Baicalein Inhibits Metastasis of Oral Squamous Cell Carcinoma Cells by Regulating ERK/ELK-1/Snail Signaling.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is associated with high recurrence and poor prognosis. Baicalin has multiple pharmacological effects, including anti-inflammatory and anti-proliferative activities. Here, we examine the effect of baicalein on OSCC metastasis and its potential mechanism of action. METHODS: SCC-4 and CAL-27 cells were treated with different concentrations of baicalein. The proliferation of OSCC cells was evaluated by Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. As for migration and metastasis, baicalein-treated OSCC cells were used for wound healing assay and Transwell assay. The levels of epithelial-mesenchymal transition-related proteins (E-cadherin, N-cadherin, vimentin) and extracellular regulated protein kinases (ERK)/ETS Transcription Factor ELK1 (ELK-1)/Snail signaling pathway-related proteins in baicalein-treated OSCC cells were evaluated by western blotting. RESULTS: The rates of cell proliferation and migration, along with the metastatic potential, of baicalein-treated cells were significantly lower than those of the control (p < 0.05), and the effects were concentration-dependent. Furthermore, compared to the control, baicalein significantly decreased the levels of N-cadherin and vimentin in SCC-4 and CAL-27 cells, and increased the E-cadherin level (p < 0.05). Mechanistically, baicalein downregulated the levels of p-ERK1/2, phospho-ETS Transcription Factor ELK1 (p-ELK-1), and Snail (p < 0.05). Finally, the ERK/ELK-1/Snail pathway inhibitor (U0126) promoted the effect of baicalein in inhibiting the migration and invasion of OSCC cells (p < 0.05). CONCLUSION: Baicalein abates the migration, invasion, and metastasis of OSCC cells through the ERK/ELK-1/Snail signaling pathway. This study provides a basis for the development of baicalein as a compound for the treatment of OSCC.

Elk1 enhances inflammatory cell infiltration and exacerbates acute lung injury/acute respiratory distress syndrome by suppressing Fcgr2b transcription.

OBJECTIVE: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with significant mortality rates. The role of Fcgr2b in the pathogenesis of ALI/ARDS is not fully elucidated. This study aimed to investigate the functions of Fcgr2b in ALI/ARDS and explore its underlying mechanisms. METHODS: Methods: In this study, rat models of ARDS and pulmonary microvascular endothelial cell (PMVEC) injury models were established through the administration of lipopolysaccharide (LPS). The expression levels of Fcgr2b and Elk1 were quantified in both LPS-induced ARDS rats and PMVECs. Subsequent gain- and loss-of-function experiments were conducted, followed by comprehensive assessments of lung tissue for pathomorphological changes, edema, glycogen storage, fibrosis, and infiltration of inflammatory cells. Additionally, bronchoalveolar lavage fluid was analyzed for T-helper 17 (Th17) cell infiltration, inflammatory response, and microvascular permeability to evaluate lung injury severity in ARDS models. Furthermore, the activity, cytotoxicity, apoptosis, and angiogenic potential of PMVECs were assessed to gauge cell injury. The interaction between Elk1 and Fcgr2b was also examined to confirm their regulatory relationship. RESULTS: In the context of LPS-induced ARDS and PMVEC injury, Fcgr2b expression was markedly reduced, whereas Elk1 expression was elevated. Overexpression of Fcgr2b led to a decrease in Th17 cell infiltration and mitigated lung tissue damage in ARDS models, in addition to reducing LPS-induced injury in PMVECs. Elk1 was found to suppress Fcgr2b transcription through the recruitment of histone 3 lysine 9 trimethylation (H3K9me3). Knockdown of Elk1 diminished Th17 cell infiltration and lung tissue damage in ARDS models, and alleviated LPS-induced injury in PMVECs, effects that were reversed upon Fcgr2b upregulation. CONCLUSION: Elk1 negatively regulates Fcgr2b transcription, thereby augmenting the inflammatory response and exacerbating lung injury in LPS-induced ALI/ARDS.

Mutant p53 and ELK1 co-drive FRA-1 expression to induce metastasis in breast cancer.

Tumor-associated p53 mutations induce activities different from wild-type p53, thus causing loss of the protein's tumor inhibition function. The cells carrying p53 mutations have more aggressive characteristics related to invasion, metastasis, proliferation, and cell survival. By comparing the gene expression profiles of mutant p53 (mutp53) and mutp53 silenced cohorts, we found that FOS-related antigen-1 (FRA-1), which is encoded by FOSL1, is a potential effector of mutp53-mediated metastasis. We demonstrate that the expression of FRA-1, a gatekeeper of mesenchymal-epithelial transition, is elevated in the presence of p53 mutations. Mechanistically, mutant p53 cooperates with the transcription factor ELK1 in binding and activating the promoter of FOSL1, thus fostering lung metastasis. This study reveals new insights into how mutant p53 contributes to metastasis in breast cancer.

Aberrant expression of CKS2 induced by ELK1 contributes to malignant progression of pancreatic cancer.

Cyclin-dependent kinase subunit 2 (CKS2) has been reported to promote various malignancies. This study investigated the functional role of CKS2 in pancreatic cancer (PC). An analysis of abnormally expressed genes and their prognostic value for PC was performed by using the Gene Expression Profiling Interactive Analysis (GEPIA) database and performing immunohistochemical staining on 64 samples of tumor tissue. CCK-8 assays, EdU staining, colony formation assays, flow cytometry, and a xenograft tumor model were used to analyze the biological function of CKS2 in PC. Our results revealed that CKS2 was expressed at significantly higher levels in PC tissues than in adjacent normal tissues, and a high level of CKS2 expression was associated with a poor prognosis for patients with PC. Moreover, functional assays revealed that CKS2 knockdown suppressed cell proliferation, induced cell cycle S phase, G2/M phase arrest, and apoptosis in vitro, and also reduced tumor growth in vivo. In addition, CKS2 knockdown increased the levels of Bax, caspase-3, P53, P21, and GADD45α expression, but decreased Bcl-2, Cyclin B1, CDK1, Cyclin A, and Cdc25C expression. CKS2 overexpression produced the opposite effects of CKS2 knockdown. Furthermore, we found that ELK1 protein regulated transcription of the CKS2 gene. In conclusion, our findings suggest that CKS2 expression is regulated by ELK1, which could possibly serve as prognostic indicator and therapeutic target for PC.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Androgen receptor knockdown enhances prostate cancer chemosensitivity by down-regulating FEN1 through the ERK/ELK1 signalling pathway.

PURPOSE: Flap endonuclease 1 (FEN1) is highly upregulated in prostate cancer and promotes the growth of prostate cancer cells. Androgen receptor (AR) is the most critical determinant of the occurrence, progression, metastasis, and treatment of prostate cancer. However, the effect of FEN1 on docetaxel (DTX) sensitivity and the regulatory mechanisms of AR on FEN1 expression in prostate cancer need to be further studied. METHODS: Bioinformatics analyses were performed using data from the Cancer Genome Atlas and the Gene Expression Omnibus. Prostate cancer cell lines 22Rv1 and LNCaP were used. FEN1 siRNA, FEN1 overexpression plasmid, and AR siRNA were transfected into cells. Biomarker expression was evaluated by immunohistochemistry and Western blotting. Apoptosis and the cell cycle were explored using flow cytometry analysis. Luciferase reporter assay was performed to verify the target relationship. Xenograft assays were conducted using 22Rv1 cells to evaluate the in vivo conclusions. RESULTS: Overexpression of FEN1 inhibited cell apoptosis and cell cycle arrest in the S phase induced by DTX. AR knockdown enhanced DTX-induced cell apoptosis and cell cycle arrest at the S phase in prostate cancer cells, which was attenuated by FEN1 overexpression. In vivo experiments showed that overexpression of FEN1 significantly increased tumour growth and weakened the inhibitory effect of DTX on prostate tumour growth, while AR knockdown enhance the sensitivity of DTX to prostate tumour. AR knockdown resulted in FEN1, pho-ERK1/2, and pho-ELK1 downregulation, and the luciferase reporter assay confirmed that ELK1 can regulate the transcription of FEN1. CONCLUSION: Collectively, our studies demonstrate that AR knockdown improves the DTX sensitivity of prostate cancer cells by downregulating FEN1 through the ERK/ELK1 signalling pathway.

Transcription factor ELK1 regulates the expression of histone 3 lysine 9 to affect developmental potential of porcine preimplantation embryos.

A series of changes occur in the early embryo that are critical for subsequent development, and the pig is an excellent animal model of human disease, so understanding the regulatory mechanisms of early embryonic development in the pig is of very importance. To find key transcription factors regulating pig early embryonic development, we first profiled the transcriptome of pig early embryos, and confirmed that zygotic gene activation (ZGA) in porcine embryos starts from 4 cell stage. Subsequent enrichment analysis of up-regulated gene motifs during ZGA revealed that the transcription factor ELK1 ranked first. The expression pattern of ELK1 in porcine early embryos was analyzed by immunofluorescence staining and qPCR, and the results showed that the transcript level of ELK1 reached the highest at the 8 cell stage, while the protein level reached the highest at 4 cell stage. To further investigate the effect of ELK1 on early embryo development in pigs, we silenced ELK1 in zygotes and showed that ELK1 silencing significantly reduced cleavage rate, blastocyst rate as well as blastocyst quality. A significant decrease in the expression of the pluripotency gene Oct4 was also observed in blastocysts from the ELK1 silenced group by immunofluorescence staining. Silencing of ELK1 also resulted in decreased H3K9Ac modification and increased H3K9me3 modification at 4 cell stage. To investigate the effect of ELK1 on ZGA, we analyzed transcriptome changes in 4 cell embryos after ELK1 silencing by RNA seq, which revealed that ELK1 silencing resulted in significant differences in the expression of a total of 1953 genes at the 4 cell stage compared with their normal counterparts, including 1106 genes that were significantly upregulated and 847 genes that were significantly downregulated. Through GO and KEGG enrichment, we found that the functions and pathways of down-regulated genes were concentrated in protein synthesis, processing, cell cycle regulation, etc., while the functions of up-regulated genes were focused on aerobic respiration process. In conclusion, this study demonstrates that the transcription factor ELK1 plays an important role in regulation of preimplantation embryo development of pigs and deficiency of ELK1 leads to abnormal epigenetic reprogramming as well as zygotic genome activation, thus adversely affecting embryonic development. This study will provide important reference for the regulation of transcription factors in porcine embryo development.

The Small Molecule Antagonist KCI807 Disrupts Association of the Amino-Terminal Domain of the Androgen Receptor with ELK1 by Modulating the Adjacent DNA Binding Domain.

The androgen receptor (AR) is a crucial coactivator of ELK1 for prostate cancer (PCa) growth, associating with ELK1 through two peptide segments (358-457 and 514-557) within the amino-terminal domain (NTD) of AR. The small-molecule antagonist 5-hydroxy-2-(3-hydroxyphenyl)chromen-4-one (KCI807) binds to AR, blocking ELK1 binding and inhibiting PCa growth. We investigated the mode of interaction of KCI807 with AR using systematic mutagenesis coupled with ELK1 coactivation assays, testing polypeptide binding and Raman spectroscopy. In full-length AR, deletion of neither ELK1 binding segment affected sensitivity of residual ELK1 coactivation to KCI807. Although the NTD is sufficient for association of AR with ELK1, interaction of the isolated NTD with ELK1 was insensitive to KCI807. In contrast, coactivation of ELK1 by the AR-V7 splice variant, comprising the NTD and the DNA binding domain (DBD), was sensitive to KCI807. Deletions and point mutations within DBD segment 558-595, adjacent to the NTD, interfered with coactivation of ELK1, and residual ELK1 coactivation by the mutants was insensitive to KCI807. In a glutathione S-transferase pull-down assay, KCI807 inhibited ELK1 binding to an AR polypeptide that included the two ELK1 binding segments and the DBD but did not affect ELK1 binding to a similar AR segment that lacked the sequence downstream of residue 566. Raman spectroscopy detected KCI807-induced conformational change in the DBD. The data point to a putative KCI807 binding pocket within the crystal structure of the DBD and indicate that either mutations or binding of KCI807 at this site will induce conformational changes that disrupt ELK1 binding to the NTD. SIGNIFICANCE STATEMENT: The small-molecule antagonist KCI807 disrupts association of the androgen receptor (AR) with ELK1, serving as a prototype for the development of small molecules for a novel type of therapeutic intervention in drug-resistant prostate cancer. This study provides basic information needed for rational KCI807-based drug design by identifying a putative binding pocket in the DNA binding domain of AR through which KCI807 modulates the amino-terminal domain to inhibit ELK1 binding.

Comparative analysis of protein expression systems and PTM landscape in the study of transcription factor ELK-1.

Post-translational modifications (PTMs) are important for protein folding and activity, and the ability to recreate physiologically relevant PTM profiles on recombinantly-expressed proteins is vital for meaningful functional analysis. The ETS transcription factor ELK-1 serves as a paradigm for cellular responses to mitogens and can synergise with androgen receptor to promote prostate cancer progression, although in vitro protein function analyses to date have largely overlooked its complex PTM landscapes. We expressed and purified human ELK-1 using mammalian (HEK293T), insect (Sf9) and bacterial (E. coli) systems in parallel and compared PTMs imparted upon purified proteins, along with their performance in DNA and protein interaction assays. Phosphorylation of ELK-1 within its transactivation domain, known to promote DNA binding, was most apparent in protein isolated from human cells and accordingly conferred the strongest DNA binding in vitro, while protein expressed in insect cells bound most efficiently to the androgen receptor. We observed lysine acetylation, a hitherto unreported PTM of ELK-1, which appeared highest in insect cell-derived ELK-1 but was also present in HEK293T-derived ELK-1. Acetylation of ELK-1 was enhanced in HEK293T cells following starvation and mitogen stimulation, and modified lysines showed overlap with previously identified regulatory SUMOylation and ubiquitination sites. Our data demonstrate that the choice of recombinant expression system can be tailored to suit biochemical application rather than to maximise soluble protein production and suggest the potential for crosstalk and antagonism between different PTMs of ELK-1.

The autism risk factor CHD8 is a chromatin activator in human neurons and functionally dependent on the ERK-MAPK pathway effector ELK1.

The chromodomain helicase DNA-binding protein CHD8 is the most frequently mutated gene in autism spectrum disorder. Despite its prominent disease involvement, little is known about its molecular function in the human brain. CHD8 is a chromatin regulator which binds to the promoters of actively transcribed genes through genomic targeting mechanisms which have yet to be fully defined. By generating a conditional loss-of-function and an endogenously tagged allele in human pluripotent stem cells, we investigated the molecular function and the interaction of CHD8 with chromatin in human neurons. Chromatin accessibility analysis and transcriptional profiling revealed that CHD8 functions as a transcriptional activator at its target genes in human neurons. Furthermore, we found that CHD8 chromatin targeting is cell context-dependent. In human neurons, CHD8 preferentially binds at ETS motif-enriched promoters. This enrichment is particularly prominent on the promoters of genes whose expression significantly changes upon the loss of CHD8. Indeed, among the ETS transcription factors, we identified ELK1 as being most highly correlated with CHD8 expression in primary human fetal and adult cortical neurons and most highly expressed in our stem cell-derived neurons. Remarkably, ELK1 was necessary to recruit CHD8 specifically to ETS motif-containing sites. These findings imply that ELK1 and CHD8 functionally cooperate to regulate gene expression and chromatin states at MAPK/ERK target genes in human neurons. Our results suggest that the MAPK/ERK/ELK1 axis potentially contributes to the pathogenesis caused by CHD8 mutations in human neurodevelopmental disorders.

BCL6 is regulated by the MAPK/ELK1 axis and promotes KRAS-driven lung cancer.

Mutational activation of KRAS is a common oncogenic event in lung cancer, yet effective therapies are still lacking. Here, we identify B cell lymphoma 6 (BCL6) as a lynchpin in KRAS-driven lung cancer. BCL6 expression was increased upon KRAS activation in lung tumor tissue in mice and was positively correlated with the expression of KRAS-GTP, the active form of KRAS, in various human cancer cell lines. Moreover, BCL6 was highly expressed in human KRAS-mutant lung adenocarcinomas and was associated with poor patient survival. Mechanistically, the MAPK/ERK/ELK1 signaling axis downstream of mutant KRAS directly regulated BCL6 expression. BCL6 maintained the global expression of prereplication complex components; therefore, BCL6 inhibition induced stalling of the replication fork, leading to DNA damage and growth arrest in KRAS-mutant lung cancer cells. Importantly, BCL6-specific knockout in lungs significantly reduced the tumor burden and mortality in the LSL-KrasG12D/+ lung cancer mouse model. Likewise, pharmacological inhibition of BCL6 significantly impeded the growth of KRAS-mutant lung cancer cells both in vitro and in vivo. In summary, our findings reveal a crucial role of BCL6 in promoting KRAS-addicted lung cancer and suggest BCL6 as a therapeutic target for the treatment of this intractable disease.

ELK1-mediated YTHDF1 drives prostate cancer progression by facilitating the translation of Polo-like kinase 1 in an m6A dependent manner.

Background: N6-methyladenosine (m6A) is one of the most prevalent mRNA modifications in mammals, and it regulates the fate of modified RNA transcripts. In the current study, we aimed to elucidate the role of YTH m6A RNA-binding protein 1 (YTHDF1), a "reader" of m6A modification, in prostate cancer tumorigenesis. Methods: We employed a multi-omics approach to detect the direct target of YTHDF1 upon manipulation of YTHDF1 expression in prostate cancer cells. Expression of YTHDF1 was also evaluated in human prostate tumors and either adjacent or paired normal tissues. Additionally, in vivo tumor growth and metastasis experimental assays were performed to evaluate the role of YTHDF1 in tumorigenesis. Finally, luciferase reporter assays and Chromatin immunoprecipitation (ChIP) were conducted to elucidate the transcriptional regulators of YTHDF1. Results: We demonstrated that polo-like kinase 1 (PLK1) is a direct target of YTHDF1. YTHDF1 facilitated the translation efficiency of PLK1 in an m6A-dependent manner by identifying the m6A-modified PLK1 mRNA and subsequently promoted the hyperactivation of the PI3K/AKT signaling pathway. Moreover, our results indicated that YTHDF1 was upregulated in prostate cancer tissue and that high YTHDF1 expression was associated with adverse prognosis in patients with prostate cancer. Furthermore, upregulation of YTHDF1 promoted prostate cancer tumorigenesis and metastasis in vitro and in vivo. Additionally, dysregulation of ETS transcription factor ELK1 activated the transcription of YTHDF1 by directly binding to its promoter region. Conclusions: Collectively, our findings suggest that the ELK1/YTHDF1/PLK1/PI3K/AKT axis is critical for prostate cancer progression and may serve as a potential therapeutic target for prostate cancer treatment.

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

BACKGROUND: Glutathione 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. METHODS: The 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. RESULTS: The 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. CONCLUSION: The 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.

Identification of ELK1 interacting peptide segments in the androgen receptor.

Prostate cancer (PCa) growth requires tethering of the androgen receptor (AR) to chromatin by the ETS domain transcription factor ELK1 to coactivate critical cell proliferation genes. Disruption of the ELK1-AR complex is a validated potential means of therapeutic intervention in PCa. AR associates with ELK1 by coopting its two ERK docking sites, through the amino-terminal domain (A/B domain) of AR. Using a mammalian two-hybrid assay, we have now functionally mapped amino acids within the peptide segments 358-457 and 514-557 in the A/B domain as required for association with ELK1. The mapping data were validated by GST (glutathione S-transferase)-pulldown and BRET (bioluminescence resonance energy transfer) assays. Comparison of the relative contributions of the interacting motifs/segments in ELK1 and AR to coactivation of ELK1 by AR suggested a parallel mode of binding of AR and ELK1 polypeptides. Growth of PCa cells was partially inhibited by deletion of the upstream segment in AR and nearly fully inhibited by deletion of the downstream segment. Our studies have identified two peptide segments in AR that mediate the functional association of AR with its two docking sites in ELK1. Identification of the ELK1 recognition sites in AR should enable further structural studies of the ELK1-AR interaction and rational design of small molecule drugs to disrupt this interaction.

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 122049 siRNA 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.

Sirt7 associates with ELK1 to participate in hyperglycemia memory and diabetic nephropathy via modulation of DAPK3 expression and endothelial inflammation.

Diabetic nephropathy (DN) is one of the most serious complications of advanced diabetes, and increases patient mortality. Recently, epigenetics-mediated hyperglycemic memory in pathological process of DN has received attention. The purpose of this study was to determine the underlying mechanism by which sirt7 modulates hyperglycemic memory in DN. In glomerular endothelial cells (GECs) cultured in high glucose and glomeruli of DN patients and rats, an increase in p65 phosphorylation and endothelial adhesion molecule levels persisted after glucose normalization but was reversed by glucose normalization associated with death-associated protein kinase-3 (DAPK3) knockout or DAPK3 inhibitor. High glucose-mediated decrease in sirt7, the deacetylase modulating H3K18-acetylation (H3K18ac), was sustained after normoglycemia. Sirt7 overexpression accompanied by glucose normalization suppressed DAPK3 expression and inflammation in GECs. Moreover, sh-sirt7-induced inflammation was inhibited by si-DAPK3. Furthermore, sirt7 and H3K18ac were located at the DAPK3 promoter region. ELK1 was found to combine with sirt7. si-ELK1 supplemented with normoglycemia inhibited high glucose-induced DAPK3 expression and inflammation in GECs. ELK1 overexpression-mediated inflammation was inhibited by si-DAPK3. In addition, ELK1 and sirt7 were located at the same promoter region of DAPK3. ELK1 overexpression enhanced DAPK3 promoter activity, which disappeared after specific binding site mutation. In vivo, sirt7 overexpression decreased inflammation and improved renal function during insulin treatment of DN rats, whereas insulin alone did not work. Our data demonstrated high glucose-mediated mutual inhibition between sirt7 and ELK1 induced DAPK3 transcription and inflammation despite normoglycemia in GECs, thus forming a vicious cycle and participating in the occurrence of hyperglycemic memory in DN.

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

BACKGROUND: Diabetic 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. METHODS: Immunohistochemistry, 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. RESULTS: Our 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. CONCLUSIONS: SETD8 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.

PURPOSE: Osteosarcoma 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. METHODS: The 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. RESULTS: LncRNA 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. CONCLUSIONS: The 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.

Highly-metastatic colorectal cancer cell released miR-181a-5p-rich extracellular vesicles promote liver metastasis by activating hepatic stellate cells and remodelling the tumour microenvironment.

Liver metastasis of colorectal cancer (CRLM) is the most common cause of CRC-related mortality, and is typically caused by interactions between CRC cells and the tumour microenvironment (TME) in the liver. However, the molecular mechanisms underlying the crosstalk between tumour-derived extracellular vesicle (EV) miRNAs and the TME in CRLM have yet to be fully elucidated. The present study demonstrated that highly metastatic CRC cells released more miR-181a-5p-rich EVs than cells which exhibit a low metastatic potential, in-turn promoting CRLM. Additionally, we verified that FUS mediated packaging of miR-181a-5p into CRC EVs, which in-turn persistently activated hepatic stellate cells (HSCs) by targeting SOCS3 and activating the IL6/STAT3 signalling pathway. Activated HSCs could secrete the chemokine CCL20 and further activate a CCL20/CCR6/ERK1/2/Elk-1/miR-181a-5p positive feedback loop, resulting in reprogramming of the TME and the formation of pre-metastatic niches in CRLM. Clinically, high levels of serum EV containing miR-181a-5p was positively correlated with liver metastasis in CRC patients. Taken together, highly metastatic CRC cells-derived EVs rich in miR-181a-5p could activate HSCs and remodel the TME, thereby facilitating liver metastasis in CRC patients. These results provide novel insight into the mechanism underlying liver metastasis in CRC.

T-cell activation decreases miRNA-15a/16 levels to promote MEK1-ERK1/2-Elk1 signaling and proliferative capacity.

While miRs have been extensively studied in the context of malignancy and tumor progression, their functions in regulating T-cell activation are less clear. In initial studies, we found reduced levels of miR-15a/16 at 3 to 18 h post-T-cell receptor (TCR) stimulation, suggesting a role for decreased levels of this miR pair in shaping T-cell activation. To further explore this, we developed an inducible miR15a/16 transgenic mouse model to determine how elevating miR-15a/16 levels during early stages of activation would affect T-cell proliferation and to identify TCR signaling pathways regulated by this miR pair. Doxycycline (DOX)-induced expression of miR-15a/16 from 0 to 18 h post-TCR stimulation decreased ex vivo T-cell proliferation as well as in vivo antigen-specific T-cell proliferation. We also combined bioinformatics and proteomics approaches to identify the mitogen-activated protein kinase kinase 1 (MEK1) (Map2k1) as a target of miR-15a/16. MEK1 targeting by miR-15a/16 was confirmed using miR mimics that decreased Map2k1 mRNA containing the 3'-UTR target nucleotide sequence (UGCUGCUA) but did not decrease Map2k1 containing a mutated control sequence (AAAAAAAA). Phosphorylation of downstream signaling molecules, extracellular signal-regulated protein kinase 1/2 (ERK1/2) and Elk1, was also decreased by DOX-induced miR-15a/16 expression. In addition to MEK1, ERK1 was subsequently found to be targeted by miR-15a/16, with DOX-induced miR-15a/16 reducing total ERK1 levels in T cells. These findings show that TCR stimulation reduces miR-15a/16 levels at early stages of T-cell activation to facilitate increased MEK1 and ERK1, which promotes the sustained MEK1-ERK1/2-Elk1 signaling required for optimal proliferation.