Super-enhancer-driven LncRNA PPARα-seRNA exacerbates glucolipid metabolism and diabetic cardiomyopathy via recruiting KDM4B.

OBJECTIVE: Aberrant glucolipid metabolism in the heart is a characteristic factor in diabetic cardiomyopathy (DbCM). Super-enhancers-driven noncoding RNAs (seRNAs) are emerging as powerful regulators in the progression of cardiac diseases. However, the functions of seRNAs in DbCM have not been fully elucidated. METHODS: Super enhancers and their associated seRNAs were screened and identified by H3K27ac ChIP-seq data in the Encyclopedia of DNA Elements (ENCODE) dataset. A dual-luciferase reporter assay was performed to analyze the function of super-enhancers on the transcription of peroxisome proliferator-activated receptor α-related seRNA (PPARα-seRNA). A DbCM mouse model was established using db/db leptin receptor-deficient mice. Adeno-associated virus serotype 9-seRNA (AAV9-seRNA) was injected via the tail vein to evaluate the role of seRNA in DbCM. The underlying mechanism was explored through RNA pull-down, RNA and chromatin immunoprecipitation, and chromatin isolation by RNA purification. RESULTS: PPARα-seRNA was regulated by super-enhancers and its levels were increased in response to high glucose and palmitic acid stimulation in cardiomyocytes. Functionally, PPARα-seRNA overexpression aggravated lipid deposition, reduced glucose uptake, and repressed energy production. In contrast, PPARα-seRNA knockdown ameliorated metabolic disorder in vitro. In vivo, overexpression of PPARα-seRNA exacerbated cardiac metabolic disorder and deteriorated cardiac dysfunction, myocardial fibrosis, and hypertrophy in DbCM. Mechanistically, PPARα-seRNA bound to the histone demethylase KDM4B (Lysine-specific demethylase 4B) and decreased H3K9me3 levels in the promoter region of PPARα, ultimately enhancing its transcription. CONCLUSIONS: Our study revealed the pivotal function of a super-enhancer-driven long noncoding RNA (lncRNA), PPARα-seRNA, in the deterioration of cardiac function and the exacerbation of metabolic abnormalities in diabetic cardiomyopathy, which recruited KDM4B to the promoter region of PPARα and repression of its transcription. This suggests a promising therapeutic strategy for the treatment of DbCM.

circBRAF promotes the progression of triple-negative breast cancer through modulating methylation by recruiting KDM4B to histone H3K9me3 and IGF2BP3 to mRNA.

Understanding the molecular characteristics of triple-negative breast cancer (TNBC) and developing more tailored treatment approaches is crucial. Circular RNAs (circRNAs), as potential therapeutic targets, remain largely unexplored in TNBC. This study utilized circRNA microarray analysis to determine the expression of circRNAs in TNBC, analyzing nine patient specimens. The characteristics of circBRAF were examined using divergent PCR primers, Sanger sequencing, fluorescence in situ hybridization (FISH) analysis, and the application of RNase and actinomycin D. The biological function of circBRAF in TNBC was further investigated through colony formation, tube formation, and transwell assays. Crucially, the mechanisms underlying the effects of circBRAF on TNBC progression were explored via RNA immunoprecipitation sequencing (RIP-seq) data, MS2 pulldown, RNA sequencing (RNA-seq) analysis, circBRAF knockdown, histone H3K9me3 modification, and Chromatin Isolation by RNA Purification (ChIRP) tests followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We focused particularly on hsa_circ_0007178, produced from exons 4-13 of the oncogene BRAF. Functional experiments revealed that circBRAF is crucial for the development of TNBC, with its knockdown preventing angiogenesis, metastasis, and cell division in vitro. Mechanistically, circBRAF interacts with KDM4B and IGF2BP3, promoting TNBC growth. Interaction of circBRAF with IGF2BP3 increased the expression of VCAN, FN1, CDCA3, or B4GALT3 by controlling mRNA stability through RNA N6-methyladenosine (m6A) modification. Furthermore, circBRAF upregulated the expression of ADAMTS14 and MMP9 through recruitment of KDM4B to enhance respective H3K9me3 modification. Furthermore, overexpression of circBRAF was able to overcome the inhibitory effects of siKDM4B and siIGF2BP3 on cell migration and invasion. Our findings suggest that circBRAF may act as an oncogene in TNBC through its specific interactions with KDM4B and IGF2BP3, implying that circBRAF could serve as a potentially effective novel therapeutic target for TNBC.

Deubiquitinase UCHL1 stabilizes KDM4B to augment VEGF signaling and confer bevacizumab resistance in clear cell renal cell carcinoma.

BACKGROUND: Bevacizumab resistance poses barriers to targeted therapy in clear cell renal cell carcinoma (ccRCC). Whether there exist epigenetic targets that modulate bevacizumab sensitivity in ccRCC remains indefinite. The focus of this study is to explore the role of UCHL1 in ccRCC. METHODS: Both in vitro and in vivo experiments were utilized to investigate the roles of UCHL1 in ccRCC. In vivo ubiquitination assays were performed to validate the posttranslational modification of KDM4B by UCHL1. Luciferase reporter and chromatin immunoprecipitation (ChIP) assays were utilized to explore KDM4B/VEGFA epigenetic regulations. RESULTS: UCHL1 was increased in ccRCC and associated with unfavorable survival outcomes in patients. UCHL1 was required for ccRCC growth and migration. Mechanistically, the wild-type UCHL1, but not C90A mutant, mediated the deubiquitination of KDM4B and thereby stabilized its proteins. KDM4B was up-regulated in ccRCC and potentiated cell growth. UCHL1 depended on KDM4B to augment ccRCC malignancies. Targeting UCHL1 suppressed tumor growth, colony formation, and migration abilities, which could be rescued by KDM4B. Furthermore, KDM4B was directly bound to the promoter region of VEGFA, abolishing repressive H3K9me3 modifications. KDM4B coordinated with HIF2α to activate VEGFA transcriptional levels. UCHL1-KDM4B axis governs VEGFA levels to sustain the angiogenesis phenotypes. Finally, a specific small-molecule inhibitor (6RK73) targeting UCHL1 remarkably inhibited ccRCC progression and further sensitized ccRCC to bevacizumab treatment. CONCLUSION: Overall, this study defined an epigenetic mechanism of UCHL1/KDM4B in activating VEGF signaling. The UCHL1-KDM4B axis represents a novel target for treating ccRCC and improving the efficacy of anti-angiogenesis therapy.

[The Regulatory Effect of RNA m6 A Methylation Modification on KDM4B Gene Expression in t (8;21) AML Cells by MeRIP-qPCR].

OBJECTIVE: To confirm the direct regulatory effect of WTAP-mediated RNA m6A modification on the KDM4B gene in t (8;21) acute myeloid leukemia (AML) cells through MeRIP combined with reverse transcription real-time quantitative PCR (RT-qPCR) technology. METHODS: The lentivirus-mediated shRNA target WTAP or KDM4B gene was used to transfect the t (8;21) AML cell lines: Kasumi-1 and SKNO-1, and cells transfected with randomly shuffled shRNA as the control. Using the Ultrapure RNA Extraction Kit (DNase I) to extract RNA. The Magna MeRIPTM m6A Kit was used to enrich methylated modified fragments, and detect the m6A methylated RNA regions by RT-qPCR, and the protein and mRNA expression levels of WTAP and KDM4B in cells were detected by Western blot and reverse transcription real-time quantitative PCR (RT-qPCR). Colony formation assays were used to detect the colony ability of cells in vitro. RESULTS: Silencing the expression of WTAP in Kasumi-1 cells, the enrichment of m6A methylation modification was significantly decreased in the 3'UTR of KDM4B mRNA(P < 0.01), and the protein(P < 0.001) and mRNA (Kasumi-1:P < 0.001; SKNO-1: P < 0.01) expression levels of KDM4B were also significantly inhibited in Kasumi-1 and SKNO-1 cells upon WTAP knockdown (all P < 0.01), accompanied by a significant decrease in the colony-forming ability of both cell lines (both P < 0.01). CONCLUSION: In t(8;21) AML cell lines, WTAP could regulate the expression of KDM4B by regulating the m6A modification of the 3'UTR of KDM4B mRNA, and silencing the expression of KDM4B could inhibit the cellular proliferation in vitro.

Uncovering Sex-Specific Epigenetic Regulatory Mechanism Involving H3k9me2 in Neural Inflammation, Damage, and Recovery in the Internal Carotid Artery Occlusion Mouse Model.

Cerebral ischemic stroke is one of the foremost global causes of death and disability. Due to inadequate knowledge in its sequential disease mechanisms, therapeutic efforts to mitigate acute ischemia-induced brain injury are limited. Recent studies have implicated epigenetic mechanisms, mostly histone lysine acetylation/deacetylation, in ischemia-induced neural damage and death. However, the role of lysine methylation/demethylation, another prevalent epigenetic mechanism in cerebral ischemia has not undergone comprehensive investigation, except a few recent reports, including those from our research cohort. Considering the impact of sex on post-stroke outcomes, we studied both male and female mice to elucidate molecular details using our recently developed Internal Carotid Artery Occlusion (ICAO) model, which induces mild to moderate cerebral ischemia, primarily affecting the striatum and ventral hippocampus. Here, we demonstrate for the first time that female mice exhibit faster recovery than male mice following ICAO, evaluated through neurological deficit score and motor coordination assessment. Furthermore, our investigation unveiled that dysregulated histone lysine demethylases (KDMs), particularly kdm4b/jmjd2b are responsible for the sex-specific variance in the modulation of inflammatory genes. Building upon our prior reportage blocking KDMs by DMOG (Dimethyloxalylglycine) and thus preventing the attenuation in H3k9me2 reduced the post-ICAO transcript levels of the inflammatory molecules and neural damage, our present study delved into investigating the differential role of H3k9me2 in the regulation of pro-inflammatory genes in female vis-à-vis male mice underlying ICAO-induced neural damage and recovery. Overall, our results reveal the important role of epigenetic mark H3k9me2 in mediating sex-specific sequential events in inflammatory response, elicited post-ICAO.

Genotype-phenotype correlation study of structural abnormalities in a fetal brain caused by a novel KDM4B variant.

BACKGROUND: Fetal ventriculomegaly (VM), a common brain structure malformation detected during prenatal ultrasound diagnosis, is associated with an increased risk of neurodevelopmental disorders (NDDs) after birth. KDM4B encodes a lysine-specific demethylase that interacts with histone H3K23me3. Variations in KDM4B are reportedly associated with human NDDs; however, only 11 such patients have been reported. Herein, we report a fetus with VM and agenesis of the corpus callosum (ACC), which suggests that KDM4B plays an important role in fetal brain development. METHODS: Fetal skin tissue and parental peripheral venous blood samples were collected. Whole-exome and Sanger sequencing were performed to analyze fetal germline variants. Human 293T cells transfected with wild-type or mutant KDM4B were used for western blotting (WB) to analyze protein expression levels. RESULTS: An insertion variant of KDM4B, NM_015015.3: c.2889_2890insGAGAGCATCACGGTGAGCTGTGGGGTGGGGCAGGGGGCGGGGGGAGGCTGGGAGCACAGTGACAACCTGTACCCC, was identified in the fetal tissue; however, the parents carried the wild-type gene. The WB results indicated significantly reduced expression of the mutant protein, likely owing to decreased stability. CONCLUSIONS: The structural abnormalities in the brain of the studied fetus may be attributed to an insertion variant of KDM4B. This study highlights the importance of screening for KDM4B variants and considering potential copy number variations when observing VM or ACC in prenatal ultrasound imaging.

KDM4B down-regulation facilitated breast cancer cell stemness via PHGDH upregulation in H3K36me3-dependent manner.

Despite recent advances have been made in clinical treatments of breast cancer, the general prognosis of patients remains poor. Therefore, it is imperative to develop a more effective therapeutic strategy. Lysine demethylase 4B (KDM4B) has been reported to participate in breast cancer development recently, but its exact biological role in breast cancer remains unclear. Here, we observed that KDM4B was down-regulated in human primary BRCA tissues and the low levels of KDM4B expression were correlated with poor survival. Gain- and loss-of-function experiments showed that KDM4B inhibited the proliferation and metastasis of breast cancer cells. Besides, knockdown of KDM4B promoted the epithelial-mesenchymal transition (EMT) and cell stemness in breast cancer cells. Mechanistically, KDM4B down-regulates PHGDH by decreasing the enrichment of H3K36me3 on the promoter region of PHGDH. Knockdown of PHGDH could significantly reversed proliferation, migration, EMT, and cell stemness induced by KDM4B silencing in breast cancer cells. Collectively, we propose a model for a KDM4B/PHGDH axis that provides novel insight into breast cancer development, which may serve as a potential factor for predicting prognosis and a therapeutic target for breast cancer.

KDM4B: A promising oncology therapeutic target.

Epigenetic modifications are significant in tumor pathogenesis, wherein the process of histone demethylation is indispensable for regulating gene transcription, apoptosis, DNA replication, and repair of damaged DNA. The lysine demethylases (KDMs) serve an essential role in the aforementioned processes, with particular emphasis on the KDM4 family, also referred to as JMJD2. Multiple studies have underscored the significance of the KDM4 family in the regulation of various biological processes including, but not limited to, the cell cycle, DNA repair mechanisms, signaling pathways, and the progression of tumor formation. Nevertheless, it is imperative to elucidate the underlying mechanism of KDM4B, which belongs to the KDM4 gene family. This review presents a comprehensive examination of the structure, mechanism, and function of KDM4B, as well as a critical analysis of the current body of research pertaining to its involvement in tumorigenesis and development. Furthermore, this review explores the potential therapeutic strategies that specifically target KDM4B.

Human phenotype caused by biallelic KDM4B frameshift variant.

KDM4B (MIM*609765, NM_015015.3, formerly JMJD2B) encodes a histone demethylase and regulates gene expression via demethylation, mainly of H3K9 tri-methylation. Heterozygous KDM4B loss-of-function variants cause autosomal dominant intellectual developmental disorder 65 (MIM#619320), which is characterized by global developmental delay, intellectual disability, language and gross motor delays, structural brain anomalies, characteristic facial features, and clinodactyly. Although the majority of reported patients have de novo pathogenic variants, some patients inherit pathogenic variants from affected parents. To our knowledge, only 23 patients with heterozygous KDM4B variants have been reported to date, and there are no reports of patients with biallelic KDM4B pathogenic variants. Herein, we report a female patient with a biallelic KDM4B frameshift variant (NM_015015.3: c.1384_1394delinsGGG, p.(Leu462Glyfs*43)) located at exon 12 of 23 protein-coding exons, which is thought to be subject to nonsense-mediated mRNA decay and no protein production. She presented developmental and language delays and a hypotonic and characteristic face. The patient's phenotype was more obvious than that of her mother, who is heterozygous for the same variant. Although declining birth rate (embryonic lethality in male mice) in homozygous knockout mice has been demonstrated, our report suggests that homozygous KDM4B frameshift variants can be viable in humans at least female.

Histone demethylase KDM4B accelerates the progression of glioblastoma via the epigenetic regulation of MYC stability.

BACKGROUND: Glioblastoma (GBM) is the most malignant and invasive human brain tumor. Histone demethylase 4B (KDM4B) is abnormally expressed in GBM, but the molecular mechanisms by which KDM4B affects the malignant tumor progression are not well defined. METHODS: GBM cell lines and xenograft tumor samples were subjected to quantitative PCR (qPCR), Western blot, immunohistochemical staining (IHC), as well as ubiquitination, immunoprecipitation (IP), and chromatin immunoprecipitation (ChIP) assays to investigate the role of KDM4B in the progression of GBM. RESULTS: Here, we report that KDM4B is an epigenetic activator of GBM progression. Abnormal expression of KDM4B is correlated with a poor prognosis in GBM patients. In GBM cell lines, KDM4B silencing significantly inhibited cell survival, proliferation, migration, and invasion, indicating that KDM4B is essential for the anchorage-independent growth and tumorigenic activity of GBM cells. Mechanistically, KDM4B silencing led to downregulation of the oncoprotein MYC and suppressed the expression of cell cycle proteins and epithelial-to-mesenchymal transition (EMT)-related proteins. Furthermore, we found that KDM4B regulates MYC stability through the E3 ligase complex SCFFBXL3+CRY2 and epigenetically activates the transcription of CCNB1 by removing the repressive chromatin mark histone H3 lysine 9 trimethylation (H3K9me3). Finally, we provide evidence that KDM4B epigenetically activates the transcription of miR-181d-5p, which enhances MYC stability. CONCLUSIONS: Our study has uncovered a KDM4B-dependent epigenetic mechanism in the control of tumor progression, providing a rationale for utilizing KDM4B as a promising therapeutic target for the treatment of MYC-amplified GBM.

Therapeutic targeting of histone lysine demethylase KDM4B blocks the growth of castration-resistant prostate cancer.

Epigenetics is an emerging mechanism for tumorigenesis. Treatment that targets epigenetic regulators is becoming an attractive strategy for cancer therapy. The role of epigenetic therapy in prostate cancer (PCa) remains elusive. Previously we demonstrated that upregulation of histone lysine demethylase KDM4B correlated with the appearance of castration resistant prostate cancer (CRPC) and identified a small molecular inhibitor of KDM4B, B3. In this study, we further investigated the role of KDM4B in promoting PCa progression and tested the efficacy of B3 using clinically relevant PCa models including PCa cell line LNCaP and 22Rv1 and xenografts derived from these cell lines. In loss and gain-functional studies of KDM4B in PCa cells, we found that overexpression of KDM4B in LNCaP cells enhanced its tumorigenicity whereas knockdown of KDM4B in 22Rv1 cells reduced tumor growth in castrated mice. B3 suppressed the growth of 22Rv1 xenografts and sensitized tumor to anti-androgen receptor (AR) antagonist enzalutamide inhibition. B3 also inhibited 22Rv1 tumor growth synergistically with rapamycin, leading to cell apoptosis. Comparative transcriptomic analysis performed on KDM4B knockdown and B3-treated 22Rv1 cells revealed that B3 inhibited both H3K9me3 and H3K27me3 demethylase activities. Our studies establish KDM4B as a target for CRPC and B3 as a potential therapeutic agent. B3 as monotherapy or in combination with other anti-PCa therapeutics offers proof of principle for the clinical translation of epigenetic therapy targeting KDMs for CRPC patients.

Methylation of the JMJD2B epigenetic regulator differentially affects its ability to coactivate the ETV1 and JUN transcription factors.

OBJECTIVES: Jumonji C domain-containing (JMJD) 2B (JMJD2B) is a transcriptional cofactor and histone demethylase that is involved in prostate cancer formation. However, how its function is regulated by posttranslational modification has remained elusive. Hence, we examined if JMJD2B would be regulated by lysine methylation. METHODS: Through in vitro methylation assays and Western blotting with methyl-lysine specific antibodies, we analyzed lysine methylation within JMJD2B. Identified methylated lysine residues were mutated to arginine residues and the respective impact on JMJD2B transcriptional activity measured with a reporter gene assay in human LNCaP prostate cancer cells. RESULTS: We discovered that JMJD2B is methylated on up to six different lysine residues. Further, we identified the suppressor of variegation 3-9/enhancer of zeste/trithorax (SET) domain-containing protein 7/9 (SET7/9) as the methyltransferase being responsible for this posttranslational modification. Mutating the methylation sites in JMJD2B to arginine residues led to diminished coactivation of the Ju-nana (JUN) transcription factor, which is a known oncogenic protein in prostate tumors. In contrast, methylation of JMJD2B had no impact on its ability to coactivate another transcription factor associated with prostate cancer, the DNA-binding protein E26 transformation-specific (ETS) variant 1 (ETV1). Consistent with a potential joint action of JMJD2B, SET7/9 and JUN in prostate cancer, the expression of JMJD2B in human prostate tumors was positively correlated with both SET7/9 and JUN levels. CONCLUSIONS: The identified SET7/9-mediated methylation of JMJD2B appears to impact its cooperation with selected interacting transcription factors in prostate cancer cells. Given the implicated roles of JMJD2B beyond prostate tumorigenesis, SET7/9-mediated methylation of JMJD2B possibly also influences the development of other cancers, while its impairment might have relevance for obesity or a global developmental delay that can be elicited by reduced JMJD2B activity.

Histone H3K9 demethylase JMJD2B/KDM4B promotes osteogenic differentiation of bone marrow-derived mesenchymal stem cells by regulating H3K9me2 on RUNX2.

Background: A variety of proteins including epigenetic factors are involved in the differentiation of human bone marrow mesenchymal stem cells. These cells also exhibited an epigenetic plasticity that enabled them to trans-differentiate from adipocytes to osteoblasts (and vice versa) after commitment. Further in-depth study of their epigenetic alterations may make sense. Methods: Chromatin Immunoprecipitation-PCR (ChIP-PCR) was used to detect the methylation enrichment status of H3K9me2 in the Runx2 promoter, alizarin red and alkaline phosphatase (ALP) staining were used to detect osteogenic differentiation and mineralization ability, western blot and quantitative RT-PCR were used to measure the differential expression of osteogenesis-related proteins and genes. Recombinant Lentivirus mediated gain-of-function and loss-of-function study. The scale of epigenetic modification was detected by laser confocal. Results: Our results showed that compared with human bone marrow mesenchymal stem cells (hBMSCs) without osteogenic differentiation treatment, hBMSCs after osteogenic differentiation significantly promoted osteogenic differentiation and mRNA expression such as JMJD2B/KDM4B, osteogenesis-related genes like Runx2 and FAM210A in hBMSCs cells, suggesting that upregulation of JMJD2B/KDM4B is involved in the promoting effect of osteogenesis. After overexpression and silencing expression of JMJD2B, we found a completely opposite and significant difference in mRNA expression of osteogenesis-related genes and staining in hBMSCs. Overexpression of JMJD2B/KDM4B significantly promoted osteogenic differentiation, suggesting that JMJD2B/KDM4B could promote osteogenesis. In addition, ChIP-PCR showed that overexpression of JMJD2B/KDM4B significantly reversed the methylation enrichment status of H3K9me2 in Runx2 promoter. Furthermore, overexpression of JMJD2B/KDM4B significantly reverses the inhibitory effect of BIX01294 on H3K9me2, suggesting that JMJD2B/KDM4B regulates the osteogenic differentiation of hBMSCs by changing the methylation status of H3K9me2 at the Runx2 promoter. Conclusions: Taken together, these results suggest that JMJD2B/ KDM4B may induce the osteogenic differentiation of hBMSCs by regulating the methylation level of H3K9me2 at the Runx2 promoter.

Epigenetic regulation of Cebpb activation by pY19-Caveolin-2 at the nuclear periphery in association with the nuclear lamina.

Here, we show that Caveolin-2 (Cav-2) is an epigenetic regulator for adipogenesis. Upon adipogenic stimulation, inner nuclear membrane (INM)-targeted pY19-Cav-2 interacted with lamin A/C to disengage the repressed Cebpb promoter from lamin A/C, which facilitated the Cebpb promoter association with lamin B1. Consequently, pY19-Cav-2 recruited lysine demethylase 4b (KDM4b) for demethylation of histone H3 lysine 9 trimethylation (H3K9me3) and histone acetyltransferase GCN5 for acetylation of H3K27, and subsequently RNA polymerase II (Pol II) on Cebpb promoter for epigenetic activation of Cebpb, to initiate adipogenesis. Cav-2 knock-down abrogated the Cebpb activation and blocked the Pparg2 and Cebpa activation. Re-expression of Cav-2 restored Cebpb activation and adipogenesis in Cav-2-deficient preadipocytes. Our data identify a new mechanism by which the epigenetic activation of Cebpb is controlled at the nuclear periphery to promote adipogenesis.

Epigenetic Regulation of NGF-Mediated Osteogenic Differentiation in Human Dental Mesenchymal Stem Cells.

Nerve growth factor (NGF) is the best-characterized neurotrophin and is primarily recognized for its key role in the embryonic development of the nervous system and neuronal cell survival/differentiation. Recently, unexpected actions of NGF in bone regeneration have emerged as NGF is able to enhance the osteogenic differentiation of mesenchymal stem cells. However, little is known regarding how NGF signaling regulates osteogenic differentiation through epigenetic mechanisms. In this study, using human dental mesenchymal stem cells (DMSCs), we demonstrated that NGF mediates osteogenic differentiation through p75NTR, a low-affinity NGF receptor. P75NTR-mediated NGF signaling activates the JNK cascade and the expression of KDM4B, an activating histone demethylase, by removing repressive H3K9me3 epigenetic marks. Mechanistically, NGF-activated c-Jun binds to the KDM4B promoter region and directly upregulates KDM4B expression. Subsequently, KDM4B directly and epigenetically activates DLX5, a master osteogenic gene, by demethylating H3K9me3 marks. Furthermore, we revealed that KDM4B and c-Jun from the JNK signaling pathway work in concert to regulate NGF-mediated osteogenic differentiation through simultaneous recruitment to the promoter region of DLX5. We identified KDM4B as a key epigenetic regulator during the NGF-mediated osteogenesis both in vitro and in vivo using the calvarial defect regeneration mouse model. In conclusion, our study thoroughly elucidated the molecular and epigenetic mechanisms during NGF-mediated osteogenesis.

Effects of demethylase KDM4B on the biological characteristics and function of yak cumulus cells in vitro.

The present study aims to investigate the expression and function of lysine-specific demethylase 4B (KDM4B) in yak cumulus cells (CCs) in order to reveal the mechanisms by which KDM4B regulates biological characteristics and function of CCs. The cellular location of KDM4B and the methylation pattern of H3K9 were detected using immunofluorescence (IF) staining in CCs. The mRNA expression levels of apoptosis-related genes (BCL-2, HAX1 and BAX) and genes related to the estrogen pathway (ESR2, CYP17 and 3B-HSD) were estimated by qRT-PCR after knockdown of KDM4B expression by siRNA in yak CCs. Then, a proliferation assay, Annexin V-FITC staining, and ELISA were utilized to explore the effects of KDM4B silencing on CCs proliferation, apoptosis, and estrogen (E2) secretion, respectively. The results showed that KDM4B is located in the nuclei of yak CCs and is distributed in a dotted pattern. Knockdown KDM4B induced a decrease in cell proliferation, an increase in apoptotic rate and a reduction in the levels of E2 secretion of CCs. Additionally, the methylation patterns of H3K9me2 and H3K9me3 were significantly increased in CCs transfected with KDM4B siRNA-1 (P < 0.05). The mRNA expression level of apoptosis promoting BAX genes was significantly upregulated, but 3B-HSD, ESR2 and anti-apoptotic HAX1 genes were significantly downregulated in transfected CCs (P < 0.05). Furthermore, the rate of embryos developing from the 2-cell stage to blastocysts was lower in the siRNA-1 transfection group than that of the control group (28.6 ± 2.9% vs 40.4 ± 2.4%, P < 0.05). In conclusion, our study indicates that KDM4B regulates the biological characteristics and physiological function of yak CCs mainly through changing the methylation patterns of H3K9 and related gene expression levels.

Targeting KDM4B that coactivates c-Myc-regulated metabolism to suppress tumor growth in castration-resistant prostate cancer.

Rationale: The progression of prostate cancer (PCa) to castration-resistant PCa (CRPC) despite continuous androgen deprivation therapy is a major clinical challenge. Over 90% of patients with CRPC exhibit sustained androgen receptor (AR) signaling. KDM4B that removes the repressive mark H3K9me3/2 is a transcriptional activator of AR and has been implicated in the development of CRPC. However, the mechanisms of KDM4B involvement in CRPC remain largely unknown. Here, we sought to demonstrate the molecular pathway mediated by KDM4B in CRPC and to provide proof-of-concept evidence that KDM4B is a potential CRPC target. Methods: CRPC cells (C4-2B or CWR22Rv1) depleted with KDM4B followed by cell proliferation (in vitro and xenograft), microarray, qRT-PCR, Seahorse Flux, and metabolomic analyses were employed to identify the expression and metabolic profiles mediated by KDM4B. Immunoprecipitation was used to determine the KDM4B-c-Myc interaction region. Reporter activity assay and ChIP analysis were used to characterize the KDM4B-c-Myc complex-mediated mechanistic actions. The clinical relevance between KDM4B and c-Myc was determined using UCSC Xena analysis and immunohistochemistry. Results: We showed that KDM4B knockdown impaired CRPC proliferation, switched Warburg to OXPHOS metabolism, and suppressed gene expressions including those targeted by c-Myc. We further demonstrated that KDM4B physically interacted with c-Myc and they were co-recruited to the c-Myc-binding sequence on the promoters of metabolic genes (LDHA, ENO1, and PFK). Importantly, KDM4B and c-Myc synergistically promoted the transactivation of the LDHA promoter in a demethylase-dependent manner. We also provided evidence that KDM4B and c-Myc are co-expressed in PCa tissue and that high expression of both is associated with worse clinical outcome. Conclusions: KDM4B partners with c-Myc and serves as a coactivator of c-Myc to directly enhance c-Myc-mediated metabolism, hence promoting CRPC progression. Targeting KDM4B is thus an alternative therapeutic strategy for advanced prostate cancers driven by c-Myc and AR.

KDM4B Overexpression Promotes the Growth, Migration, and Invasion of Rheumatoid Arthritis Fibroblast-Like Synoviocytes by Activating STAT3 Pathway.

In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) present a unique aggressive phenotype and have a passive response to the inflammatory microenvironment, which are critical for the disease's progression. KDM4B, as a histone demethylase, functions as an oncogenic factor in many cancers and is implicated in osteoclastogenesis as well as pro-inflammatory cytokine release in inflammatory diseases. However, the effects of KDM4B on RA FLS have not been reported. To investigate this issue, our study determined the expression of KDM4B in RA FLS using RT-qPCR and western blot. The effects of KDM4B on RA FLS viability, apoptosis, migration, and invasion were detected by MTT, flow cytometry, transwell migration, and invasion assays. Furthermore, the interaction of KDM4B with STAT3 signaling was studied by western blot, MTT, flow cytometry, transwell migration, and invasion assays. The experimental results showed that KDM4B expression was upregulated in RA synovial tissues and FLS as compared to healthy control tissues and normal FLS. Knockdown of KDM4B obviously suppressed RA FLS viability, migration and invasion, and induced apoptosis. In addition, knockdown of KDM4B in RA FLS decreased the expression of p-STAT3 and MMP-9 but increased cleaved caspase-3 expression compared with the control group. Moreover, KDM4B overexpression could promote cell growth, migration and invasion, and suppress apoptosis in RA FLS by activating STAT3 signaling. Therefore, these findings provide new insight for understanding the pathogenesis of RA and indicate that KDM4B may have a potential to be an effective therapeutic target for RA.

Role for the Histone Demethylase KDM4B in Rhabdomyosarcoma via CDK6 and CCNA2: Compensation by KDM4A and Apoptotic Response of Targeting Both KDM4B and KDM4A.

Histone demethylases are epigenetic modulators that play key roles in regulating gene expression related to many critical cellular functions and are emerging as promising therapeutic targets in a number of tumor types. We previously identified histone demethylase family members as overexpressed in the pediatric sarcoma, rhabdomyosarcoma. Here we show high sensitivity of rhabdomyosarcoma cells to a pan-histone demethylase inhibitor, JIB-04 and identify a key role for the histone demethylase KDM4B in rhabdomyosarcoma cell growth through an RNAi-screening approach. Decreasing KDM4B levels affected cell cycle progression and transcription of G1/S and G2/M checkpoint genes including CDK6 and CCNA2, which are bound by KDM4B in their promoter regions. However, after sustained knockdown of KDM4B, rhabdomyosarcoma cell growth recovered. We show that this can be attributed to acquired molecular compensation via recruitment of KDM4A to the promoter regions of CDK6 and CCNA2 that are otherwise bound by KDM4B. Furthermore, upfront silencing of both KDM4B and KDM4A led to RMS cell apoptosis, not seen by reducing either alone. To circumvent compensation and elicit stronger therapeutic responses, our study supports targeting histone demethylase sub-family proteins through selective poly-pharmacology as a therapeutic approach.

MicroRNA-15a Carried by Mesenchymal Stem Cell-Derived Extracellular Vesicles Inhibits the Immune Evasion of Colorectal Cancer Cells by Regulating the KDM4B/HOXC4/PD-L1 Axis.

The relevance of microRNA-15a (miR-15a) to autoimmunity has been reported. Herein, we intended to probe the potential roles of miR-15a shuttled by adipose-derived mesenchymal stem cells (adMSCs)-derived extracellular vesicles (Evs) in colorectal cancer (CRC). Initially, CRC cells were treated with interferon gamma (IFN-γ) to screen out differentially expressed genes by transcriptome sequencing. Following a 24-h co-culture with 20 µM adMSCs-derived Evs, CRC cell viability, migration, invasion, and apoptosis were assessed. After the determination of histone lysine demethylase 4B (KDM4B) as our target, its regulatory miRNA was predicted by the bioinformatics websites and verified by dual-luciferase and RNA pull-down assays. Intriguingly, KDM4B downregulated homeobox C4 (HOXC4) expression, while HOXC4 bound to the promoter sequence of programmed death-ligand 1 (PD-L1). Thus, we conducted rescue experiments to study the role of KDM4B and HOXC4. Finally, we evaluated the effects of adMSCs on CRC cell growth and immune evasion through in vivo tumorigenesis experiments. AdMSCs-derived Evs overexpressing miR-15a repressed proliferation, migration, and invasion, while it promoted the apoptosis of CRC cells via downregulation of KDM4B. These in vivo findings were reproduced in vitro on CRC immune evasion. Collectively, adMSCs-derived Evs overexpressing miR-15a restricted the immune evasion of CRC via the KDM4B/HOXC4/PD-L1 axis.