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Recent studies have shed light on disrupted collagen signaling in Gliomas, yet the regulatory landscape remains largely unexplored. This study enquired into the role of polycomb repressive complex-2 (PRC2)-mediated H3K27me3 modification, a key epigenetic factor in glioma. Using in-house data, we identified miRNAs downregulated in glioblastoma (GBM) with the potential to regulate Collagen VI family genes. Notably, miR-3189 emerged as a prime PRC2 target. Its expression was significantly downregulated in Indian GBM patients as well as other glioma cohorts. Mechanistic insights, involving Luciferase assays, mutagenesis, and Western blot analysis, confirmed direct targeting of Collagen VI member COL6A2 by miR-3189-3p. Functional assays demonstrated that miR-3189-3p restrained GBM malignancy by inhibiting proliferation, migration, and epithelial-mesenchymal transition (EMT). Conversely, COL6A2 overexpressed in GBM patients, countered miR-3189, and promoted the malignant phenotype. Gene set enrichment analysis highlighted EMT enrichment in GBM patients with elevated COL6A2 expression, carrying prognostic implications. This study uncovers intricate interactions between two epigenetic regulators-H3K27me3 and miR-3189-working synergistically to modulate Collagen VI gene; thus, influencing the malignancy of GBM. Targeting this H3K27me3|miR-3189-3p|COL6A2 axis presents a potential therapeutic avenue against GBM., (© 2024 Wiley Periodicals LLC.)

Bone marrow-derived CD34-positive (CD34+) endothelial progenitor cells (EPCs) has unique functions in the mechanism of compensatory lung growth (CLG). The content of this study is mainly to describe the effect of microRNA (miR)-155 in the mechanisms of EPCs and CLG. Our study found that transfection of miR-155 mimic could promote EPC proliferation, migration and tube formation, while transfection of miR-155 inhibitor had the opposite effect. It was also found that transfection of pc-JARID2 inhibited EPC proliferation, migration and tube formation, while transfection of si-JARID2 had the opposite effect. miR-155 can target and negatively regulate JARID2 expression. Overexpression of JARID2 weakened the promoting effects of miR-155 mimic on EPC proliferation, migration, and tubular formation, while silencing JARID2 weakened the inhibitory effects of miR-155 inhibitors on EPC proliferation, migration, and tubular formation. Transplantation of EPCs transfected with miR-155 mimic into the left lung model effectively increased lung volume, total alveolar number, diaphragm surface area, and lung endothelial cell number, while transplantation of EPCs co-transfected with miR-155 mimic and pc-JARID2 reversed this phenomenon. Overall, we found that miR-155 activates CD34+ EPC by targeting negative regulation of JARID2 and promotes CLG., Competing Interests: The authors declare that they have no competing interests., (Copyright: © 2024 Zhao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

HOX transcript antisense intergenic RNA (HOTAIR) is a long non-coding RNA (lncRNA) which is increasingly being perceived as a tremendous molecular mediator of brain pathophysiology at multiple levels. Epigenetic regulation of target gene expression carried out by HOTAIR is thorough modulation of chromatin modifiers; histone methyltransferase polycomb repressive complex 2 (PRC2) and histone demethylase lysine-specific demethylase 1 (LSD1). Incidentally, HOTAIR was the first lncRNA shown to elicit sponging of specific microRNA (miRNA or miR) species in a trans-acting manner. It has been extensively studied in various cancers, including gliomas and is regarded as a prominent pro-tumorigenic and pro-oncogenic lncRNA. Indeed, the expression of HOTAIR may serve as glioma grade predictor and prognostic biomarker. The objective of this timely review is not only to outline the multifaceted pathogenic roles of HOTAIR in the development and pathophysiology of gliomas and brain cancers, but also to delineate the research findings implicating it as a critical regulator of overall brain pathophysiology. While the major focus is on neuro-oncology, wherein HOTAIR represents a particularly potent underlying pathogenic player and a suitable therapeutic target, mechanisms underlying the regulatory actions of HOTAIR in neurodegeneration, traumatic, hypoxic and ischemic brain injuries, and neuropsychiatric disorders are also presented., (© 2024. The Author(s).)

The histone methyltransferase enhancer of zeste homolog 2 (EZH2) is overexpressed in a variety of malignancies including prostate cancer (PCa) and may play important roles in tumor progression. Gene copy number gains, enhanced transcription, and a few circRNAs have been reported to upregulate EZH2. It was not known whether EZH2 itself generates circRNAs that promote its own expression. We here report the identification of circEZH2 E2/E3 that is derived from exons 2 and 3 of the EZH2 gene and overexpressed in PCa. We show that circEZH2 E2/E3 functions as a dual inhibitor for both miR363 and miR708 that target the EZH2 3'UTR and CDS, respectively, resulting in the upregulation of EZH2 expression and hence the downregulation of EZH2-repressed genes (e.g., CDH1 and DAB2IP), and enhancement of PCa cell proliferation, migration, invasion, and xenograft PCa growth. Overexpression of circEZH2 E2/E3 is significantly correlated with higher tumor grade, tumor progression, and unfavorable progression-free and disease-specific survival in PCa patients. These findings show a novel autoenhancing EZH2-circEZH2 E2/E3 -miR363/miR708-EZH2 regulatory loop, by which circEZH2 E2/E3 plays important roles in PCa tumorigenesis and progression by upregulating EZH2, and may have potential diagnostic, prognostic, and therapeutic uses in PCa management., (© 2022 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Recent studies by us and others have shown that enhancer of zeste homolog-2 (EZH2), a histone methyltransferase, in glial cells regulates the genesis of neuropathic pain by modulating the production of proinflammatory cytokines and chemokines. In this review, we summarize recent advances in this research area. EZH2 is a subunit of polycomb repressive complex 2 (PRC2), which primarily serves as a histone methyltransferase to catalyze methylation of histone 3 on lysine 27 (H3K27), ultimately resulting in transcriptional repression. Animals with neuropathic pain exhibit increased EZH2 activity and neuroinflammation of the injured nerve, spinal cord, and anterior cingulate cortex. Inhibition of EZH2 with DZNep or GSK-126 ameliorates neuroinflammation and neuropathic pain. EZH2 protein expression increases upon activation of Toll-like receptor 4 and calcitonin gene-related peptide receptors, downregulation of miR-124-3p and miR-378 microRNAs, or upregulation of Lncenc1 and MALAT1 long noncoding RNAs. Genes suppressed by EZH2 include suppressor of cytokine signaling 3 (SOCS3), nuclear factor (erythroid-derived 2)-like-2 factor (NrF2), miR-29b-3p, miR-146a-5p, and brain-specific angiogenesis inhibitor 1 (BAI1). Pro-inflammatory mediators facilitate neuronal activation along pain-signaling pathways by sensitizing nociceptors in the periphery, as well as enhancing excitatory synaptic activities and suppressing inhibitory synaptic activities in the CNS. These studies collectively reveal that EZH2 is implicated in signaling pathways known to be key players in the process of neuroinflammation and genesis of neuropathic pain. Therefore, targeting the EZH2 signaling pathway may open a new avenue to mitigate neuroinflammation and neuropathic pain.

Long intergenic non-coding RNA 00852 (LINC00852) has been shown to promote the progression of many different cancers including prostate cancer. However, the involved mechanism in promoting the proliferation, migration and invasion of prostate cancer cells has not been reported. In this study, we found that LINC00852 was highly expressed in the tissue of prostate cancer using quantitative reverse transcription PCR (qRT-PCR). CCK-8 assay, colony formation experiment, Transwell migration and invasion experiments were performed to prove that the up-regulation of LINC00852 could promote the proliferation, migration and invasion of prostate cancer cells in vitro. Xenograft tumors experiments in nude mice confirmed that up-regulation of LINC00852 promoted the proliferation of prostate cancer cells in vivo. Bioinformatics predictions and dual-luciferase reporter gene assay showed that miR-29a-3p binds to the 3'-untranslated region of JARID2, and the enhancement of miR-29a-3p could reverse the effect of LINC00852 overexpression in vitro. Moreover, the results of qRT-PCR and western blot showed that LINC00852 could regulate the expression of JARID2 through miR-29a-3p induction. In summary, we demonstrated that LINC00852 played a key role in promoting the prostate cancer, and LINC00852/miR-29a-3p/JARID2 axis could be used as a target for prostate cancer treatment., (© 2022. The Author(s).)

Asthmatic airway inflammation is divided into two typical endotypes: Th2-mediated eosinophilic and Th1- or Th17-mediated neutrophilic airway inflammation. The miRNA miR-155 has well-documented roles in the regulation of adaptive T-cell responses and innate immunity. However, no specific cell-intrinsic role has yet been elucidated for miR-155 in T cells in the course of Th2-eosinophilic and Th17-neutrophilic airway inflammation using actual in vivo asthma models. Here, using conditional KO (miR155 ΔCD4 cKO) mice that have the specific deficiency of miR-155 in T cells, we found that the specific deficiency of miR-155 in T cells resulted in fully suppressed Th2-type eosinophilic airway inflammation following acute allergen exposure, as well as greatly attenuated the Th17-type neutrophilic airway inflammation induced by repeated allergen exposure. Furthermore, miR-155 in T cells appeared to regulate the expression of several different target genes in the functional activation of CD4 + Th2 and Th17 cells. To be more precise, the deficiency of miR-155 in T cells enhanced the expression of c-Maf, SOCS1, Fosl2 and Jarid2 in the course of CD4 + Th2 cell activation, while C/EBPβ was highly enhanced in CD4 + Th17 cell activation in the absence of miR-155 expression. Conclusively, our data revealed that miR-155 could promote Th2 and Th17-mediated airway inflammation via the regulation of several different target genes, depending on the context of asthmatic diseases. Therefore, these results provide valuable insights into actual understanding of specific cell-intrinsic role of miR-155 in eosinophilic and neutrophilic airway inflammation for the development of fine-tune therapeutic strategies., (© 2022 John Wiley & Sons Ltd.)

The Polycomb Repressive Complex (PRC) proteins, EZH2 and EZH1 regulate many biological processes by generating the repressive H3K27me3 modifications in the chromatin. However, the factors that regulate the EZH1/EZH2 functions are poorly studied. We identify that the 3'UTRs of EZH2 and EZH1 mRNAs contain the binding sites for the miRNA, miR-150. MicroRNA-150 (miR-150) controls numerous biological processes including cell proliferation, differentiation and pathogenesis of a variety of diseases including cancer. We find that miR-150 regulates the levels of EZH1 and EZH2 through various experimental investigations. Since EZH2 is known to form a repressive complex with other epigenetic repressors especially DNMT3A and DNMT3B, we investigated whether miR-150 also regulates the DNMT3A and DNMT3B levels. We report that miR-150 regulates DNMT3A and DNMT3B levels through direct and indirect mechanisms respectively. Since these epigenetic repressors promote cell proliferation, we investigated the effect of miR-150 perturbation on HEK293 cell proliferation. We found that miR-150 inhibits cell proliferation and induces S-phase arrest by increasing the levels of tumor suppressors and decreasing the cell cycle regulators. Collectively, our study shows that miR-150 act as a tumor suppressor by down-regulating the oncogenic major epigenetic repressors and controls cell proliferation., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The detailed function of ARAP1-AS1, the antisense RNA of Arf-GAP with Rho-GAP domain, ANK repeat and PH domain-containing protein 1 (ARAP1), in lung adenocarcinoma (LUAD) has not been clearly elucidated and required further investigation. Our study is committed to exploring the role of ARAP1-AS1 in LUAD. Gene expression in LUAD was measured by real-time quantitative polymerase-chain reaction (RT-qPCR). The influence of ARAP1-AS1 on LUAD cell malignant behaviors was evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay, Transwell invasion assay and wound healing assay. Subcellular fractionation assay detected the cellular localization of ARAP1-AS1 in LUAD. The protein levels were subjected to western blotting. RNA immunoprecipitation (RIP) and luciferase reporter assay were employed to verify the interaction between ARAP1-AS1, ARAP1 and enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2). Our investigation identified that ARAP1-AS1 was upregulated in LUAD cells and tissues. ARAP1-AS1 silencing repressed LUAD cell growth and migration. Furthermore, ARAP1-AS1 knockdown altered the expression of its sense mRNA, ARAP1. ARAP1-AS1 could recruit EZH2 to inhibit ARAP1 expression. Additionally, the downregulation of ARAP1 reversed ARAP1-AS1 downregulation-induced repression of cell growth and migration in LUAD. In conclusion, ARAP1-AS1 recruited EZH2 to silence ARAP1, facilitating cell proliferation, migration and invasion in LUAD. Our study demonstrated the possibility of ARAP1-AS1 to be a novel therapeutic target for LUAD. [Figure: see text].

The functions of exosomes in allergic diseases including asthma have aroused increasing concerns. This paper focuses on the effects of exosomes derived from human bone marrow-mesenchymal stem cells (hBM-MSCs) on the proliferation of bronchial smooth muscle cells in asthma and the mechanism involved. Exosomes were extracted from hBM-MSCs and identified. Human BSMCs were induced with transforming growth factor (TGF)-β1 to mimic an asthma-like condition in vitro and then treated with exosomes. A mouse model with asthma was induced by ovalbumin (OVA) and treated with exosomes for in vivo study. The hBM-MSC-derived exosomes significantly reduced the abnormal proliferation and migration of TGF-β1-treated BSMCs. microRNA (miR)-188 was the most enriched miRNA in exosomes according the microarray analysis, and JARID2 was identified as a mRNA target of miR-188. Either downregulation of miR-188 or upregulation of JARID2 blocked the protective effects of exosomes on BSMCs. JARID2 activated the Wnt/β-catenin signaling pathway. In the asthmatic mice, hBM-MSC-derived exosomes reduced inflammatory cell infiltration, mucus production, and collagen deposition in mouse lung tissues. In conclusion, this study suggestes that hBM-MSC-derived exosomes suppress proliferation of BSMCs and lung injury in asthmatic mice through the miR-188/JARID2/Wnt/β-catenin axis. This study may provide novel insights into asthma management.

Bladder cancer (BC), as one of the most common cancers around the world, begins in the inner side of the bladder and is inclined to spread to the remaining parts of the body. Extensive documents have shown that long noncoding RNAs function as stimuli in various cancer types. With regard to LINC00649, there is limited investigation on its role previously. In our research, we discovered that LINC00649 was considerably highly expressed in BC cells and the lack of LINC00649 would cause inactivity in cellular proliferation, migration, and invasion. miR-16-5p turned out to be competitively incorporated by LINC00649 in the upstream or JARID2 downstream. In BC cells, LINC00649 was found to bind with miR-16-5p to increase the expression of JARID2. Overly expressed JARID2 was found to reverse the LINC00649 shortage-mediated suppressive impacts on the cellular process of BC cells. Concisely, it was the first study on the molecular mechanism of LINC00649 in BC. This work detected that LINC00649 enhanced cell proliferation, migration, and invasion of BC cells by acting as a sponge of miR-16-5p and upregulating JARID2, providing novel insight into understating BC., (© 2020 S. Karger AG, Basel.)

Objective: Alzheimer's disease (AD) is a neurological disease. Dexmedetomidine (Dex) has been evidenced to exert neuroprotective effects on multiple neurological diseases, while the function of microRNA(miR)- 214-5p on Dex-mediated AD progression via targeting the suppressor of zest 12 (SUZ12) remains unclear. This study obligates to investigate the regulatory functions of Dex, miR-214-5p and SUZ12 on AD., Methods: The expression of miR-214-5p and SUZ12 in APPswe/PS1dE9 mice (hereinafter referred to as AD mice) was examined. Thereafter, the AD mice were treated with Dex or increased miR-214-5p or reduced SUZ12 to determine the spatial memory ability, apoptosis of hippocampal neurons and the contents of serum inflammatory and oxidative stress factors of AD mice. Finally, the target relationship between miR-214-5p and SUZ12 was detected., Results: MiR-214-5p was reduced and SUZ12 was elevated in AD mice. Dex administration reduced the apoptosis of hippocampal neurons, the contents of serum inflammatory factor and oxidative stress, and attenuated the cognitive impairment of AD mice accompanied by up-regulated miR-214-5p and down-regulated SUZ12, and the overexpression of miR-214-5p or reduction of SUZ12 could effectively enhance the Dex-treated effects on AD mice. MiR-214-5p targeted SUZ12., Conclusion: Dex may have a potential neuroprotective effect on AD via the miR-214-5p/SUZ12 axis. This study provides novel therapeutic targets for AD treatment., (Copyright © 2021. Published by Elsevier Inc.)

Cardiac hypertrophy is imposed much pressure on heart and threatening our live. Previous study suggested that dysregulation of Celf1 is largely connecting to neonatal cardiac dysfunction. Hence, we aimed to explore the precise function and probable regulatory mechanism upstream of Celf1in cardiac hypertrophy. Here, Ang-II treatment was implemented to stimulate hypertrophic phenotypes inH9C2 and MCM cells. Immunofluorescence assay was conducted to measure the surface area of cardiomyocytes. And qRT-PCR assay was conducted to investigate gene expression. Moreover, western blot assay was conducted to probe the protein levels. Results uncovered that Celf1 expression was increased dependent on elevated Ang-II concentration, and that inhibited Celf1 could relieve the Ang-II-caused cardiac hypertrophy. Significantly, Celf1was found to be targeted by miR-129-5p but then released via the sponging role of circ-Jarid2. Furthermore, circ-Jarid2 was found to promote cardiac hypertrophy, whereas miR-129-5p played suppressing parts in hypertrophic cardiomyocytes. Moreover, we verified circ-Jarid2 contributed to cardiac hypertrophy via miR-129-5p/Celf1 axis both in vitro and in vivo. In conclusion, circ-Jarid2/miR-129-5p/Celf1 axis aggravates cardiac hypertrophy, which provides new ideas for developing treatment strategies for patients with cardiac hypertrophy., (© 2020. The Author(s), under exclusive licence to Springer Nature Limited.)

How organisms control when to transition between different stages of development is a key question in biology. In plants, epigenetic silencing by Polycomb repressive complex 1 (PRC1) and PRC2 plays a crucial role in promoting developmental transitions, including from juvenile-to-adult phases of vegetative growth. PRC1/2 are known to repress the master regulator of vegetative phase change, miR156, leading to the transition to adult growth, but how this process is regulated temporally is unknown. Here we investigate whether transcription factors in the VIVIPAROUS/ABI3-LIKE (VAL) gene family provide the temporal signal for the epigenetic repression of miR156. Exploiting a novel val1 allele, we found that VAL1 and VAL2 redundantly regulate vegetative phase change by controlling the overall level, rather than temporal dynamics, of miR156 expression. Furthermore, we discovered that VAL1 and VAL2 also act independently of miR156 to control this important developmental transition. In combination, our results highlight the complexity of temporal regulation in plants., Competing Interests: The authors have declared that no competing interests exist.

Unmasking the complex regulatory pathways that mediate the malignant phenotypes of cancer cells can provide novel targets for therapies that could limit the recurrence and metastasis of gastric cancer (GC). Herein, we intended to clarify the role of embryonic ectoderm development protein (EED), microRNA-228-5p (miR-338-5p), methyltransferase like 3 (METTL3) and CUB domain containing protein 1 (CDCP1) in GC. Differentially expressed miRNAs and their target genes were extracted by in silico analysis. The studies revealed high expression of EED in GC tissues and cell lines and it high expression in GC patients was shown to be associated with poor prognosis. The chromatin immunoprecipitation assay identified that EED methylated miR-338-5p to inhibit its expression. EED knockdown could restrain the proliferative and invasive abilities of GC cells by inducing miR-338-5p. Furthermore, miR-338-5p targeted m6A methylase METTL3, while METTL3 amplified the translation of CDCP1 via m6A activity which led to accelerated proliferation and invasion of GC cells. Moreover, in vivo experiments validated that EED promoted the progression of GC through mediating the miR-338-5p/METTL3/CDCP1 axis. Collectively, EED downregulated miR-338-5p through histone methylation, which in turn impaired miR-338-5p-dependent METTL3 inhibition and enhanced CDCP1 translation, therefore contributing to the development of GC.

Activation of trimethylation of histone 3 lysine 27 (H3K27me3) by EZH2, a component of the Polycomb repressive complex 2 (PRC2), is suggested to play a role in endometriosis. However, the mechanism by which this complex is dysregulated in endometriosis is not completely understood. Here, using eutopic and ectopic tissues, as well as peritoneal fluid (PF) from IRB-approved and consented patients with and without endometriosis, the expression of PRC2 complex components, JARID2, miR-155 (known regulators of EZH2), and a key inflammatory modulator, FOXP3, was measured. A higher expression of EZH2, H3K27me3, JARID2, and FOXP3 as well as miR-155 was noted in both the patient tissues and in endometrial PF treated cells. Gain-or-loss of function of miR-155 showed an effect on the PRC2 complex but had little effect on JARID2 expression, suggesting alternate pathways. Chromatin immunoprecipitation followed by qPCR showed differential expression of PRC2 complex proteins and its associated binding partners in JARID2 vs. EZH2 pull down assays. In particular, endometriotic PF treatment increased the expression of PHF19 ( p = 0.0474), a gene silencer and co-factor that promotes PRC2 interaction with its targets. Thus, these studies have identified the potential novel crosstalk between miR-155-PRC2 complex-JARID2 and PHF19 in endometriosis, providing an opportunity to test other epigenetic targets in endometriosis.

Purpose: Malignant gliomas remain significant challenges in clinic and pose dismal prognosis on patients. In this study, we focused on growth arrest-specific 5 (GAS5), a tumor suppressive long non-coding RNA in glioma, explored its crosstalk with miR-424, and examined their biological functions in glioma., Methods: Expressions of GAS5 and miR-424 were measured using qRT-PCR. The regulation of GAS5 on miR-424 expression was examined in GAS5-overexpressing glioma cells by combining methylation-specific PCR, western blotting, and RNA immunoprecipitation. Functional significance of GAS5 and miR-424 on in vitro cell proliferation, apoptosis, migration, invasion, and in vivo tumor growth was examined using colony formation, flow cytometry, wound healing, transwell assay, and the xenograft model, respectively. The potential targeting of AKT3 by miR-424 was investigated using luciferase reporter assay., Results: GAS5 and miR-424 were significantly down-regulated in glioma cells. GAS5 directly interacted with enhancer of zeste homolog 2 (EZH2), stimulated the formation of polycomb repressive complex 2 (PRC2), reduced the levels of DNA methyltransferases (Dnmts), alleviated promoter methylation of miR-424, and promoted miR-424 expression. Functionally, GAS5, by up-regulating miR-424, inhibited cell proliferation, migration, and invasion, while increased apoptosis of glioma cells in vitro, and suppressed xenograft growth in vivo. miR-424 directly inhibited AKT3 and altered the expressions of AKT3 targets, cyclinD1, c-Myc, Bax, and Bcl-2, which might contribute to its tumor suppressive activities., Conclusions: GAS5, by inhibiting methylation and boosting expression of miR-424, inhibits AKT3 signaling and suppresses multiple malignant phenotypes. Therefore, stimulating GAS5/miR-424 signaling may benefit the treatment of glioma.

Background: Th subsets particularly T helper 17 and regulatory T cells play a critical role in immune balance in colonic mucosa of Inflammatory Bowel Disease. Recent studies have indicated miR-155 is overexpressed in the colonic mucosa in IBD patients. Thus, whether and how miR-155 influences Th17/Treg cell balance in IBD patients is worthy of researching., Methods: We divided mice into four groups: the mice oral administration of 3.0 % DSS in fresh drinking water for 7 days except normal group. In this period, starting from the fifth day, the miR-155 and NC antagomir group were carried out by intraperitoneal injection of miR-155 antagomirs and corresponding negative controls. In vitro, we isolated naïve CD4 + T cells and divided into two groups: the cells were transfected with mmu-miR-155-5p inhibitor or corresponding negative controls and then induced differentiation., Results: We found miR-155 antagomir can reach colon tissues in DSS-induced colitis and indeed ameliorated DSS-induced experimental colitis. Subsequently, we proved the levels of Th17 cells in spleens and Mesenteric lymph nodes and its associated IL-6, IL-17A and RORγt in colonic tissues were dramatically decreased and TGF-β1 raised in DSS + miR-155 antagomir group. However, miR-155 antagomir significantly increased the expression of Tregs. In vitro, we found miR-155 inhibitor could improve the Tregs but decrease Th17 cells. Finally, we dig out that Jarid2 was apparently improved by miR-155 antagomir, Wnt/β-catenin and its associated T cell factor-4 (TCF-4) and Cyclin D1 expression were positively correlated with Jarid2., Conclusion: Silencing of miR-155 attenuates DSS-induced colitis by regulating Th17/Treg cell balance and Jarid2/Wnt/β-catenin participated in the process., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020. Published by Elsevier Masson SAS.)

Recent advancement in understanding cancer etiology has highlighted epigenetic deregulation as an important phenomenon leading to poor prognosis in glioblastoma (GBM). Polycomb repressive complex 2 (PRC2) is one such important epigenetic modifier reportedly altered in GBM. However, its defined mechanism in tumorigenesis still remains elusive. In present study, we analyzed our in-house ChIPseq data for H3k27me3 modified miRNAs and identified miR-490-3p to be the most common target in GBM with significantly downregulated expression in glioma patients in both TCGA and GBM patient cohort. Our functional analysis delineates for the first time, a central role of PRC2 catalytic unit EZH2 in directly regulating expression of this miRNA and its host gene CHRM2 in GBM. In accordance, cell line treatment with EZH2 siRNA and 5-azacytidine also confirmed its coregulation by CpG and histone methylation based epigenetic mechanisms. Furthermore, induced overexpression of miR-490-3p in GBM cell lines significantly inhibited key hallmarks including cellular proliferation, colony formation and spheroid formation, as well as epithelial-to-mesenchymal transition (EMT), with downregulation of multiple EMT transcription factors and promigratory genes (MMP9, CCL5, PIK3R1, ICAM1, ADAM17 and NOTCH1). We also for the first time report TGFBR1 and TGIF2 as two direct downstream effector targets of miR-490-3p that are also deregulated in GBM. TGIF2, a novel target, was shown to promote migration and EMT that could partially be rescued by miR-490-3p overexpression. Overall, this stands as a first study that provides a direct link between epigenetic modulator EZH2 and oncogenic TGF-β signaling involving novel miR-490-3p/TGIF2/TGFBR1 axis, that being targetable might be promising in developing new therapeutic intervention strategies for GBM., (© 2019 UICC.)

A growing body of evidence implicates aberrant expression of microRNAs (miRNAs) and dysregulation of mRNA translation in the development and growth of cancer cells. However, little is known about the mechanisms of action of miRNAs in glioma, the most common form of adult‑onset malignant brain tumor. In the present study, the expression and function of miR‑767‑5p were examined in human glioblastoma multiforme (GBM) tissue specimens and cell lines. miR‑767‑5p expression levels were analyzed by quantitative reverse‑transcription PCR; cell proliferation was assessed by CCK‑8, colony formation and 5‑ethynyl‑2'‑deoxyuridine (EDU) assays; the cell cycle phase and apoptosis were detected by flow cytometry; and cell invasiveness was analyzed using wound healing and Transwell invasion assays. It was revealed found that miR‑767‑5p was significantly upregulated in GBM tissues (n=18) compared with normal brain tissues (n=8) and in 6 GBM cell lines compared with normal human astrocytes. Ectopic expression of miR‑767‑5p suppressed proliferation, colony formation, and migration, and promoted cell cycle arrest and apoptosis in GBM cell lines in vitro, and inhibited GBM tumor growth in a mouse xenograft model. Bioinformatics analysis identified the PRC2 component suppressor of zeste‑12 (SUZ12) as a putative target of miR‑767‑5p. Co‑transfection of miR‑767‑5p inhibited the activity of a luciferase reporter construct driven by the wild‑type 3' untranslated region of SUZ12 mRNA, but this was abolished by mutation of the putative miR‑767‑5p‑binding sites. Consistent with the possibility that miR‑767‑5p acts by regulating SUZ12 expression, it was revealed that the inhibitory effects of miR‑767‑5p on GBM cell phenotypes were reversed by overexpression of SUZ12. Our results indicated that forced upregulation of miR‑767‑5p may represent a novel therapeutic strategy for glioma patients by targeting SUZ12.

T cell senescence and exhaustion are major barriers to successful cancer immunotherapy. Here we show that miR-155 increases CD8 + T cell antitumor function by restraining T cell senescence and functional exhaustion through epigenetic silencing of drivers of terminal differentiation. miR-155 enhances Polycomb repressor complex 2 (PRC2) activity indirectly by promoting the expression of the PRC2-associated factor Phf19 through downregulation of the Akt inhibitor, Ship1. Phf19 orchestrates a transcriptional program extensively shared with miR-155 to restrain T cell senescence and sustain CD8 + T cell antitumor responses. These effects rely on Phf19 histone-binding capacity, which is critical for the recruitment of PRC2 to the target chromatin. These findings establish the miR-155-Phf19-PRC2 as a pivotal axis regulating CD8 + T cell differentiation, thereby paving new ways for potentiating cancer immunotherapy through epigenetic reprogramming of CD8 + T cell fate.

The reduced expression of cardiac sarco-endoplasmic reticulum Ca 2+ ATPase (SERCA2a) is a hallmark of heart failure. We previously showed that miR-25 is a crucial transcriptional regulator of SERCA2a in the heart. However, the precise mechanism of cardiac miR-25 regulation is largely unknown. Literatures suggested that miR-25 is regulated by the transcriptional co-factor, sine oculis homeobox homolog 1 (Six1), which in turn is epigenetically regulated by polycomb repressive complex 2 (PRC 2) in cardiac progenitor cells. Therefore, we aimed to investigate whether Six1 and PRC2 are indeed involved in the regulation of the miR-25 level in the setting of heart failure. Six1 was up-regulated in the failing hearts of humans and mice. Overexpression of Six1 led to adverse cardiac remodeling, whereas knock-down of Six1 attenuated pressure overload-induced cardiac dysfunction. The adverse effects of Six1 were ameliorated by knock-down of miR-25. The epigenetic repression on the Six1 promoter by PRC2 was significantly reduced in failing hearts. Epigenetic repression of Six1 is relieved through a reduction of PRC2 activity in heart failure. Six1 up-regulates miR-25, which is followed by reduction of cardiac SERCA2a expression. Collectively, these data showed that the PRC2-Six1-miR-25 signaling axis is involved in heart failure. Our finding introduces new insight into potential treatments of heart failure., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Gene expression can be regulated at multiple levels, but it is not known if and how there is broad coordination between regulation at the transcriptional and post-transcriptional levels. Transcription factors and chromatin regulate gene expression transcriptionally, whereas microRNAs (miRNAs) are small regulatory RNAs that function post-transcriptionally. Systematically identifying the post-transcriptional targets of miRNAs and the mechanism of transcriptional regulation of the same targets can shed light on regulatory networks connecting transcriptional and post-transcriptional control. We used individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) for the RNA-induced silencing complex (RISC) component AGO2 and global miRNA depletion to identify genes directly targeted by miRNAs. We found that Polycomb repressive complex 2 (PRC2) and its associated histone mark, H3K27me3, is enriched at hundreds of miRNA-repressed genes. We show that these genes are directly repressed by PRC2 and constitute a significant proportion of direct PRC2 targets. For just over half of the genes corepressed by PRC2 and miRNAs, PRC2 promotes their miRNA-mediated repression by increasing expression of the miRNAs that are likely to target them. miRNAs also repress the remainder of the PRC2 target genes, but independently of PRC2. Thus, miRNAs post-transcriptionally reinforce silencing of PRC2-repressed genes that are inefficiently repressed at the level of chromatin, by either forming a feed-forward regulatory network with PRC2 or repressing them independently of PRC2., (© 2019 Shivram et al.; Published by Cold Spring Harbor Laboratory Press.)

Aging is a prominent risk factor for neurodegenerative disease. Defining gene expression mechanisms affecting healthy brain aging should lead to insight into genes that modulate susceptibility to disease. To define such mechanisms, we have pursued analysis of miR-34 mutants in Drosophila. The miR-34 mutant brain displays a gene expression profile of accelerated aging, and miR-34 upregulation is a potent suppressor of polyglutamine-induced neurodegeneration. We demonstrate that Pcl and Su(z)12, two components of polycomb repressive complex 2, (PRC2), are targets of miR-34, with implications for age-associated processes. Because PRC2 confers the repressive H3K27me3 mark, we hypothesize that miR-34 modulates PRC2 activity to relieve silencing of genes promoting healthful aging. Gene expression profiling of the brains of hypomorphic mutants in Enhancer of zeste (E(z)), the enzymatic methyltransferase component of PRC2, revealed a younger brain transcriptome profile and identified the small heat shock proteins as key genes reduced in expression with age.

The ability to convert human somatic cells efficiently to neurons facilitates the utility of patient-derived neurons for studying neurological disorders. As such, ectopic expression of neuronal microRNAs (miRNAs), miR-9/9 ∗ and miR-124 (miR-9/9 ∗ -124) in adult human fibroblasts has been found to evoke extensive reconfigurations of the chromatin and direct the fate conversion to neurons. However, how miR-9/9 ∗ -124 break the cell fate barrier to activate the neuronal program remains to be defined. Here, we identified an anti-neurogenic function of EZH2 in fibroblasts that acts outside its role as a subunit of Polycomb Repressive Complex 2 to directly methylate and stabilize REST, a transcriptional repressor of neuronal genes. During neuronal conversion, miR-9/9 ∗ -124 induced the repression of the EZH2-REST axis by downregulating USP14, accounting for the opening of chromatin regions harboring REST binding sites. Our findings underscore the interplay between miRNAs and protein stability cascade underlying the activation of neuronal program., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Dysregulation of human trophoblast invasion and differentiation with placental hypoxia can result in preeclampsia, a hypertensive disorder of pregnancy. Herein, we characterized the role and regulation of miR-1246, which is markedly induced during human syncytiotrophoblast differentiation. miR-1246 targets GSK3β and AXIN2, inhibitors of WNT/β-catenin signaling, which is crucial for placental development, and is predicted to target JARID2, which promotes silencing of developmentally regulated genes. Human cytotrophoblasts cultured in 20% O2 spontaneously differentiate to syncytiotrophoblast with induction of hCYP191A/aromatase, a marker of differentiation. miR-1246 was induced >150-fold during syncytiotrophoblast differentiation in 20% O2, whereas targets-GSK3β, AXIN2, and JARID2-were significantly decreased. However, when cytotrophoblasts were cultured in 2% O2, miR-1246 and aromatase induction were prevented. miR-1246 was significantly decreased in placentas of women with severe preeclampsia, whereas AXIN2, GSK3β, and JARID2 were increased, compared with normotensive subjects. To identify factors that regulate miR-1246, we investigated the redox-regulated transcription factor NRF2, which has predicted binding sites in the miR-1246 promoter. Intriguingly, NRF2 messenger RNA was upregulated during syncytiotrophoblast differentiation and significantly reduced by hypoxia and in preeclamptic placentas. Moreover, NRF2 knockdown in cytotrophoblasts inhibited induction of miR-1246 and hCYP19A1, as well as transcription factors C/EBPβ and PPARγ, which are implicated in placental differentiation. Using chromatin immunoprecipitation-quantitative polymerase chain reaction, we found that binding of endogenous NRF2 to the miR-1246 and hCYP191A promoters increased during syncytiotrophoblast differentiation. Thus, NRF2 promotes syncytiotrophoblast differentiation by inducing C/EBPβ, PPARγ, hCYP19A1, and miR-1246, which targets WNT inhibitors and JARID2 and is dysregulated in preeclampsia.

The frequently dysregulated Wnt/β-catenin signaling in different malignancies, by activation of its own or orchestration with other co-factors, regulates various oncogenic or tumor-suppressive genes. Among these genes, miRNAs, which are negative posttranscriptional regulators, are also embedded in the Wnt signaling network. Different from the Wnt-induced oncogenic miRNAs, the specific mechanism underlying the Wnt-repressed tumor-suppressive miRNAs is much less understood. In our study, firstly by analyzing a ChIP-seq dataset against TCF4, the core transcription factor for initiation of Wnt signaling in colorectal cancer (CRC) cells, we screened out several tumor-suppressive miRNAs potentially regulated by Wnt signaling. Then through siRNA-mediated knock-down tests and protein and chromatin immunoprecipitations, we found the TCF4-β-catenin complex can recruit the histone trimethylation complex PRC2 as a co-repressor while binding to the TCF4-binding element (TBE) in the promoter regions of miR-145, miR-132 and miR-212. Thus, upon Wnt signaling activation, the PRC2-mediated trimethylation of histone H3 at lysine 27 increases at these promoter regions, leading to decreased miRNA levels. Furthermore, we found that by targeting TCF4 and SUZ12, the key components of the negative regulation complexes, the tumor-suppressive miR-145 co-repressed by Wnt signaling and histone trimethylation, forms double-negative regulation loops with its negative regulators in CRC cells. And the inverse associations between miR-145 and its targets/negative regulators have also been demonstrated in nude mice and clinical samples. Collectively, we elucidated the detailed molecular mechanism of how dysregulated Wnt/β-catenin signaling and tumor-suppressive miRNAs reciprocally regulate each other in CRC cells., (© 2017 UICC.)

MicroRNAs (miRNAs) play an important role in regulating immune system function by mRNA destabilisation or inhibition of translation. Recently, miR-155 was detected to be significantly up-regulated in colonic tissues of patients with active UC. However, it is unknown whether miR-155 is involved in the pathogenesis of UC and how it influences immune response in dextran sulfate sodium (DSS)-induced colitis mice. Here, we investigated the role of miR-155 in UC. Firstly, through bioinformatics analysis and luciferase report assay, we found Jarid2 was a direct target of miR-155; then, we carried out in situ hybridization, immunofluorescence and flow cytometry, and revealed that miR-155 levels were increased, Jarid2 levels were decreased and the frequency of Th17 cells was elevated in DSS-induced mice; we also used lentiviral vector to deliver miR-155 inhibition sequences to silence miR-155 that was effectively taken up by epithelial cells. MiR-155 inhibition attenuated DSS-induced colonic damage and inhibited Th17 cells differentiation. This study suggests that miR-155 plays a host-damaging role during DSS-induced colitis mice and induces Th17 differentiation by targeting Jarid2., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Rationale: Direct reprogramming of cardiac fibroblasts to cardiomyocytes has recently emerged as a novel and promising approach to regenerate the injured myocardium. We have previously demonstrated the feasibility of this approach in vitro and in vivo using a combination of 4 microRNAs (miR-1, miR-133, miR-208, and miR-499) that we named miR combo. However, the mechanism of miR combo mediated direct cardiac reprogramming is currently unknown., Objective: Here, we investigated the possibility that miR combo initiated direct cardiac reprogramming through an epigenetic mechanism., Methods and Results: Using a quantitative polymerase chain reaction array, we found that histone methyltransferases and demethylases that regulate the trimethylation of H3K27 (H3K27me3), an epigenetic modification that marks transcriptional repression, were changed in miR combo-treated fibroblasts. Accordingly, global H3K27me3 levels were downregulated by miR combo treatment. In particular, the promoter region of cardiac transcription factors showed decreased H3K27me3 as revealed by chromatin immunoprecipitation coupled with quantitative polymerase chain reaction. Inhibition of H3K27 methyltransferases or of the PRC2 (Polycomb Repressive Complex 2) by pharmaceutical inhibition or siRNA reduced the levels of H3K27me3 and induced cardiogenic markers at the RNA and protein level, similarly to miR combo treatment. In contrast, knockdown of the H3K27 demethylases Kdm6A and Kdm6B restored the levels of H3K27me3 and blocked the induction of cardiac gene expression in miR combo-treated fibroblasts., Conclusions: In summary, we demonstrated that removal of the repressive mark H3K27me3 is essential for the induction of cardiac reprogramming by miR combo. Our data not only highlight the importance of regulating the epigenetic landscape during cell fate conversion but also provide a framework to improve this technique., (© 2017 American Heart Association, Inc.)

Thousands of long non-coding RNAs (lncRNAs) have been identified in mammals, many of which represent important regulators of gene expression. However, the mechanisms used by lncRNAs to control transcription remain largely uncharacterized. Here, we report on HOTAIRM1, a promising lncRNA biomarker in leukemia and solid tumors. We find that HOTAIRM1 contributes to three-dimensional chromatin organization changes required for the temporal collinear activation of HOXA genes. We show that distinct HOTAIRM1 variants preferentially associate with either UTX/MLL or PRC2 complexes to modulate the levels of activating and silencing marks at the bivalent domain. HOTAIRM1 contributes to physical dissociation of chromatin loops at the cluster proximal end, which delays recruitment of the histone demethylase UTX and transcription of central HOXA genes. Interestingly, we find overall proximal HOXA gene activation without chromatin conformation changes by HOTAIRM1 in a different cell type. Our results reveal a previously unappreciated relationship between chromatin structure, architecture and lncRNA function.

Histone methylation is implicated in a number of biological and pathological processes, including cancer development. In this study, we investigated the molecular mechanism for the recruitment of Polycomb repressive complex-2 (PRC2) and its accessory component, JARID2, to chromatin, which regulates methylation of lysine 27 of histone H3 (H3K27), during epithelial-mesenchymal transition (EMT) of cancer cells. The expression of MEG3 long noncoding RNA (lncRNA), which could interact with JARID2, was clearly increased during transforming growth factor-β (TGF-β)-induced EMT of human lung cancer cell lines. Knockdown of MEG3 inhibited TGF-β-mediated changes in cell morphology and cell motility characteristic of EMT and counteracted TGF-β-dependent changes in the expression of EMT-related genes such as CDH1, ZEB family, and the microRNA-200 family. Overexpression of MEG3 influenced the expression of these genes and enhanced the effects of TGF-β in their expressions. Chromatin immunoprecipitation (ChIP) revealed that MEG3 regulated the recruitment of JARID2 and EZH2 and histone H3 methylation on the regulatory regions of CDH1 and microRNA-200 family genes for transcriptional repression. RNA immunoprecipitation and chromatin isolation by RNA purification assays indicated that MEG3 could associate with JARID2 and the regulatory regions of target genes to recruit the complex. This study demonstrated a crucial role of MEG3 lncRNA in the epigenetic regulation of the EMT process in lung cancer cells., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Whole transcriptome analyses in many organisms have revealed that most transcribed RNAs do not encode proteins. These non-coding RNAs likely contribute to the regulation of gene expression during the development of multicellular organisms. In eukaryotes, the roles of small RNAs, one class of non-coding RNAs, in transcriptional and post-transcriptional regulation have been well characterized. However, the functions of a second class of non-coding RNAs, long intergenic noncoding (linc) RNAs, are relatively unknown, especially in plants. Recent advances in RNA-seq and tiling microarray technologies have revealed the presence of many lincRNAs across plant species. This review focuses on the functions of lincRNAs that have been recently reported in plants. One of the most well characterized functions of lincRNAs is to epigenetically regulate gene expression by recruiting proteins for chromosome modification to specific loci. Second, lincRNAs are known to inhibit the physical interaction between microRNAs (miRNAs) and their target mRNAs thus controling protein levels of the target mRNAs. Lastly, lincRNAs control alternative splicing by binding and sequestering the proteins required for alternative splicing.

The role of microRNA-214-3p (miR-214-3p) in cardiac fibrosis was not well illustrated. The present study aimed to investigate the expression and potential target of miR-214-3p in angiotensin II (Ang-II)-induced cardiac fibrosis. MiR-214-3p was markedly decreased in the fibrotic myocardium of a mouse Ang-II infusion model, but was upregulated in Ang-II-treated mouse myofibroblasts. Cardiac fibrosis was shown attenuated in Ang-II-infused mice received tail vein injection of miR-214-3p agomir. Consistently, miR-214-3p inhibited the expression of Col1a1 and Col3a1 in mouse myofibroblasts in vitro. MiR-214-3p could bind the 3'-UTRs of enhancer of zeste homolog 1 (EZH1) and -2, and suppressed EZH1 and -2 expressions at the transcriptional level. Functionally, miR-214-3p mimic, in parallel to EZH1 siRNA and EZH2 siRNA, could enhance peroxisome proliferator-activated receptor-γ (PPAR-γ) expression and inhibited the expression of Col1a1 and Col3a1 in myofibroblasts. In addition, enforced expression of EZH1 and -2, and knockdown of PPAR-γ resulted in the increase of Col1a1 and Col3a1 in myofibroblasts. Moreover, the NF-κB signal pathway was verified to mediate Ang-II-induced miR-214-3p expression in myofibroblasts. Taken together, our results revealed that EZH1 and -2 were novel targets of miR-214-3p, and miR-214-3p might be one potential miRNA for the prevention of cardiac fibrosis.

We aimed to determine the specific miRNA profile of tumor budding cells and investigate the potential role of miR-320a in invasion and metastasis of tongue squamous cell carcinoma (TSCC). We collected tumor budding cells and paired central tumor samples from five TSCC specimens with laser capture microdissection and examined the specimens using a miRNA microarray. The specific miRNA signature of tumor budding cells was identified. We found that miR-320a was dramatically decreased in tumor budding cells. Knockdown of miR-320a significantly enhanced migration and invasion of TSCC cell lines. Suz12 was shown to be a direct target of miR-320a. Similar results were also observed in nude mouse models. Multivariate analysis indicated that miR-320a was an independent prognostic factor. Kaplan-Meier analysis demonstrated that decreased miR-320a and high intensity of tumor budding were correlated with poor survival rate, especially in the subgroup with high-intensity tumor budding and low expression of miR-320a. We concluded that decreased expression of miR-320a could promote invasion and metastasis of tumor budding cells by targeting Suz12 in TSCC. A combination of tumor budding and miR-320a may serve as an index to identify an aggressive sub-population of TSCC cells with high metastatic potential.

Fetal liver hematopoietic stem cells (HSCs) seed bone marrow (BM) and undergo reprograming into adult-type HSCs that are largely quiescent and restricted in their self-renewal activity. Here we report that in the absence of the polycomb-group gene Ezh2, a cohort of fetal-specific genes, including let-7 target genes, were activated in BM hematopoietic stem/progenitor cells (HSPCs), leading to acquisition of fetal phenotypes by BM HSPCs, such as enhanced self-renewal activity and production of fetal-type lymphocytes. The Lin28b/let-7 pathway determines developmentally timed changes in HSPC programs. Of note, many of the fetal-specific let-7 target genes, including Lin28, appear to be transcriptionally repressed by Ezh2-mediated H3K27me3 in BM HSPCs, and Ezh2 loss results in their ectopic expression, particularly in hematologic malignancies that develop in the absence of Ezh2. These findings suggest that Ezh2 cooperates with let-7 microRNAs in silencing the fetal gene signature in BM HSPCs and restricts their transformation., (Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Enhancer of zeste homolog 2 (EZH2) catalyses histone H3 lysine 27 trimethylation (H3K27me3) to silence tumour-suppressor genes in hepatocellular carcinoma (HCC) but the process of locus-specific recruitment remains elusive. Here we investigated the transcription factors involved and the molecular consequences in HCC development. The genome-wide distribution of H3K27me3 was determined by chromatin immunoprecipitation coupled with high-throughput sequencing or promoter array analyses in HCC cells from hepatitis B virus (HBV) X protein transgenic mouse and human cell models. Transcription factor binding site analysis was performed to identify EZH2-interacting transcription factors followed by functional characterization. Our cross-species integrative analysis revealed a crucial link between Yin Yang 1 (YY1) and EZH2-mediated H3K27me3 in HCC. Gene expression analysis of human HBV-associated HCC specimens demonstrated concordant overexpression of YY1 and EZH2, which correlated with poor survival of patients in advanced stages. The YY1 binding motif was significantly enriched in both in vivo and in vitro H3K27me3-occupied genes, including genes for 15 tumour-suppressive microRNAs. Knockdown of YY1 reduced not only global H3K27me3 levels, but also EZH2 and H3K27me3 promoter occupancy and DNA methylation, leading to the transcriptional up-regulation of microRNA-9 isoforms in HCC cells. Concurrent EZH2 knockdown and 5-aza-2'-deoxycytidine treatment synergistically increased the levels of microRNA-9, which reduced the expression and transcriptional activity of nuclear factor-κB (NF-κB). Functionally, YY1 promoted HCC tumourigenicity and inhibited apoptosis of HCC cells, at least partially through NF-κB activation. In conclusion, YY1 overexpression contributes to EZH2 recruitment for H3K27me3-mediated silencing of tumour-suppressive microRNAs, thereby activating NF-κB signalling in hepatocarcinogenesis., (Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Chemotherapeutic insensitivity remains a major obstacle to treating osteosarcoma effectively. Recently, increasing evidence has suggested that microRNAs (miRNAs) are involved in drug resistance. However, the effect of miR-138 on cisplatin chemoresistance in osteosarcoma has not been reported. We used real-time PCR to detect the expression of mature miR-138 in osteosarcoma tissues and cell lines. Cell proliferation, invasion, and migration assays were used to observe changes to the osteosarcoma malignant phenotype. MiR-138 was downregulated in osteosarcoma tissues and cell lines, and miR-138 overexpression negatively regulated osteosarcoma cell proliferation, migration, and invasion. We also verified that EZH2 is a direct target of miR-138. Furthermore, enhancing EZH2 expression reduced the inhibitory effects of miR-138 on osteosarcoma. Proliferation, apoptosis assays and caspase-3 activity assay confirmed that elevated miR-138 expression enhanced osteosarcoma cell chemosensitivity to cisplatin by targeting EZH2. In conclusion, the present study demonstrates that miR-138 acts as a tumor suppressor by enhancing osteosarcoma cell chemosensitivity and supports its potential application for treating osteosarcoma in the future.

Accumulating evidence demonstrates the important roles of microRNAs (miRNAs) in tumor development and progression. miR-26a has been reported to be downregulated in several types of cancers including hepatocellular carcinoma, but the underlying mechanism of how miR-26a is repressed remains largely unknown. In the present study, we performed western blot analysis, qRT-PCR, luciferase reporter assay and chromatin immunoprecipitation assay to investigate the relationship between miR-26a and the enhancer of zest homologue 2 (EZH2). CCK-8 assay and colony formation assay were carried out to explore the effect of miR-26a on HCC cells proliferation. We demonstrated that miR-26a was epigenetically repressed by EZH2-mediated H3K27 trimethylation within the miR-26a promoter. Moreover, we confirmed that EZH2 was also a direct target of miR-26a in HCC cells, thus, creating a double-negative feedback loop. Furthermore, miR-26a restoration increased the expressions of its host genes (CTDSPL and CTDSP2). Overexpression of EZH2 abrogated miR-26a induction of CTDSPL and CTDSP2. Restoring the balance of the double-negative feedback loop by miR-26a overpression or EZH2 silence significantly inhibited HCC cell growth. Overexpression of EZH2 rescued the growth inhibition effect of miR-26a. These findings suggest that an imbalanced double-negative feedback loop between EZH2 and miR-26a exists in HCC cells, which contributes to miR-26a deregulation and regulates tumor cells proliferation.

Laryngeal squamous cell carcinoma (LSCC) is a common malignant tumor of the otolaryngeal region and accounts for 1-2% of all malignancies diagnosed worldwide. miR-340 down-regulation and EZH2 up-regulation have been frequently identified in multiple cancers, but the role of miR-340 and EZH2 in LSCC has not been explored. In this study, we investigated the regulative role of miR-340 in EZH2 expression and LSCC progression. The results showed that EZH2 was up-regulated and miR-340 was down-regulated in both Hep-2 cells and LSCC tissues. Molecularly, our results confirmed that miR-340 directly targeted EZH2 gene and inhibited EZH2 expression. MTT assay and BrdU assay showed that miR-340 transfection reduced the cell proliferation ability of Hep-2 cells. The transwell assay indicated that the invasion and migration ability of Hep-2 cells was dramatically inhibited by miR-340 transfection. In addition, miR-340 transfection induced cell apoptosis with concomitant enhancement of Bax, increase of Caspase-3 expression and activity, and reduction of Bcl-2 expression in Hep-2 cells. Both miR-340 transfection and EZH2 knockdown induced p27 expression and suppressed PI3K/Akt activation in Hep-2 cells. Strikingly, EZH2 knockdown reduced cell proliferation, and EZH2 overexpression significantly rescued the miR-340-mediated suppressive effect on cell proliferation. Moreover, miR-340 could obviously induce the inhibition of Hep-2 cell-derived tumor growth and EZH2/p27 expression ratio in vivo. Taken together, these data suggest that miR-340 impedes LSCC progression by targeting EZH2 with the possible mechanism to enhance the expression of anti-oncogene p27 and suppress PI3K/Akt activation, providing a novel target and a potential therapeutic pathway against LSCC., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Although the tumor suppressive role of miR-101 is well documented in hepatocellular carcinoma (HCC), how the expression of miR-101 itself is regulated remains elusive. In the present study, we demonstrated that the miR-101 precursor pre-miR-101-1 could be regulated by an important epigenetic regulator, the enhancer of zeste homolog 2 (EZH2). Reporter gene assays revealed that ectopic expression of EZH2 inhibited the transcriptional activities of miR-101-1 promoter. Subsequent analyses revealed that miR-101-1 directly represses the expression of EZH2, and miR-101-1 and EZH2 form a reciprocal negative feedback loop as indicated by the fact that ectopic mature miR-101 could induce endogenous pre-miR-101-1 expression. This mature miR-101-induced pre-miR-101 expression was specific to pre-miR-101-1 and depended on EZH2 activities. Moreover, our results also demonstrated that similar antitumor effects can be achieved either by ectopic miR-101 or EZH2 silencing in HCC cells. These findings show that elevated EZH2 contributes to miR-101 deregulation in HCC and highlight the coordinated role of miR-101 and EZH2 in hepatocarcinogenesis.

Background: Our previous study reported that microRNA-26a (miR-26a) inhibited tumor progression by inhibiting tumor angiogenesis and intratumoral macrophage infiltration in hepatocellular carcinoma (HCC). The direct roles of miR-26a on tumor cell invasion remain poorly understood. In this study, we aim to explore the mechanism of miR-26a in modulating epithelial-mesenchymal transition (EMT) in HCC., Methods: In vitro cell morphology and cell migration were compared between the hepatoma cell lines HCCLM3 and HepG2, which were established in the previous study. Overexpression and down-regulation of miR-26a were induced in these cell lines, and Western blot and immunofluorescence assays were used to detect the expression of EMT markers. Xenograft nude mouse models were used to observe tumor growth and pulmonary metastasis. Immunohistochemical assays were conducted to study the relationships between miR-26a expression and enhancer of zeste homolog 2 (EZH2) and E-cadherin expression in human HCC samples., Results: Down-regulation of miR-26a in HCCLM3 and HepG2 cells resulted in an EMT-like cell morphology and high motility in vitro and increased in tumor growth and pulmonary metastasis in vivo. Through down-regulation of EZH2 expression and up-regulation of E-cadherin expression, miR-26a inhibited the EMT process in vitro and in vivo. Luciferase reporter assay showed that miR-26a directly interacted with EZH2 messenger RNA (mRNA). Furthermore, the expression of miR-26a was positively correlated with E-cadherin expression and inversely correlated with EZH2 expression in human HCC tissue., Conclusions: miR-26a inhibited the EMT process in HCC by down-regulating EZH2 expression.

The tumor suppressor p53 and the transcriptional repressor Enhancer of Zeste Homolog 2 (EZH2) have both been implicated in the regulation of epithelial-mesenchymal transition (EMT) and tumor metastasis via their impacts on microRNA expression. Here, we report that mutant p53 (mutp53) promotes EMT in endometrial carcinoma (EC) by disrupting p68-Drosha complex assembly. Overexpression of mutp53 has the opposite effect of wild-type p53 (WTp53), repressing miR-26a expression by reducing pri-miR-26a-1 processing in p53-null EC cells. Re-expression of miR-26a in mutp53 EC cells decreases cell invasion and promotes mesenchymal-epithelial transition (MET). Rescuing miR-26a expression also inhibits EZH2, N-cadherin, Vimentin, and Snail expression and induces E-cadherin expression both in vitro and in vivo. Moreover, patients with higher serum miR-26a levels have a better survival rate. These results suggest that p53 gain-of-function mutations accelerate EC tumor progression and metastasis by interfering with Drosha and p68 binding and pri-miR-26a-1 processing, resulting in reduced miR-26a expression and EZH2 overexpression.

Bladder cancer (BC) is a highly prevalent disease, ranking fifth in the most common cancers worldwide. Various miRNAs have recently emerged as potential prognostic biomarkers in cancer. The miR-200 family, which repressed the epithelial-to-mesenchymal transition (EMT), is repressed in multiple advanced cancers. However, its expression and function in BC is still poorly understood. Here we show that miR-200 family displays increased expression, probably due to the activation of specific oncogenic signaling pathways, and reduced promoter methylation, in BC compared to normal bladder samples. Furthermore, we show that the expression of these miRNAs is decreased in high grade and stage tumors, and the down-regulation is associated with patient's poor clinical outcome. Our data indicate that the miR-200 family plays distinct roles in Non-Muscle (NMIBC) and Muscle-Invasive BC (MIBC). In MIBC, miR-200 expression post transcriptionally regulates EMT-promoting transcription factors ZEB1 and ZEB2, whereas suppresses BMI1 expression in NMIBC. Interestingly, we show that increased EZH2 and/or BMI1 expression repress the expression of miR-200 family members. Collectively, these findings support a model of BC progression through a coordinated action between the Polycomb Repression Complex (PRC) members repressing the miR-200 expression, which ultimately favors invasive BC development. Since pharmacological inhibition of EZH2 in BC cell lines lead to increased miR-200 expression, our findings may support new therapeutic strategies for BC clinical management.

Lung cancer is regarded as the leading cause of cancer-related deaths, and cigarette smoking is one of the strongest risk factors for the development of lung cancer. However, the mechanisms for cigarette smoke-induced lung carcinogenesis remain unclear. The present study investigated the effects of an miRNA (miR-217) on levels of an lncRNA (MALAT1) and examined the role of these factors in the epithelial-mesenchymal transition (EMT) induced by cigarette smoke extract (CSE) in human bronchial epithelial (HBE) cells. In these cells, CSE caused decreases of miR-217 levels and increases in lncRNA MALAT1 levels. Over-expression of miR-217 with a mimic attenuated the CSE-induced increase of MALAT1 levels, and reduction of miR-217 levels by an inhibitor enhanced expression of MALAT1. Moreover, the CSE-induced increase of MALAT1 expression was blocked by an miR-217 mimic, indicating that miR-217 negatively regulates MALAT1 expression. Knockdown of MALAT1 reversed CSE-induced increases of EZH2 (enhancer of zeste homolog 2) and H3K27me3 levels. In addition to the alteration from epithelial to spindle-like mesenchymal morphology, chronic exposure of HBE cells to CSE increased the levels of EZH2, H3K27me3, vimentin, and N-cadherin and decreased E-cadherin levels, effects that were reversed by MALAT1 siRNA or EZH2 siRNA. The results indicate that miR-217 regulation of EZH2/H3K27me3 via MALAT1 is involved in CSE-induced EMT and malignant transformation of HBE cells. The posttranscriptional silencing of MALAT1 by miR-217 provides a link, through EZH2, between ncRNAs and the EMT and establishes a mechanism for CSE-induced lung carcinogenesis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The oncoprotein EZH2, as a histone H3K27 methyltransferase, is frequently overexpressed in various cancer types. However, the mechanisms underlying its role in urinary bladder cancer (UBC) cells have not yet fully understood. Herein, we reported that honokiol, a biologically active biphenolic compound isolated from the Magnolia officinalis inhibited human UBC cell proliferation, survival, cancer stemness, migration, and invasion, through downregulation of EZH2 expression level, along with the reductions of MMP9, CD44, Sox2 and the induction of tumor suppressor miR-143. Either EZH2 overexpression or miR-143 inhibition could partially reverse honokiol-induced cell growth arrest and impaired clonogenicity. Importantly, it was first revealed that EZH2 could directly bind to the transcriptional regulatory region of miR-143 and repress its expression. Furthermore, honokiol treatment on T24 tumor xenografts confirmed its anticancer effects in vivo, including suppression tumor growth and tumor stemness, accompanied by the dysregulation of EZH2 and miR-143 expressions. Our data suggest a promising therapeutic option to develop drugs targeting EZH2/miR-143 axis, such as honokiol, for bladder cancer treatment.

Increasing evidence reveals that aberrant expression of microRNA contributes to the development and progression of colon cancer, but the roles of microRNA-506 (miR-506) in colon cancer remain elusive. Here, we demonstrated that miR-506 was down-regulated in colon cancer tissue and cells and that miR-506 expression was inversely correlated with EZH2 expression, tumor size, lymph node invasion, TNM stage and metastasis. A high level of miR-506 identified patients with a favorable prognosis. In vitro and in vivo experiments confirmed that miR-506 inhibits the proliferation and metastasis of colon cancer, and a luciferase reporter assay confirmed that EZH2 is a direct and functional target of miR-506 via the 3'UTR of EZH2. The restoration of EZH2 expression partially reversed the proliferation and invasion of miR-506-overexpressing colon cancer cells. Moreover, we confirmed that the miR-506-EZH2 axis inhibits proliferation and metastasis by activating/suppressing specific downstream tumor-associated genes and the Wnt/β-catenin signaling pathway. Taking together, our study sheds light on the role of miR-506 as a suppressor for tumor growth and metastasis and raises the intriguing possibility that miR-506 may serve as a new potential marker for monitoring and treating colon cancer.

Our previous study demonstrated that enhancer of zeste homolog 2 (EZH2) overexpression may be associated with aggressive tumor progression and poor prognosis in human astrocytoma. The aim of this study was to investigate the underlying mechanisms of EZH2 on astrocytoma tumorigenesis. An online program miRWalk (http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/) was used to predict possible microRNAs (miRNAs) that might target EZH2 messenger RNA (mRNA). Then the functions of the miRNA-EZH2 mRNA axis in astrocytoma cell proliferation, invasion, and migration were also assessed. We further evaluated the clinical value of the miRNA-EZH2 mRNA axis in astrocytomas. As a result, we identified EZH2 as a target gene of miR-144. In addition, forced expression of miR-144 suppressed astrocytoma cell proliferation, invasion, and migration by down-regulating EZH2. Moreover, miR-144 down-regulation and EZH2 mRNA up-regulation were both significantly associated with advanced World Health Organization grades and low Karnofsky performance status score of astrocytoma patients. Importantly, survival analysis identified the combined expression of miR-144 and EZH2 (miR-144/EZH2) as an independent prognostic factor for overall survival in astrocytoma patients. In conclusion, miR-144 may function as a tumor suppressor by regulating EZH2 expression, and miR-144/EZH2 expression may be a highly sensitive marker for the prognosis in astrocytoma patients., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: To investigate the effect of miRNA-101 on the expression of the enhancer of zeste homolog 2 (EXH2) in human androgen-independent prostated cancer LNCaP cells., Methods: We divided LNCaP cells into a blank control, a negative control, and a miRNA-l01 transfection group, constructed the vector by transfecting synthetic miRNA-101 mimics into the LNCaP cells, and evaluated the efficiency of transfection by fluorescence microscopy. Then we determined the expression level of EZH2 mRNA by qRT-PCR in the three groups of cells and that of the EZH2 protein in the negative control and transfection groups by Western blot., Results: Green fluorescence signals were observed in over 70% of the LNCaP cells in the transfection group after 24 hours of transfection. At 72 hours, the expression of miRNA-101 was significantly upregulated in the transfected cells (P < 0.01), that of EZH2 mRNA was remarkably lower in the transfection group (0.01 ± 0.10) than in the blank control (0.95 ± 0.40) and negative control (0.86 ± 0.30) groups (both P < 0.01), and that of the EZH2 protein was increased in the negative control but decreased in the transfection group with the extension of culture time., Conclusion: miRNA-101, with its inhibitory effect on the expression of EZH2 in LNCaP cells, is a potential biotherapeutic for prostate cancer.

microRNA-217 (miR-217) is frequently dysregulated in cancer. Here, we report that miR-217 levels were lower in tumor tissue compared with the adjacent normal tissue. Low levels of miR-217 were associated with aggressive tumor phenotypes and poor overall survival in gastric cancer patients. The ectopic expression of miR-217 inhibited cell proliferation, migration and invasion in vitro and tumor growth and metastasis in vivo, whereas knockdown of endogenous miR-217 increased cell proliferation and invasion. Further experiments revealed that Polycomb group protein enhancer of zeste homolog 2 (EZH2) was a direct target of miR-217 in gastric cancer cells. Knockdown of EZH2 mimicked the tumor-suppressive effects of miR-217 in gastric cancer cells, whereas the reintroduction of EZH2 abolished its effects. Taken together, these results demonstrated that miR-217 may be used as a prognostic marker, and the newly identified miR-217-EZH2 axis may be a potential target in the development of therapeutic strategies for gastric cancer patients.

It is suggested that microRNAs play important roles in the development of various cancers. Here, we showed that miR-137 is downregulated in glioblastoma (GBM) cell lines and that low levels of miR-137 are associated with a poor prognostic phenotype of GBM patients. Ectopic expression of miR-137 significantly inhibited GBM cell proliferation and angiogenesis. In addition, ectopic expression of miR-137 inhibited tumor growth and angiogenesis in a SCID mouse xenograft model. EZH2 was identified as a direct target of miR-137 by using luciferase reporter and Western blot assays, and EZH2 overexpression can rescue the inhibitory effect of miR-137 on cell proliferation and angiogenesis. Furthermore, tumor samples from GBM patients showed an inverse relationship between miR-137 and EZH2 levels. Our results suggest that miR-137 may serve as a biomarker in GBM, and the modulation of its activity may represent a novel therapeutic strategy for the treatment of GBM patients.