induced the cells apoptosis. After transfection of siRNA targeting JARID1B at 0, 30, 60, 120 nmmol/L for 24 hours, the apoptotic rate were (11.0 ± 3.6)%, (35.2 ± 5.1)%, (52.7 ± 3.8)%, and (62.0 ± 5.7)% respectively (F = 70.27, P < 0.01). The protein expression of P27 was upregulated, and Bcl-2, procaspase-9, procaspase-3, c-myc was down regulated.</p><p><b>CONCLUSIONS</b>JARID1B siRNA upregulates histone methylated H3K4. It reduces HL-60 cells proliferation and apoptosis by up regulating the p27 expression and down regulating the Bcl-2, procaspase-9, procaspase-3, c-myc expression. It might be a new therapeutic targeting for human leukemia.</p>

Effect of Jumonji domain-containing protein-3 on the proliferation and migration of lung cancer cell line / 生物医学工程学杂志

For investigating the effect of Jumonji domain-containing protein-3 (JMJD3) on the behavior of lung cancer cell line, A549 proliferation was measured with EDU staining and flow cytometer after JMJD3 expression plasmid and pcDNA3. 1 transfection at 48h. The migration ability of A549 was tested at the same time. The expression of p21 mRNA was measured with RT-PCR. The results showed that JMJD3 transfection increased the EDU positive cells ratio (JMJD3: 40.75% +/- 2.07%, control: 20.97% +/- 1.5%, P < 0.001). G1 phase cell ration also increased after JMJD3 transfection (JMJD3:47. 80% +/- 1.85%, control: 54.60% +/- 0.95%, P = 0.005). The mRNA expression of p21 decreased in JMJD3 group (JMJD3: 35. 89% +/- 3.71%, control: 91.78% +/- 3.74%, P < 0.001). The distances of migration were (0.47 +/- 0.27) cm and (0.96 +/- 0.40) cm after 24h and 48h with JMJD3 tranfection, compared to (0.57 +/- 0.22)cm and (1.08 +/- 0.33)cm in control, respectively (P > 0.05). JMJD3 promoted the proliferation of A549 and decreased the G1 cell numbers, decreased the p21 mRNA, but had no effect on A549 migration.