Elk-1 Expression Research Articles

Conditioned medium-enriched umbilical cord mesenchymal stem cells: a potential therapeutic strategy for spinal cord injury, unveiling transcriptomic and secretomic insights.

Spinal cord injury (SCI) leads to significant destruction of nerve tissue, causing the degeneration of axons and the formation of cystic cavities. This study aimed to examine the characteristics of human umbilical cord-derived mesenchymal stem cells (HUCMSCs) cultured in a serum-free conditioned medium (CM) and assess their effectiveness in a well-established hemitransection SCI model. In this study, HUCMSCs cultured medium was collected and characterized by measuring IL-10 and identifying proteomics using mass spectroscopy. This collected serum-free CM was further used in the experiments to culture and characterize the HUMSCs. Later, neuronal cells derived from CM-enriched HUCMSC were tested sequentially using an injectable caffeic acid-bioconjugated gelatin (CBG), which was further transplanted in a hemitransection SCI model. In vitro, characterization of CM-enriched HUCMSCs and differentiated neuronal cells was performed using flow cytometry, immunofluorescence, electron microscopy, and post-transplant analysis using immunohistology analysis, qPCR, in vivo bioluminescence imaging, and behavioral analysis using an infrared actimeter. The cells that were cultured in the conditioned media produced a pro-inflammatory cytokine called IL-10. Upon examining the secretome of the conditioned media, the Kruppel-like family of KRAB and zinc-finger proteins (C2H2 and C4) were found to be activated. Transcriptome analysis also revealed an increased expression of ELK-1, HOXD8, OTX2, YY1, STAT1, ETV7, and PATZ1 in the conditioned media. Furthermore, the expression of Human Stem-101 confirmed proliferation during the first 3 weeks after transplantation, along with the migration of CBG-UCNSC cells within the transplanted area. The gene analysis showed increased expression of Nestin, NeuN, Calb-2, Msi1, and Msi2. The group that received CBG-UCNSC therapy showed a smooth recovery by the end of week 2, with most rats regaining their walking abilities similar to those before the spinal cord injury by week 5. In conclusion, the CBG-UCNSC method effectively preserved the integrity of the transplanted neuronal-like cells and improved locomotor function. Thus, CM-enriched cells can potentially reduce biosafety risks associated with animal content, making them a promising option for clinical applications in treating spinal cord injuries.

BRCA1 and ELK-1 regulate neural progenitor cell fate in the optic tectum in response to visual experience in Xenopus laevis tadpoles.

In developing Xenopus tadpoles, the optic tectum begins to receive patterned visual input while visuomotor circuits are still undergoing neurogenesis and circuit assembly. This visual input regulates neural progenitor cell fate decisions such that maintaining tadpoles in the dark increases proliferation, expanding the progenitor pool, while visual stimulation promotes neuronal differentiation. To identify regulators of activity-dependent neural progenitor cell fate, we profiled the transcriptomes of proliferating neural progenitor cells and newly differentiated neurons using RNA-Seq. We used advanced bioinformatic analysis of 1,130 differentially expressed transcripts to identify six differentially regulated transcriptional regulators, including Breast Cancer 1 (BRCA1) and the ETS-family transcription factor, ELK-1, which are predicted to regulate the majority of the other differentially expressed transcripts. BRCA1 is known for its role in cancers, but relatively little is known about its potential role in regulating neural progenitor cell fate. ELK-1 is a multifunctional transcription factor which regulates immediate early gene expression. We investigated the potential functions of BRCA1 and ELK-1 in activity-regulated neurogenesis in the tadpole visual system using in vivo time-lapse imaging to monitor the fate of GFP-expressing SOX2+ neural progenitor cells in the optic tectum. Our longitudinal in vivo imaging analysis showed that knockdown of either BRCA1 or ELK-1 altered the fates of neural progenitor cells and furthermore that the effects of visual experience on neurogenesis depend on BRCA1 and ELK-1 expression. These studies provide insight into the potential mechanisms by which neural activity affects neural progenitor cell fate.

The Anticancer Effects of Marine Carotenoid Fucoxanthin through Phosphatidylinositol 3-Kinase (PI3K)-AKT Signaling on Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that lacks specific targets such as estrogen, progesterone, and HER2 receptors. TNBC affects one in eight women in the United States, making up 15-20% of breast cancer cases. Patients with TNBC can develop resistance to chemotherapy over time, leading to treatment failure. Therefore, finding other options like natural products is necessary for treatment. The advantages of using natural products sourced from plants as anticancer agents are that they are less toxic, more affordable, and have fewer side effects. These products can modulate several cellular processes of the tumor microenvironment, such as proliferation, migration, angiogenesis, cell cycle arrest, and apoptosis. The phosphatidyl inositol 3-kinase (PI3K)-AKT signaling pathway is an important pathway that contributes to the survival and growth of the tumor microenvironment and is associated with these cellular processes. This current study examined the anticancer effects of fucoxanthin, a marine carotenoid isolated from brown seaweed, in the MDA-MB-231 and MDA-MB-468 TNBC cell lines. The methods used in this study include a cytotoxic assay, PI3K-AKT signaling pathway PCR arrays, and Wes analysis. Fucoxanthin (6.25 µM) + TNF-α (50 ng/mL) and TNF-α (50 ng/mL) showed no significant effect on cell viability compared to the control in both MDA-MB-231 and MDA-MB-468 cells after a 24 h treatment period. PI3K-AKT signaling pathway PCR array studies showed that in TNF-α-stimulated (50 ng/mL) MDA-MB-231 and MDA-MB-468 cells, fucoxanthin (6.25 µM) modulated the mRNA expression of 12 genes, including FOXO1, RASA1, HRAS, MAPK3, PDK2, IRS1, EIF4EBP1, EIF4B, PTK2, TIRAP, RHOA, and ELK1. Additionally, fucoxanthin significantly downregulated the protein expression of IRS1, EIF4B, and ELK1 in MDA-MB-231 cells, and no change in the protein expression of EIF4B and ELK1 was shown in MDA-MB-468 cells. Fucoxanthin upregulated the protein expression of RHOA in both cell lines. The modulation of the expression of genes and proteins of the PI3K-AKT signaling pathway may elucidate fucoxanthin's effects in cell cycle progression, apoptotic processes, migration, and proliferation, which shows that PI3K-AKT may be the possible molecular mechanism for fucoxanthin's effects. In conclusion, the results obtained in this study elucidate fucoxanthin's molecular mechanisms and indicate that fucoxanthin may be considered a promising candidate for breast cancer-targeted therapy.

MicroRNA-10a enrichment in factor VIIa–released endothelial extracellular vesicles: potential mechanisms

BackgroundFactor VIIa induces the release of extracellular vesicles (EVs) from endothelial cells (EEVs). Factor VIIa–released EEVs are enriched with microRNA-10a (miR10a) and elicit miR10a-dependent cytoprotective responses. ObjectivesTo investigate mechanisms by which FVIIa induces miR10a expression in endothelial cells and sorts miR10a into the EVs. MethodsActivation of Elk-1 and TWIST1 expression was analyzed by immunofluorescence microscopy and immunoblot analysis. Small interfering RNA silencing approach was used to knock down the expression of specific genes in endothelial cells. EVs secreted from endothelial cells or released into circulation in mice were isolated by centrifugation and quantified by nanoparticle tracking analysis. Factor VIIa or EVs were injected into mice; mice were challenged with lipopolysaccharides to assess the cytoprotective effects of FVIIa or EVs. ResultsFVIIa activation of ERK1/2 triggered the activation of Elk-1, which led to the induction of TWIST1, a key transcription factor involved in miR10a expression. Factor VIIa also induced the expression of La, a small RNA-binding protein. Factor VIIa–driven acid sphingomyelinase (ASM) activation and the subsequent activation of the S1P receptor pathway were responsible for the induction of La. Silencing of ASM or La significantly reduced miR10a levels in FVIIa-released EEVs without affecting the cellular expression of miR10a. Factor VIIa–EEVs from ASM knocked-down cells failed to provide cytoprotective responses in cell and murine model systems. Administration of FVIIa protected wild-type but not ASM−/− mice against lipopolysaccharide-induced inflammation and vascular leakage. ConclusionOur data suggest that enhanced cellular expression of miR10a coupled with La-dependent sorting of miR10a is responsible for enriching FVIIa-released EVs with miR10a.

Probiotic Properties and Safety Evaluation of Lactobacillus plantarum HY7718 with Superior Storage Stability Isolated from Fermented Squid.

The aim of this study was to identify new potential probiotics with improved storage stability and to evaluate their efficacy and safety. Sixty lactic acid bacteria strains were isolated from Korean traditional fermented foods, and their survival was tested under extreme conditions. Lactobacillus plantarum HY7718 (HY7718) showed the greatest stability during storage. HY7718 also showed a stable growth curve under industrial conditions. Whole genome sequencing revealed that the HY7718 genome comprises 3.26 Mbp, with 44.5% G + C content, and 3056 annotated Protein-coding DNA sequences (CDSs). HY7718 adhered to intestinal epithelial cells and was tolerant to gastric fluids. Additionally, HY7718 exhibited no hemolytic activity and was not resistant to antibiotics, confirming that it has probiotic properties and is safe for consumption. Additionally, we evaluated its effects on intestinal health using TNF-induced Caco-2 cells. HY7718 restored the expression of tight junction proteins such as zonular occludens (ZO-1, ZO-2), occludin (OCLN), and claudins (CLDN1, CLDN4), and regulated the expression of myosin light-chain kinase (MLCK), Elk-1, and nuclear factor kappa B subunit 1 (NFKB1). Moreover, HY7718 reduced the secretion of proinflammatory cytokines such as interleukin-6 (IL-6) and IL-8, as well as reducing the levels of peroxide-induced reactive oxygen species. In conclusion, HY7718 has probiotic properties, is safe, is stable under extreme storage conditions, and exerts positive effects on intestinal cells. These results suggest that L. plantarum HY7718 is a potential probiotic for use as a functional supplement in the food industry.

ELK1/KIFC1 axis promotes breast cancer cell proliferation by regulating glutathione metabolism.

KIFC1 exerts an important function in centrosome aggregation in breast cancer (BC) cells and a variety of other cancer cells, but its potential mechanisms in BC pathogenesis are yet fully elucidated. The aim of this study was to investigate the effects of KIFC1 on BC progression and its underlying mechanisms. Expression of ELK1 and KIFC1 in BC was analyzed by The Cancer Genome Atlas database and quantitative real-time polymerase chain reaction. Cell proliferative capacity was examined by CCK-8 and colony formation assays, respectively. Glutathione (GSH)/glutathione disulfide (GSSG) ratio and GSH level were measured using the kit. Expression of GSH metabolism-related enzymes (G6PD, GCLM, and GCLC) was detected by western blot. Intracellular reactive oxygen species (ROS) levels were measured by the ROS Assay Kit. The transcription factor ELK1 upstream of KIFC1 was identified by hTFtarget, KnockTFv2 database and Pearson correlation. Their interaction was validated by dual-luciferase reporter assay and chromatin immunoprecipitation. This study demonstrated the upregulation of ELK1 and KIFC1 in BC and found that ELK1 could bind to the KIFC1 promoter to promote KIFC1 transcription. KIFC1 overexpression increased cell proliferation and intracellular GSH levels, while decreasing intracellular ROS levels. The addition of the GSH metabolism inhibitor BSO attenuated the promotion of BC cell proliferation induced by KIFC1 overexpression. In addition, KIFC1 overexpression reversed the inhibitory effect of knockdown of ELK1 on BC cell proliferation. ELK1 was a transcriptional factor of KIFC1. ELK1/KIFC1 axis reduced ROS level by increasing GSH synthesis, thus facilitating BC cell proliferation. Current observations suggest that ELK1/ KIFC1 may be a potential therapeutic target for BC treatment.

SRF and SRF Cofactor mRNA Expression Is Differentially Regulated by BDNF Stimulation in Cortical Neurons.

Serum response factor (SRF) is a transcription factor that plays essential roles in multiple brain functions in concert with SRF cofactors such as ternary complex factor (TCF) and megakaryoblastic leukemia (MKL)/myocardin-related transcription factor (MRTF), which comprises MKL1/MRTFA and MKL2/MRTFB. Here, we stimulated primary cultured rat cortical neurons with brain-derived neurotrophic factor (BDNF) and investigated the levels of SRF and SRF cofactor mRNA expression. We found that SRF mRNA was transiently induced by BDNF, whereas the levels of SRF cofactors were differentially regulated: mRNA expression of Elk1, a TCF family member, and MKL1/MRTFA were unchanged, while in contrast, mRNA expression of MKL2/MRTFB was transiently decreased. Inhibitor experiments revealed that BDNF-mediated alteration in mRNA levels detected in this study was mainly due to the extracellular signal-regulated protein kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway. Collectively, BDNF mediates the reciprocal regulation of SRF and MKL2/MRTFB at the mRNA expression level through ERK/MAPK, which may fine-tune the transcription of SRF target genes in cortical neurons. Accumulating evidence regarding the alteration of SRF and SRF cofactor levels detected in several neurological disorders suggests that the findings of this study might also provide novel insights into valuable therapeutic strategies for the treatment of brain diseases.

A-Kinase Anchor Protein 95 Is Involved in ERK1/2-Elk-1 Signal Transduction in Colon Cancer.

To assess A-kinase anchor protein 95 (AKAP95), B-Raf, extracellular regulated protein kinases 1/2 (ERK1/2), and Elk-1 expression in colon cancer tissue, and characterize AKAP95 associations with B-Raf, ERK1/2, Elk-1, and colon cancer clinicopathological indices. The immunohistochemistry streptavidin-perosidase (SP) method was used to determine protein expression levels in 64 colon cancer and 32 para-carcinoma tissue specimens. (1) Positive AKAP95 expression rates in colon cancer tissue were higher when compared with para-carcinoma tissue (92.19% vs. 59.38%, P < 0.05). Similar findings were determined for B-Raf (76.56% vs. 25%, P < 0.05), ERK1/2 (90.63% vs. 31.25%, P < 0.05), and Elk-1 levels (92.19% vs. 40.63%, P < 0.05). (2) No significant associations were identified between AKAP95, B-Raf, ERK1/2, and Elk-1 protein expression and degree of differentiation, histological type, and lymph node metastasis in colon cancer samples (P > 0.05); however, in The Cancer Genome Atlas and Gene Expression Omnibus datasets, AKAP95 was closely related to immune infiltration, and highly expressed AKAP95 was negatively associated with overall survival and relapse free survival rates in colon cancer patients. (3) Correlations were observed between AKAP95 and ERK1/2, AKAP95 and Elk-1, B-Raf and ERK1/2, B-Raf and Elk-1, and ERK1/2 and Elk-1 (all P < 0.05), but no correlation was observed between AKAP95 and B-Raf (P > 0.05). AKAP95 may affect immune infiltration levels in colon cancer by participating in ERK1/2-Elk-1 signal transduction.

Silencing circ_0074371 inhibits the progression of sepsis-induced acute kidney injury by regulating miR-330-5p/ELK1 axis.

Sepsis-induced acute kidney injury (AKI) is a common in clinic. Circular RNAs (circRNAs) play significant roles in ameliorating AKI. The purpose of this study was aimed to identify the role of circ_0074371 and the potential action mechanism in sepsis-induced AKI. AKI patients and healthy individual serum samples were collected and the relative expression of circ_0074371 was measured by real-time polymerase chain reaction (RT-PCR). HK2 cells were treated with different dose (0, 2.5, 5 and 10μg/ml) lipopolysaccharide (LPS) to establish the AKI cell model. The cell viability and apoptosis of HK2 cells were detected using cell counting kit-8 (CCK-8) and flow cytometry, respectively. The contents of malondialdehyde (MDA), and superoxide dismutase (SOD) were evaluated using the relative commercial kits. The IL-1β and TNF-α levels in cell culture supernatants were measured by ELISA. The interaction relationship between miR-330-5p and circ_0074371 or ELK1 was predicted by Targetscan database and further confirmed by the dual-luciferase reporter assay system. The circ_0074371 expression was up-regulated in sepsis patients and LPS-induced HK2 cells. Silencing circ_0074371 promoted HK2 cells viability and inhibited the HK2 cells apoptosis. miR-330-5p inhibitor weakened circ_0074371 inhibitor-induced cell viability, apoptosis and oxidative stress. Further mechanism analysis showed that circ_0074371 acted as a sponge for miR-330-5p to increase ELK1 expression level. Importantly, miR-330-5p downregulation or ELK1 upregulation reversed the action of circ_0074371 knockdown on LPS-induced HK2 cells. Knockdown of circ_0074371 ameliorated LPS-induced HK2 cells apoptosis, inflammation and oxidative stress via regulating miR-330-5p/ELK1, opening a new window into the pathogenesis AKI.

GPX4 suppresses ferroptosis to promote malignant progression of endometrial carcinoma via transcriptional activation by ELK1

BackgroundGlutathione Peroxidase 4 (GPX4) is a key protein that inhibits ferroptosis. However, its biological regulation and mechanism in endometrial cancer (EC) have not been reported in detail.MethodsThe expression of GPX4 in EC tissues was determined by TCGA databases, qRT-PCR, Western blot, and immunohistochemistry (IHC). The effects of GPX4 on EC cell proliferation, migration, apoptosis, and tumorigenesis were studied in vivo and in vitro. In addition, ETS Transcription Factor ELK1 (ELK1) was identified by bioinformatics methods, dual-luciferase reporter assay, and chromatin immunoprecipitation (ChIP). Pearson correlation analysis was used to evaluate the association between ELK1 and GPX4 expression.ResultsThe expression of GPX4 was significantly up-regulated in EC tissues and cell lines. Silencing GPX4 significantly inhibited the proliferation, migration ability, induced apoptosis, and arrested the cell cycle of Ishikawa and KLE cells. Knockdown of GPX4 accumulated intracellular ferrous iron and ROS, disrupted MMP, and increased MDA levels. The xenograft tumor model also showed that GPX4 knockdown markedly reduced tumor growth in mice. Mechanically, ELK1 could bind to the promoter of GPX4 to promote its transcription. In addition, the expression of ELK1 in EC was positively correlated with GPX4. Rescue experiments confirmed that GPX4 knockdown could reverse the strengthens of cell proliferation and migration ability and the lower level of Fe2+ and MDA caused by upregulating ELK1.ConclusionThe results of the present study suggest that ELK1 / GPX4 axis plays an important role in the progress of EC by promoting the malignant biological behavior and inducing ferroptosis of EC cells, which provides evidence for investigating the potential therapeutic strategies of endometrial cancer.

Elk1/miR-462-731 Feedback Loop Regulates Macrophages Polarization and Phagocytosis in Grass Carp (Ctenopharyngodon idella).

MicroRNA clusters are microRNAs (miRNAs) that are distributed in close proximity on chromosomes. In this study, we report a miRNA cluster identified from grass carp (Ctenopharyngodon idella), miR-462-731, which plays a positive role in host antibacterial immunity. The expression of miR-462-731 was disrupted after infection by Aeromonas hydrophila. Transcription factor ETS transcription factor ELK1 was identified to bind to the promoter of the miR-462-731 cluster and suppress its expression. In addition, miR-731 negatively regulates the expression of elk1, forms an elk1/miR-462-731 double negative feedback loop. In addition, we found that miR-731 directly targets ezrin a (ezra), participates in inducing PI3K/AKT signaling in macrophage, to induce macrophage polarization to the M1 phenotype with stronger phagocytosis. Our results demonstrate a novel elk1/miR-462-731 feedback loop. The data deepen our understanding of the relationship between macrophage polarization and phagocytosis in teleost fish.

LncRNA 122049 suppresses apoptosis of renal tubular epithelial cells in ischemic AKI by targeting the miR-330-5p/ELK1 axis.

Several studies have reported that long non-coding RNAs (LncRNAs) were associated with the progression of acute kidney injury (AKI). However, the role and regulation mechanism of lncRNA122049 in ischemic AKI remains unknown. In the present study, we found that lncRNA 122049 protected against the ischemia/reperfusion (I/R) induced apoptosis in BUMPT cells. Mechanistically, the lncRNA 122049 directly sponged miR-330-5p, then increased the expression of ELK1(ETS transcription factor ELK1) to decrease renal cell apoptosis. In addition, miR-330-5p inhibitor completely reversed the pro-apoptotic effect of LncRNA 122049siRNA on I/R-induced BUMPT cells apoptosis. Finally, overexpression of lncRNA 122049 attenuated ischemic mice AKI via targeting of the miR-330-5p/ELK1 axis. Collectively, the data demonstrated that LncRNA 122049 prevented the I/R-induced renal cell apoptosis via regulation of the miR-330-5p/ELK1 axis, which brings new insights into the pathogenesis and potential targeted treatment of ischemic AKI.

The SETD8/ELK1/bach1 complex regulates hyperglycaemia-mediated EndMT in diabetic nephropathy

BackgroundDiabetic nephropathy (DN), the most common microvascular complication in patients with diabetes, induces kidney failure. Previous research showed that endothelial-to-mesenchymal transition (EndMT) of human glomerular endothelial cells (HGECs) is involved in the progression of DN. Moreover, SET domain-containing protein 8 (SETD8), ETS-domain containing protein (ELK1) and BTB and CNC homology 1 (bach1) all participate in endothelial injury. In this study, we hypothesize that the SETD8/ELK1/bach1 functional axis is involved in mediating EndMT in diabetic nephropathy.MethodsImmunohistochemistry, Western blotting and qPCR were performed to determine the protein and mRNA levels of genes in HGECs and the kidney tissues of participants and rats. Immunofluorescence, Co-IP and GST pulldown assays were performed to verify the direct interaction between SETD8 and ELK1. ChIP and dual-luciferase assays were performed to determine the transcriptional regulation of bach1 and Snail. AVV-SETD8 injection in rat kidney was used to verify the potential protective effect of SETD8 on DN.ResultsOur current study showed that hyperglycaemia triggered EndMT by increasing Snail expression both in vitro and in vivo. Moreover, high glucose increased bach1 expression in HGECs, positively regulating Snail and EndMT. As a transcription factor, ELK1 was augmented and participated in hyperglycaemia-induced EndMT via modulation of bach1 expression. Moreover, ELK1 was found to associate with SETD8. Furthermore, SETD8 negatively regulated EndMT by cooperating with bach1 to regulate Snail transcription. Furthermore, histone H4-Lys-20 monomethylation (H4K20me1), which is downstream of SETD8, was accompanied by ELK1 localization at the same promoter region of bach1. ELK1 overexpression enhanced bach1 promoter activity, which disappeared after specific binding site deletion. Mutual inhibition between ELK1 and SETD8 was found in HGECs. In vivo, SETD8 overexpression decreased ELK1 and bach1 expression, as well as EndMT. Moreover, SETD8 overexpression improved the renal function of rats with DN.ConclusionsSETD8 cooperates with ELK1 to regulate bach1 transcription, thus participating in the progression of DN. In addition, SETD8 interacts with bach1 to modulate Snail transcription, thus inducing EndMT in DN. SETD8 plays a core role in the SETD8/ELK1/bach1 functional axis, which participates in hyperglycaemia-mediated EndMT in DN, and SETD8 may be a potential therapeutic target for DN.Trial registration ChiCTR, ChiCTR2000029425. 2020/1/31, http://www.chictr.org.cn/showproj.aspx?proj=48548

Knockdown of lncRNA LINC00662 suppresses malignant behaviour of osteosarcoma cells via competition with miR-30b-3p to regulate ELK1 expression

PurposeOsteosarcoma is a type of bone malignancy that mainly occurred in teenagers. This investigation is aimed to clarify the effect of long non-coding RNA (lncRNA) LINC00662 on the proliferation, migration, and invasion in osteosarcoma and explore the underlying action mechanisms.MethodsThe mRNA expression of LINC00662 was determined by real-time quantitative polymerase chain reaction. Cell proliferation, migration, and invasion were evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, wound healing, and transwell assays, respectively. A dual-luciferase reporter assay was used to validate the target relationships Between microRNA (miR)-30b-3p and LINC00662/ ETS domain-containing protein 1 (ELK1). Western blotting was performed to determine the protein expression of ELK1. Xenograft model was established to evaluate the effects of LINC00662 silencing on tumor growth in vivo.ResultsLncRNA LINC00662 and ELK1 were significantly increased, while miR-30b-3p was reduced in osteosarcoma tissues. The results of functional experiments indicated that transfection of small hairpin (sh)-LINC00662 and miR-30b-3p mimics repressed the migration, invasion, and proliferation of osteosarcoma cells. LncRNA LINC00662 also appeared to sponge miR-30b-3p in order to affect the expression of ELK1. Simultaneously, there were weak negative correlations between the expression of miR-30b-3p and LINC00662/ELK1 in osteosarcoma tissues. Rescue experiments suggested that ELK1 overexpression and downregulation of miR-30b-3p reversed the suppressive effects of sh-LINC00662 on the cell migration, invasion, and proliferation in osteosarcoma.ConclusionsThe current study indicated that knockdown of LINC00662 repressed cell migration, invasion, and proliferation through sponging miR-30b-3p to regulate the expression of ELK1 in osteosarcoma. These results may uncover a promising target for the treatment of osteosarcoma.

Chemical Inhibition of Apurinic-Apyrimidinic Endonuclease 1 Redox and DNA Repair Functions Affects the Inflammatory Response via Different but Overlapping Mechanisms.

The presence of oxidized DNA lesions, such as 7,8-dihydro-8-oxoguanine (8-oxoG) and apurinic/apyrimidinic sites (AP sites), has been described as epigenetic signals that are involved in gene expression control. In mammals, Apurinic-apyrimidinic endonuclease 1/Redox factor-1 (APE1/Ref-1) is the main AP endonuclease of the base excision repair (BER) pathway and is involved in active demethylation processes. In addition, APE1/Ref-1, through its redox function, regulates several transcriptional factors. However, the transcriptional control targets of each APE1 function are not completely known. In this study, a transcriptomic approach was used to investigate the effects of chemical inhibition of APE1/Ref-1 redox or DNA repair functions by E3330 or methoxyamine (MX) in an inflammatory cellular model. Under lipopolysaccharide (LPS) stimulation, both E3330 and MX reduced the expression of some cytokines and chemokines. Interestingly, E3330 treatment reduced cell viability after 48 h of the treatment. Genes related to inflammatory response and mitochondrial processes were downregulated in both treatments. In the E3330 treatment, RNA processing and ribosome biogenesis genes were downregulated, while they were upregulated in the MX treatment. Furthermore, in the E3330 treatment, the cellular stress response was the main upregulated process, while the cellular macromolecule metabolic process was observed in MX-upregulated genes. Nuclear respiratory factor 1 (NRF1) was predicted to be a master regulator of the downregulated genes in both treatments, while the ETS transcription factor ELK1 (ELK1) was predicted to be a master regulator only for E3330 treatment. Decreased expression of ELK1 and its target genes and a reduced 28S/18S ratio were observed, suggesting impaired rRNA processing. In addition, both redox and repair functions can affect the expression of NRF1 and GABPA target genes. The master regulators predicted for upregulated genes were YY1 and FLI1 for the E3330 and MX treatments, respectively. In summary, the chemical inhibition of APE1/Ref-1 affects gene expression regulated mainly by transcriptional factors of the ETS family, showing partial overlap of APE1 redox and DNA repair functions, suggesting that these activities are not entirely independent. This work provides a new perspective on the interaction between APE1 redox and DNA repair activity in inflammatory response modulation and transcription.

Decreased MicroRNA-150 Exacerbates Neuronal Apoptosis in the Diabetic Retina.

Diabetic retinopathy (DR) is a chronic complication associated with diabetes and the number one cause of blindness in working adults in the US. More than 90% of diabetic patients have obesity-associated type 2 diabetes (T2D), and 60% of T2D patients will develop DR. Photoreceptors undergo apoptosis shortly after the onset of diabetes, which contributes to the retinal dysfunction and microvascular complications leading to vision impairment. However, how diabetic insults cause photoreceptor apoptosis remains unclear. In this study, obesity-associated T2D mice and cultured photoreceptors were used to investigate how decreased microRNA-150 (miR-150) and its downstream target were involved in photoreceptor apoptosis. In the T2D retina, miR-150 was decreased with its target ETS-domain transcription factor (ELK1) and phosphorylated ELK1 at threonine 417 (pELK1T417) upregulated. In cultured photoreceptors, treatments with palmitic acid (PA), to mimic a high-fat environment, decreased miR-150 but upregulated ELK1, pELK1T417, and the translocation of pELK1T417 from the cytoplasm to the cell nucleus. Deletion of miR-150 (miR-150−/−) exacerbates T2D- or PA-induced photoreceptor apoptosis. Blocking the expression of ELK1 with small interfering RNA (siRNA) for Elk1 did not rescue PA-induced photoreceptor apoptosis. Translocation of pELK1T417 from cytoplasm-to-nucleus appears to be the key step of diabetic insult-elicited photoreceptor apoptosis.

Anti-tumor effect of trametinib in bladder cancer organoid and the underlying mechanism

ABSTRACT Bladder cancer (BC), a main neoplasm of urinary tract, is usually inoperable and unresponsive to chemotherapy. As a novel experimental model for muscle-invasive BC, we previously established a culture method of dog BC organoids. In the present study, the detailed in vitro and in vivo anti‐tumor effects of trametinib were investigated by using this model. In each BC organoid strain, epidermal growth factor receptor (EGFR)/ERK signaling was upregulated compared with normal bladder cells. Trametinib even at a low concentration inhibited the cell viability of BC organoids and the activation of ERK through decreasing expression of c-Myc, ELK1, SIK1, and PLA2G4A. Trametinib arrested cell cycle of BC with few apoptosis. Dual treatment of BC organoids with trametinib and YAP inhibitor, verteporfin extremely inhibited the cell viability with apoptosis induction. Moreover, trametinib induced basal to luminal differentiation of BC organoids by upregulating luminal markers and downregulating basal ones. In vivo, trametinib decreased the tumor growth of BC organoids in mice and the xenograft-derived organoids from trametinib-administered mice showed enhanced sensitivity to carboplatin due to MSH2 upregulation. Our data suggested a new strategy of trametinib-YAP inhibitor or trametinib-carboplatin combination as a promising treatment of BC.

Long noncoding RNA HBBP1 enhances γ-globin expression through the ETS transcription factor ELK1

β-Thalassemia is an autosomal recessive genetic disease caused by defects in the production of adult hemoglobin (HbA, α2β2), which leads to an imbalance between α- and non-α-globin chains. Reactivation of γ-globin expression is an effective strategy to treat β-thalassemia patients. Previously, it was demonstrated that hemoglobin subunit beta pseudogene 1 (HBBP1) is associated with elevated fetal hemoglobin (HbF, α2γ2) in β-thalassemia patients. However, the mechanism underlying HBBP1-mediated HbF production is unknown. In this study, using bioinformatics analysis, we found that HBBP1 is involved in γ-globin production, and then preliminarily confirmed this finding in K562 cells. When HBBP1 was overexpressed, γ-globin expression was increased at the transcript and protein levels in HUDEP-2 cells. Next, we found that ETS transcription factor ELK1 (ELK1) binds to the HBBP1 proximal promoter and significantly promotes its activity. Moreover, the synthesis of γ-globin was enhanced when ELK1 was overexpressed in HUDEP-2 cells. Surprisingly, ELK1 also directly bound to and activated the γ-globin proximal promoter. Furthermore, we found that HBBP1 and ELK1 can interact with each other in HUDEP-2 cells. Collectively, these findings suggest that HBBP1 can induce γ-globin by enhancing ELK1 expression, providing some clues for γ-globin reactivation in β-thalassemia.

ETS-Domain Transcription Factor Elk-1 Regulates Stemness Genes in Brain Tumors and CD133+ BrainTumor-Initiating Cells.

Elk-1, a member of the ternary complex factors (TCFs) within the ETS (E26 transformation-specific) domain superfamily, is a transcription factor implicated in neuroprotection, neurodegeneration, and brain tumor proliferation. Except for known targets, c-fos and egr-1, few targets of Elk-1 have been identified. Interestingly, SMN, SOD1, and PSEN1 promoters were shown to be regulated by Elk-1. On the other hand, Elk-1 was shown to regulate the CD133 gene, which is highly expressed in brain-tumor-initiating cells (BTICs) and used as a marker for separating this cancer stem cell population. In this study, we have carried out microarray analysis in SH-SY5Y cells overexpressing Elk-1-VP16, which has revealed a large number of genes significantly regulated by Elk-1 that function in nervous system development, embryonic development, pluripotency, apoptosis, survival, and proliferation. Among these, we have shown that genes related to pluripotency, such as Sox2, Nanog, and Oct4, were indeed regulated by Elk-1, and in the context of brain tumors, we further showed that Elk-1 overexpression in CD133+ BTIC population results in the upregulation of these genes. When Elk-1 expression is silenced, the expression of these stemness genes is decreased. We propose that Elk-1 is a transcription factor upstream of these genes, regulating the self-renewal of CD133+ BTICs.

A novel circular RNA circPPFIA1 promotes laryngeal squamous cell carcinoma progression through sponging miR-340-3p and regulating ELK1 expression

ABSTRACT Abnormal expression of circular RNA (circRNA) is closely related to the occurrence and development of many cancers. By screening the expression of circRNA, we identified a novel circRNA termed as has_circ_0023326 in laryngeal squamous cell carcinoma (LSCC). We verified the expression of circPPFIA1 and found that it was upregulated in LSCC tissues compared to the adjacent normal tissues. Functional studies were carried out to detect the effect of circPPFIA1 expression on the phenotype of LSCC cells. These results suggest that circPPFIA1 knockdown can suppress the proliferation, migration, and invasion of LSCC cells, while circPPFIA1 overexpression can promote these processes. Mechanistically, miR-340-3p was predicted to be the target miRNA sponged by circPPFIA1 as confirmed through the luciferase assay and rescue experiments. In addition, miR-340-3p was found to target ELK1 and inhibit its expression. Taken together, circPPFIA1 promotes the progression of LSCC via the miR-340-3p/ELK1 signaling axis, which may serve as a novel prognostic or therapeutic target for LSCC.