Investigating the synergy of Shikonin and Valproic acid in inducing apoptosis of osteosarcoma cells via ROS-mediated EGR1 expression.

BACKGROUND: Osteosarcoma is the most prevalent malignant bone tumour with a poor prognosis. Shikonin (SHK) is derived from the traditional Chinese medicine Lithospermum that has been extensively studied for its notable anti-tumour effects, including for osteosarcoma. However, its application has certain limitations. Valproic acid (VPA) is a histone deacetylase inhibitor (HDACI) that has recently been employed as an adjunctive therapeutic agent that allows chromatin to assume a more relaxed state, thereby enhancing anti-tumour efficacy. PURPOSE: This study was aimed to investigate the synergistic anti-tumour efficacy of SHK in combination with VPA and elucidate its underlying mechanism. METHODS/STUDY DESIGN: CCK-8 assays were utilized to calculate the combination index. Additional assays, including colony formation, acridine orange/ethidium bromide double fluorescent staining, and flow cytometry, were employed to evaluate the effects on osteosarcoma cells. Wound healing and transwell assays were utilized to assess cell mobility. RNA sequencing, PCR, and Western blot analyses were conducted to uncover the underlying mechanism. Rescue experiments were performed to validate the mechanism of apoptotic induction. The impact of SHK and VPA combination treatment on primary osteosarcoma cells was also assessed. Finally, in vivo experiments were conducted to validate its anti-tumour effects and mechanism. RESULTS: The combination of SHK and VPA synergistically inhibited the proliferation and migration of osteosarcoma cells in vitro and induced apoptosis in these cells. Through a comprehensive analysis involving RNA sequencing, PCR, Western blot, and rescue experiments, we have substantiated our hypothesis that the combination of SHK and VPA induced apoptosis via the ROS-EGR1-Bax axis. Importantly, our in vivo experiments corroborated these findings, demonstrating the potential of the SHK and VPA combination as a promising therapeutic approach for osteosarcoma. CONCLUSION: The combination of SHK and VPA exerted an anti-tumour effect by inducing apoptosis through the ROS-EGR1-Bax pathway. Repurposing the old drug VPA demonstrated its effectiveness as an adjunctive therapeutic agent for SHK, enhancing its anti-tumour efficacy and revealing its potential value. Furthermore, our study expanded the application of natural compounds in the anti-tumour field and overcame some of their limitations through combination therapy. Finally, we enhanced the understanding of the mechanistic pathways linking reactive oxygen species (ROS) accumulation and apoptosis in osteosarcoma cells. Additionally, we elucidated the role of EGR1 in osteosarcoma cells, offering novel strategies and concepts for the treatment of osteosarcoma.

Lentinan induces apoptosis of mouse hepatocellular carcinoma cells through the EGR1/PTEN/AKT signaling axis.

Lentinan (LNT) isolated from Lentinus edodes is a vital host defense potentiator previously utilized as an adjuvant in cancer therapy. The present study investigated the effect of LNT on the mouse hepatocellular carcinoma (HCC) cell line Hepa1­6 and its possible mechanism. Mouse HCC apoptosis and its potential associated mechanism were then explored using in vitro and in vivo approaches. For in vitro approaches, the effect of LNT on the proliferation of Hepa1­6 cells was investigated by Cell Counting Kit­8 assay. Annexin V­FITC staining and flow cytometry were applied to explore HCC apoptosis. Western blotting was used to analyze related proteins, such as EGR1, phosphatase and tensin homolog (PTEN), phosphorylated protein kinase B (p­Akt), protein kinase B (Akt), B lymphocyte­2 (Bcl­2), Bcl2 family­associated X protein (Bax), etc. Cellular immunofluorescence staining was employed to assess the localization and expression of EGR1 and PTEN in nuclear and cytoplasmic fractions of Hepa1­6 cells. The association between EGR1 and PTEN was explored by EGR1 overexpression in cell lines. For in vivo methods, a mouse model of diethylnitrosamine (DEN)­induced primary liver cancer was established using C57BL/6 mice to investigate the inhibitory effect of LNT on liver cancer. Histopathology of liver tissue from mice was detected by hematoxylin­eosin staining and immunohistochemical assay. In vitro and in vivo results showed that LNT can inhibit the proliferation and promote the apoptosis of mouse HCC cells. Besides, LNT increased the expression of EGR1 in Hepa1­6 cells, which is translocated to the nucleus to function as a transcriptional factor. EGR1 then activates the expression of the tumor suppressor PTEN, thereby inhibiting the activation of the AKT signaling pathway. These data revealed a novel anti­tumor mechanism by which LNT can induce apoptosis to inhibit mouse HCC progression through the EGR1/PTEN/AKT axis. These results provide a scientific basis for the potential use of LNT in drug development and clinical applications associated with primary liver cancer.

Polyrhachis vicina Roger Alleviates Memory Impairment in a Rat Model of Alzheimer's Disease Through the EGR1/BACE1/APP Axis.

Memory deficits and loss are the earliest and most prominent features of Alzheimer's disease (AD). This study was aimed to clarify the mechanistic basis of an active fraction of Polyrhachis vicina Roger (AFPR) on the memory abilities of AD rat models, which involves early growth response 1 (EGR1) expression and ß-secretase 1 (BACE1)-mediated deposition of amyloid ß peptide (Aß). An AD rat model was developed by Aß25-35, which was further treated with AFPR alone or in combination with lentiviral EGR1. The Morris water maze test and HE and Fluoro-Jade C staining were adopted to observe the memory behaviors, hippocampus neuron morphology, and Aß deposition. Aß25-35-induced SK-N-SH and HT22 neurons were subjected to AFPR for in vitro experiments on neuronal viability and apoptosis. AFPR improved the impaired memory function, preserved the neuron structure, and suppressed Aß deposition in AD rat models. Further, the expression of APP pathway-related proteins was downregulated by AFPR in both rat and cellular models. Moreover, AFPR inhibited the Aß25-35-induced neuronal apoptosis. AFPR suppressed the expression of EGR1, downregulated the BACE1 expression via impeding the binding of EGR1 to the BACE1 promoter, and thus blocked the activation of the APP signaling, ultimately protecting neurons. Notably, the aforementioned effects of AFPR were in a concentration-dependent manner; among three doses, 3.65, 15.6, and 30 mg/(kg·d), high-dose AFPR exhibited the most appreciable effects. In conclusion, AFPR inhibited the BACE1 expression by repressing the binding of EGR1 to the promoter of BACE1, thereby suppressing the Aß deposition and improving the memory function of AD rats.

Key Disease Mechanisms Linked to Alzheimer's Disease in the Entorhinal Cortex.

Alzheimer's disease (AD) is a chronic, neurodegenerative brain disorder affecting millions of Americans that is expected to increase in incidence with the expanding aging population. Symptomatic AD patients show cognitive decline and often develop neuropsychiatric symptoms due to the accumulation of insoluble proteins that produce plaques and tangles seen in the brain at autopsy. Unexpectedly, some clinically normal individuals also show AD pathology in the brain at autopsy (asymptomatic AD, AsymAD). In this study, SWItchMiner software was used to identify key switch genes in the brain's entorhinal cortex that lead to the development of AD or disease resilience. Seventy-two switch genes were identified that are differentially expressed in AD patients compared to healthy controls. These genes are involved in inflammation, platelet activation, and phospholipase D and estrogen signaling. Peroxisome proliferator-activated receptor γ (PPARG), zinc-finger transcription factor (YY1), sterol regulatory element-binding transcription factor 2 (SREBF2), and early growth response 1 (EGR1) were identified as transcription factors that potentially regulate switch genes in AD. Comparing AD patients to AsymAD individuals revealed 51 switch genes; PPARG as a potential regulator of these genes, and platelet activation and phospholipase D as critical signaling pathways. Chemical-protein interaction analysis revealed that valproic acid is a therapeutic agent that could prevent AD from progressing.

Inhibition of EGR-1-dependent MMP1 transcription by ethanol extract of Ageratum houstonianum in HaCaT keratinocytes.

Matrix metalloproteinase 1 (MMP-1) initiates the breakdown of matrix networks by cleaving fibrillar collagen during the pathophysiological progression of skin aging. Ageratum houstonianum ethanol extract (AHE) has been used as a traditional herbal medicine to treat external wounds and skin diseases. However, the mechanism of action underlying A. houstonianum-mediated modulation of skin aging has not been investigated. In this study, we evaluated the effect of AHE on MMP-1 expression in HaCaT keratinocytes. Gene expression was analyzed by Reverse transcription-PCR (RT-PCR), Quantitative real-time PCR (Q-PCR), gene promoter-reporter assay, and immunoblotting. We found that AHE abrogated TNFα-induced MMP1 expression at the transcriptional level via the suppression of ERK1/2 mitogen-activated protein kinase (MAPK)-mediated Early Growth Response 1 (EGR1) expression. We also demonstrated that ß-caryophyllene, a cannabinoid receptor 2 (CB2) agonist, is a functional component of the AHE that inhibits TNFα-induced EGR-1 and MMP1 expression. AHE exerts inhibitory activity on TNFα-induced MMP1 expression at the transcription level through EGR-1 downregulation in keratinocytes. ß-Caryophyllene is a bioactive ingredient of AHE that is responsible for the inhibition of TNFα-induced EGR1 expression. ß-Caryophyllene can be used as a potential agent to prevent inflammation-induced skin aging.

The anxiety and ethanol intake controlling GAL5.1 enhancer is epigenetically modulated by, and controls preference for, high-fat diet.

Excess maternal fat intake and obesity increase offspring susceptibility to conditions such as chronic anxiety and substance abuse. We hypothesised that environmentally modulated DNA methylation changes (5mC/5hmC) in regulatory regions of the genome that modulate mood and consumptive behaviours could contribute to susceptibility to these conditions. We explored the effects of environmental factors on 5mC/5hmC levels within the GAL5.1 enhancer that controls anxiety-related behaviours and alcohol intake. We first observed that 5mC/5hmC levels within the GAL5.1 enhancer differed significantly in different parts of the brain. Moreover, we noted that early life stress had no significant effect of 5mC/5hmC levels within GAL5.1. In contrast, we identified that allowing access of pregnant mothers to high-fat diet (> 60% calories from fat) had a significant effect on 5mC/5hmC levels within GAL5.1 in hypothalamus and amygdala of resulting male offspring. Cell transfection-based studies using GAL5.1 reporter plasmids showed that 5mC has a significant repressive effect on GAL5.1 activity and its response to known stimuli, such as EGR1 transcription factor expression and PKC agonism. Intriguingly, CRISPR-driven disruption of GAL5.1 from the mouse genome, although having negligible effects on metabolism or general appetite, significantly decreased intake of high-fat diet suggesting that GAL5.1, in addition to being epigenetically modulated by high-fat diet, also actively contributes to the consumption of high-fat diet suggesting its involvement in an environmentally influenced regulatory loop. Furthermore, considering that GAL5.1 also controls alcohol preference and anxiety these studies may provide a first glimpse into an epigenetically controlled mechanism that links maternal high-fat diet with transgenerational susceptibility to alcohol abuse and anxiety.

Gene expression analysis in epileptic hippocampi reveals a promoter haplotype conferring reduced aldehyde dehydrogenase 5a1 expression and responsiveness.

Increasing evidence indicates the pathogenetic relevance of regulatory genomic motifs for variability in the manifestation of brain disorders. In this context, cis-regulatory effects of single nucleotide polymorphisms (SNPs) on gene expression can contribute to changing transcript levels of excitability-relevant molecules and episodic seizure manifestation in epilepsy. Biopsy specimens of patients undergoing epilepsy surgery for seizure relief provide unique insights into the impact of promoter SNPs on corresponding mRNA expression. Here, we have scrutinized whether two linked regulatory SNPs (rs2744575; 4779C > G and rs4646830; 4854C > G) located in the aldehyde dehydrogenase 5a1 (succinic semialdehyde dehydrogenase; ALDH5A1) gene promoter are associated with expression of corresponding mRNAs in epileptic hippocampi (n = 43). The minor ALDH5A1-GG haplotype associates with significantly lower ALDH5A1 transcript abundance. Complementary in vitro analyses in neural cell cultures confirm this difference and further reveal a significantly constricted range for the minor ALDH5A1 haplotype of promoter activity regulation through the key epileptogenesis transcription factor Egr1 (early growth response 1). The present data suggest systematic analyses in human hippocampal tissue as a useful approach to unravel the impact of epilepsy candidate SNPs on associated gene expression. Aberrant ALDH5A1 promoter regulation in functional terms can contribute to impaired γ-aminobutyric acid homeostasis and thereby network excitability and seizure propensity.

Chrysoeriol Prevents TNFα-Induced CYP19 Gene Expression via EGR-1 Downregulation in MCF7 Breast Cancer Cells.

Estrogen overproduction is closely associated with the development of estrogen receptor-positive breast cancer. Aromatase, encoded by the cytochrome P450 19 (CYP19) gene, regulates estrogen biosynthesis. This study aimed to identify active flavones that inhibit CYP19 expression and to explore the underlying mechanisms. CYP19 expression was evaluated using reverse transcription PCR, quantitative real-time PCR, and immunoblot analysis. The role of transcription factor early growth response gene 1 (EGR-1) in CYP19 expression was assessed using the short-hairpin RNA (shRNA)-mediated knockdown of EGR-1 expression in estrogen receptor-positive MCF-7 breast cancer cells. We screened 39 flavonoids containing 26 flavones and 13 flavanones using the EGR1 promoter reporter activity assay and observed that chrysoeriol exerted the highest inhibitory activity on tumor necrosis factor alpha (TNFα)-induced EGR-1 expression. We further characterized and demonstrated that chrysoeriol inhibits TNFα-induced CYP19 expression through inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated EGR-1 expression. Chrysoeriol may be beneficial as a dietary supplement for the prevention of estrogen receptor-positive breast cancer, or as a chemotherapeutic adjuvant in the treatment of this condition.

Anti-proliferative activity of A. Oxyphylla and its bioactive constituent nootkatone in colorectal cancer cells.

BACKGROUND: A. oxyphylla extract is known to possess a wide range of pharmacological activites. However, the molecular mechanism of A. oxyphylla and its bioactive compound nootkatone in colorectal cancer is unknown. METHODS: Our study aims to examine the role of A. oxyphylla and its bioactive compound nootkatone, in tumor suppression using several in vitro assays. RESULTS: Both A. oxyphylla extract and nootkatone exhibited antiproliferative activity in colorectal cancer cells. A. oxyphylla displayed antioxidant activity in colorectal cancer cells, likely mediated via induction of HO-1. Furthermore, expression of pro-apoptotic protein NAG-1 and cell proliferative protein cyclin D1 were increased and decreased respectively in the presence of A. oxyphylla. When examined for anticancer activity, nootkatone treatment resulted in the reduction of colony and spheroid formation. Correspondingly, nootkatone also led to increased NAG-1 expression and decreased cyclin D1 expression. The mechanism by which nootkatone suppresses cyclin D1 involves protein level regulation, whereas nootkatone increases NAG-1 expression at the transcriptional level. In addition to having PPARγ binding activity, nootkatone also increases EGR-1 expression which ultimately results in enhanced NAG-1 promoter activity. CONCLUSION: In summary, our findings suggest that nootkatone is an anti-tumorigenic compound harboring antiproliferative and pro-apoptotic activity.

Fasting-mimicking diet and hormone therapy induce breast cancer regression.

Approximately 75% of all breast cancers express the oestrogen and/or progesterone receptors. Endocrine therapy is usually effective in these hormone-receptor-positive tumours, but primary and acquired resistance limits its long-term benefit1,2. Here we show that in mouse models of hormone-receptor-positive breast cancer, periodic fasting or a fasting-mimicking diet3-5 enhances the activity of the endocrine therapeutics tamoxifen and fulvestrant by lowering circulating IGF1, insulin and leptin and by inhibiting AKT-mTOR signalling via upregulation of EGR1 and PTEN. When fulvestrant is combined with palbociclib (a cyclin-dependent kinase 4/6 inhibitor), adding periodic cycles of a fasting-mimicking diet promotes long-lasting tumour regression and reverts acquired resistance to drug treatment. Moreover, both fasting and a fasting-mimicking diet prevent tamoxifen-induced endometrial hyperplasia. In patients with hormone-receptor-positive breast cancer receiving oestrogen therapy, cycles of a fasting-mimicking diet cause metabolic changes analogous to those observed in mice, including reduced levels of insulin, leptin and IGF1, with the last two remaining low for extended periods. In mice, these long-lasting effects are associated with long-term anti-cancer activity. These results support further clinical studies of a fasting-mimicking diet as an adjuvant to oestrogen therapy in hormone-receptor-positive breast cancer.

Time course of photo-induced Egr-1 expression in the hypothalamus of a seasonally breeding songbird.

Many seasonally-breeding species use daylength to time reproduction. Light-induced release of progonadal hormones involves a complex cascade of responses both inside and outside the brain. In this study, we used induction of early growth response 1 (Egr-1), the protein product of an immediate early gene, to evaluate the time course of such responses in male white-throated sparrows (Zonotrichia albicollis) exposed to a single long day. Induction of Egr-1 in the pars tuberalis began ∼11 h after dawn. This response was followed ∼6 h later by dramatic induction in the tuberal hypothalamus, including in the ependymal cells lining the third ventricle. At approximately the same time, Egr-1 was induced in dopaminergic and vasoactive intestinal peptide neurons in the tuberal hypothalamus and in dopaminergic neurons of the premammillary nucleus. We noted no induction in gonadotropin-releasing hormone (GnRH) neurons until 2 h after dawn the following morning. Overall, our results indicate that Egr-1 responses in GnRH neurons occur rather late during photostimulation, compared with responses in other cell populations, and that such induction may reflect new synthesis related to GnRH depletion rather than stimulation by light cues.

Balance between asymmetry and abundance in multi-domain DNA-binding proteins may regulate the kinetics of their binding to DNA.

DNA sequences are often recognized by multi-domain proteins that may have higher affinity and specificity than single-domain proteins. However, the higher affinity to DNA might be coupled with slower recognition kinetics. In this study, we address this balance between stability and kinetics for multi-domain Cys2His2- (C2H2-) type zinc-finger (ZF) proteins. These proteins are the most prevalent DNA-binding domain in eukaryotes and C2H2 type zinc-finger proteins (C2H2-ZFPs) constitute nearly one-half of all known and predicted transcription factors in human. Extensive contact with DNA via tandem ZF domains confers high stability on the sequence-specific complexes. However, this can limit target search efficiency, especially for low abundance ZFPs. Earlier, we found that asymmetrical distribution of electrostatic charge among the three ZF domains of the low abundance transcription factor Egr-1 facilitates its DNA search process. Here, on a diverse set of 273 human C2H2-ZFP comprised of 3-15 tandem ZF domains, we find that, in many cases, electrostatic charge and binding specificity are asymmetrically distributed among the ZF domains so that neighbouring domains have different DNA-binding properties. For proteins containing 3-6 ZF domains, we show that the low abundance proteins possess a higher degree of non-specific asymmetry and vice versa. Our findings suggest that where the electrostatics of tandem ZF domains are similar (i.e., symmetrical), the ZFPs are more abundant to optimize their DNA search efficiency. This study reveals new insights into the fundamental determinants of recognition by C2H2-ZFPs of their DNA binding sites in the cellular landscape. The importance of electrostatic asymmetry with respect to binding site recognition by C2H2-ZFPs suggests the possibility that it may also be important in other ZFP systems and reveals a new design feature for zinc finger engineering.

Low temperature induces von-willebrand factor expression via increased early growth response 1 transcriptional activity in splenic sinusoidal endothelial cells.

von Willebrand factor (vWF) is a large plasma glycoprotein that plays an important role in hemostasis by forming molecular bridges with platelets following vascular injury. Previously, we reported that hypothermia enhanced vWF production in the spleen, which resulted in the activation of the platelet pool in a hypothermia-induced murine model. However, the mechanisms that regulate vWF expression under hypothermic conditions remain unclear. In this study, we focused on vWF expression under hypothermic conditions in splenic endothelial cell culture. Human splenic endothelial cells (HSEC) were incubated at 20 °C for 1 h. Total RNA was extracted from the cells, and cDNA microarray gene expression analysis was performed. Genes that may be associated with vWF expression in low temperature culture conditions were then selected for further analysis. Gene expression analysis showed that low temperature conditions increased the expression of FOS and EGR1. We then hypothesized that these factors upregulate vWF mRNA expression in HSEC. The transcriptional inhibitors of EGR1 significantly inhibited vWF mRNA expression in HSEC cultured at a low temperature. Our analysis revealed that low temperatures enhance the gene expression of EGR1, which transcriptionally increases vWF expression. This acute-phase reaction may play an important role in platelet activation in the spleen during hypothermia.

Active fraction of Polyrhachis vicina Rogers (AFPR) suppressed breast cancer growth and progression via regulating EGR1/lncRNA-NKILA/NF-κB axis.

Breast cancer (BC) is a major contributor of cancer-associated mortality in women. It is essential to find new therapeutic targets and drugs. Polyrhachis vicina Rogers is one of the Traditional Chinese Medicine (TCM). Our previous studies have shown an active fraction of Polyrhachis vicina Rogers (AFPR) has significant anti-inflammatory activity, suggesting its anti-cancer effect. Here, we aimed to explore the inhibitory effects of AFPR on BC and reveal its mechanism. The effects of AFPR on BC were examined by cell proliferation assay, wound healing assay, invasion assay and xenograft assay. Microarray sequencing, qRT-PCR, Western blot, chromatin immunoprecipitation assay and luciferase reporter assay were performed to investigate the regulation of AFPR on related genes and underlying mechanisms. As a result, AFPR suppressed BC cell growth, migration and invasion and inhibited tumor growth. LncRNA NKILA was most prominently upregulated in AFPR-treated MCF7 cells. AFPR inactivated NF-κB signaling pathway via regulating NKILA. Furthermore, AFPR regulated the expression of NKILA by inhibiting its transcript suppressor EGR1. This study firstly indicated that AFPR was a potential inhibitor of BC development via regulating EGR1/NKILA/NF-κB axis.

Herbal formula Yangyinjiedu induces lung cancer cell apoptosis via activation of early growth response 1.

Traditional Chinese Medicine (TCM) has been extensively used in clinical practices and proven to be effective against cancer. However, the underlying mechanisms remain to be investigated. In this study, we examined the anticancer activities of Chinese herbal formula Yangyinjiedu (YYJD) and found that YYJD exhibits cytotoxicity against lung cancer cells. Transcriptome analysis indicated that 2178 genes were differentially expressed (P < 0.05) upon YYJD treatment, with 1464 being (67.2%) up-regulated. Among these, we found that the tumour suppressor early growth response 1 (EGR1) is the most activated. We demonstrated that EGR1 contributes to YYJD-induced apoptosis in A549. Through dissecting EGR1-associated transcriptional network, we identified 275 genes as EGR1 direct targets, some targets are involved in apoptosis. Lastly, we observed that YYJD enhances EGR1 expression and induces cell death in tumour xenografts. Collectively, these findings suggest that YYJD exerts its anticancer activities through EGR1 activation, thus providing the evidence for its potential clinical application for lung cancer patients.

Effect of Mongolian warm acupuncture on the gene expression profile of rats with insomnia.

BACKGROUND: The mechanism of Mongolian warm acupuncture (MWA) for the treatment of insomnia has not been previously reported. OBJECTIVE: To investigate the effect of MWA on gene expression profile in the p-chlorophenylalanine (PCPA)-induced rat model of insomnia. METHODS: A rat model of insomnia was established and the animals were divided into five groups: control, PCPA (untreated), PCPA+estazolam, PCPA+MA (manual acupuncture), and PCPA+MWA. The rats were euthanased at 7 days after treatment, and hypothalamic tissue was harvested to extract total RNA for the analysis of gene expression profile. Micro-array and Partek Genomics Suite analysis system were used to analyse differential expression of genes between groups. Furthermore, ingenuity pathways analysis was used to analyse the main regulators. RESULTS: After treatment, in rats with improved sleep, micro-array data from the follow-up phase compared with baseline showed that MWA down-regulated 11 genes compared with the control group and 16 genes compared with the PCPA group. Six genes were selected following the micro-array detection to perform quantitative polymerase chain reaction (qPCR) verification, and the results showed that the coincidence rate was up to 90%, which verified the reliability of the microarray results. Compared with the PCPA group, transcription levels of Egr 1, Btg2 and BDNF in the PCPA+MWA group were up-regulated (P<0.05). CONCLUSION: In combination, the findings of this study suggests that MWA is efficacious at improving sleep in an experimental rat model of insomnia.

The lncRNA MIR2052HG regulates ERα levels and aromatase inhibitor resistance through LMTK3 by recruiting EGR1.

BACKGROUND: Our previous genome-wide association study using the MA.27 aromatase inhibitors adjuvant trial identified SNPs in the long noncoding RNA MIR2052HG associated with breast cancer-free interval. MIR2052HG maintained ERα both by promoting AKT/FOXO3-mediated ESR1 transcription and by limiting ubiquitin-mediated ERα degradation. Our goal was to further elucidate MIR2052HG's mechanism of action. METHODS: RNA-binding protein immunoprecipitation assays were performed to demonstrate that the transcription factor, early growth response protein 1 (EGR1), worked together with MIR2052HG to regulate that lemur tyrosine kinase-3 (LMTK3) transcription in MCF7/AC1 and CAMA-1 cells. The location of EGR1 on the LMTK3 gene locus was mapped using chromatin immunoprecipitation assays. The co-localization of MIR2052HG RNA and the LMTK3 gene locus was determined using RNA-DNA dual fluorescent in situ hybridization. Single-nucleotide polymorphisms (SNP) effects were evaluated using a panel of human lymphoblastoid cell lines. RESULTS: MIR2052HG depletion in breast cancer cells results in a decrease in LMTK3 expression and cell growth. Mechanistically, MIR2052HG interacts with EGR1 and facilitates its recruitment to the LMTK3 promoter. LMTK3 sustains ERα levels by reducing protein kinase C (PKC) activity, resulting in increased ESR1 transcription mediated through AKT/FOXO3 and reduced ERα degradation mediated by the PKC/MEK/ERK/RSK1 pathway. MIR2052HG regulated LMTK3 in a SNP- and aromatase inhibitor-dependent fashion: the variant SNP increased EGR1 binding to LMTK3 promoter in response to androstenedione, relative to wild-type genotype, a pattern that can be reversed by aromatase inhibitor treatment. Finally, LMTK3 overexpression abolished the effect of MIR2052HG on PKC activity and ERα levels. CONCLUSIONS: Our findings support a model in which the MIR2052HG regulates LMTK3 via EGR1, and LMTK3 regulates ERα stability via the PKC/MEK/ERK/RSK1 axis. These results reveal a direct role of MIR2052HG in LMTK3 regulation and raise the possibilities of targeting MIR2052HG or LMTK3 in ERα-positive breast cancer.

Furanodienone overcomes temozolomide resistance in glioblastoma through the downregulation of CSPG4-Akt-ERK signalling by inhibiting EGR1-dependent transcription.

Glioblastoma multiforme (GBM) is a highly aggressive type of brain tumour. Patients with GBM respond poorly to chemotherapy and have poor survival outcomes. Neuron-glial antigen 2 (NG2), also known as chondroitin sulphate proteoglycan 4 (CSPG4), has been shown to contribute to critical processes, such as cell survival, proliferation, and chemotherapy resistance, during glioma progression. In this study, we found that furanodienone (FUR), a diene-type sesquiterpene isolated from the rhizomes of Rhizoma curcumae, exhibited a potential cytotoxic effect on temozolomide (TMZ)-resistant GBM cells in vitro by inhibiting CSPG4 and related signalling pathways. Studies investigating the mechanism demonstrated that FUR suppressed CSPG4-Akt-ERK signalling, inflammatory responses, and cytokine levels but activated caspase-dependent pathways and mitochondrial dysfunction. Furthermore, an immunofluorescence assay and a dual-luciferase reporter assay revealed that inhibition of EGR1-mediated transcription might have contributed to the FUR-dependent blockade of CSPG4 signalling and glioma cell survival. These results established a link between FUR-induced CSPG4 inhibition and the suppression of EGR1-dependent transcription. Attenuation of ERK1/2 and cytokine signalling might have generated the EGR1-dependent negative feedback loop of the CSPG4 pathway during FUR-induced apoptosis. These findings suggested that FUR could be a therapeutic candidate for the treatment of malignant glioma via targeting CSPG4 signalling.

Dynamic gene regulation by nuclear colony-stimulating factor 1 receptor in human monocytes and macrophages.

Despite their location at the cell surface, several receptor tyrosine kinases (RTK) are also found in the nucleus, as either intracellular domains or full length proteins. However, their potential nuclear functions remain poorly understood. Here we find that a fraction of full length Colony Stimulating Factor-1 Receptor (CSF-1R), an RTK involved in monocyte/macrophage generation, migrates to the nucleus upon CSF-1 stimulation in human primary monocytes. Chromatin-immunoprecipitation identifies the preferential recruitment of CSF-1R to intergenic regions, where it co-localizes with H3K4me1 and interacts with the transcription factor EGR1. When monocytes are differentiated into macrophages with CSF-1, CSF-1R is redirected to transcription starting sites, colocalizes with H3K4me3, and interacts with ELK and YY1 transcription factors. CSF-1R expression and chromatin recruitment is modulated by small molecule CSF-1R inhibitors and altered in monocytes from chronic myelomonocytic leukemia patients. Unraveling this dynamic non-canonical CSF-1R function suggests new avenues to explore the poorly understood functions of this receptor and its ligands.

Influence of the Klotho/FGF23/Egr1 signaling pathway on calcium-phosphorus metabolism in diabetic nephropathy and the intervention of Shenyuan granules.

This study aimed to explore the effects of Shenyuan granules on the Klotho/FGFR23/Egr1 signaling pathway and calcium-phosphorus metabolism in diabetic mice models with impairment of renal function. Streptozotocin-induced diabetic nephropathy (DN) mice models were randomly divided into three groups: Shenyuan granules group (n=10), model control group (n=10), and blank control group (n=10). Corresponding drugs were given by gavage for 8 weeks. Blood glucose and serum creatinine (SCr), urea nitrogen (BUN), calcium (Ca), phosphorus (P) and mLAB were detected before and after administration. Moreover, RT-qPCR was performed to detect the expression of CYP24 and CYP27 mRNA in kidney tissue. Blood FGF23 was detected by ELISA. Western-blot and immunohistochemistry were performed to detect the expressions of Klotho, FGFR1, Egr1, CYP24, CYP27, ERK1/2 and p-ERK1/2. Compared with the blank control group, in the model control group serum FGF23,P, SCr and 24-hour proteinuria levels increased (P<0.05), serum Ca significantly decreased (P<0.05), expressionss of Egr1, CYP24, CYP27 and p-ERK1/2 were up-regulated (P<0.05), and the expressions of Klotho and FGFR1 were down-regulated (P<0.05). After treatment, compared with the model control group, in the Shenyuan granule group serum FGF23, P, SCr levels decreased (P<0.05), serum Ca increased (P<0.05), expressions of Egr-1, CYP24, CYP27 and p-ERK1/2 were down-regulated (P<0.05), and the expressions of Klotho and FGFR1 were up-regulated (P<0.05). Shenyuan granules may partly intervene in the expressions of CYP24 and CYP27 through the Klotho/FGF23/Egr1 signaling pathway, thereby improving calcium and phosphorus metabolism and alleviating renal injury in diabetic nephropathy.