Sp1 Expression Research Articles

Pan-cancer analysis of Sp1 with a focus on immunomodulatory roles in gastric cancer

BackgroundSp1, a transcription factor, regulates essential cellular processes and plays important tumorigenic roles across diverse cancers. However, comprehensive pan-cancer analyses of its expression and potential immunomodulatory roles remain unexplored.MethodsUtilizing bioinformatics tools and public datasets, we examined the expression of Sp1 across normal tissues, tumors, and immune cells, and screened for pre- and post-transcriptional modifications, including genetic alterations, DNA methylation, and protein phosphorylation, affecting its expression or function. The association of Sp1 expression with immune cell infiltration, tumor mutational burden, and immune checkpoint signaling was also investigated. Single-cell transcriptome data was used to assess Sp1 expression in immune cells in gastric cancer (GC), and findings were corroborated using immunohistochemistry and multiplex immunofluorescence in an immunotherapy-treated patient cohort. The prognostic value of Sp1 in GC patients receiving immunotherapy was evaluated with Cox regression models.ResultsElevated Sp1 levels were observed in various cancers compared to normal tissues, with notable prominence in GC. High Sp1 expression correlated with advanced stage, poor prognosis, elevated tumor mutational burden (TMB), and microsatellite instability (MSI) status, particularly in GC. Significant correlations between Sp1 levels and CD8+ T cell and the M1 phenotype of tumor-associated macrophages were further detected upon multiplex immunofluorescence in GC samples. Interestingly, we verified that GC patients with higher Sp1 levels exhibited improved response to immunotherapy. Moreover, Sp1 emerged as a prognostic and predictive biomarker for GC patients undergoing immunotherapy.ConclusionsOur pan-cancer analysis sheds light on the multifaceted role of Sp1 in tumorigenesis and underscores its potential as a prognostic and predictive biomarker for patients with GC undergoing immunotherapy.

Jia Wei Qingxin Lotus Seed Drink ameliorates epithelial mesenchymal transition injury in diabetic kidney disease via inhibition of JMJD1C/SP1/ZEB1 signaling pathway

BackgroundDiabetic kidney disease (DKD) is one of the most common microvascular complications in patients with diabetes mellitus. In this condition, renal tubular epithelial mesenchymal transition (EMT) is an important factor accelerating the progression of DKD and a major cause of renal fibrosis and end-stage renal disease. However, the therapeutic effect is unsatisfactory because of the lack of effective drugs. Jia Wei Qingxin Lotus Seed Drink (QISD) is a traditional Chinese medicine compound formula that has shown to be effective in the clinical treatment of DKD. However, the potential of QISD in DKD-EMT treatment has yet to be fully explored. PurposeThis study aimed to investigate the role of QISD in ameliorating DKD-EMT injury and its mechanism. MethodsThe active ingredients of QISD were identified via ultra-performance liquid chromatography-mass spectrometry/mass spectrometry (UHPLC-MS/MS). A DKD mouse model was constructed by high-fat diet feeding and intraperitoneal injection of STZ (60 mg/kg), and QISD (14.46, 28.92, and 57.84 g/kg/day) was administered by gavage for 12 consecutive weeks. Dapagliflozin (1 mg/kg/d) was used as a positive control. Renal pathological damage was observed by HE, PAS, and Masson staining. The expression levels of EMT-related proteins and pathway proteins were detected via immunohistochemistry, RT-qPCR, and western blot. In in vitro experiments, EMT injury was induced in human kidney tubular epithelial cells (HK-2) by using lipopolysaccharide (LPS). A combination of CCK8 assay, wound healing assay, small-molecule inhibitor intervention, and overexpression lentiviral transfection was used to investigate the effects of QISD on cell migration ability, adhesion ability, fibrotic factor formation, and mesenchymal properties. ResultsAnimal experiments showed that QISD improved blood glucose, body weight, symptoms of excessive drinking and eating, and renal pathological injury in mice, reduced extracellular matrix deposition, delayed renal EMT injury, and inhibited the activation of the histone demethylase JMJD1C. UHPLC-MS/MS and molecular docking indicated that baicalin, wogonoside, oroxylin A-7-O-β-D-glucuronide, and glulisine A found in QISD could bind to JMJD1C. The ameliorating effect of QISD on DKD-EMT injury might be related to JMJD1C. The improvement of DKD-EMT injury by QISD was accompanied by the reduction of SP1 and ZEB1 expression. The SP1 overexpression not only reversed the therapeutic effect of JIB-04, an inhibitor of JMJD1C, on DKD-EMT but also exacerbated the expression of ZEB1 and downstream EMT-related factors. Thus, QISD might affect the expression of the epithelial marker E-cadherin by inhibiting the JMJD1C/SP1/ZEB1 signaling pathway, consequently preventing the transformation of epithelial cells to mesenchymal cells and ameliorating DKD-EMT injury. ConclusionThis study was the first to demonstrate that QISD might ameliorate DKD-EMT injury by inhibiting the JMJD1C/SP1/ZEB1 signaling pathway. These findings provide strong pharmacologic evidence for the clinical use of QISD in the treatment of DKD.

MiR-24-3p modulates cardiac function in doxorubicin -induced heart failure via the Sp1/PI3K signaling pathway

PurposeThe goal of this research was to explore the role of miR-24-3p in heart failure (HF), with a focus on its impact on the specificity protein 1 (Sp1)/phosphoinositide 3-kinase (PI3K) pathway. MethodsHF rat and HF cell models were established using doxorubicin(Dox). Cardiac function was assessed through echocardiography, while histological changes were observed via hematoxylin-eosin (HE) staining. To further investigate the underlying mechanisms, HF cell models were treated with either an Sp1 inhibitor or a PI3K inhibitor. Additionally, models with miR-24-3p overexpression or silencing were constructed. N-terminal pro-brain natriuretic peptide (NT-proBNP) levels were determined by ELISA. Cell apoptosis was evaluated using TUNEL staining, and lactate dehydrogenase (LDH) levels were measured by colorimetry. Reactive oxygen species (ROS) production was analyzed using flow cytometry. Related gene and protein expressions were assessed via qRT-PCR and Western blotting. Finally, the relationship between miR-24-3p and Sp1 was confirmed through dual-luciferase assays. ResultsDox treatment increased the left ventricular internal diameter (LVIDd) while decreasing ejection fraction (EF) and fractional shortening (FS), leading to disorganized cardiomyocyte arrangement, cellular edema, and necrosis in rats. In HF rats, NT-proBNP, Caspase-3, and miR-24-3p expression levels were elevated, whereas Sp1 and PI3K mRNA and protein expression levels were decreased. Similarly, Dox-induced damage in H9c2 cardiomyocytes resulted in increased NT-proBNP, apoptosis, Caspase-3, LDH, ROS, and miR-24-3p expression, along with decreased Sp1 and PI3K expression. Treatment with either Sp1 or PI3K inhibitors exacerbated the Dox-induced cardiomyocyte damage, further elevating NT-proBNP, apoptosis, Caspase-3, LDH, ROS, and miR-24-3p expression levels. Notably, Sp1 inhibition reduced PI3K expression, and PI3K inhibition, in turn, suppressed Sp1 expression. Overexpression of miR-24-3p worsened Dox-induced cardiomyocyte damage, characterized by increased NT-proBNP, apoptosis, Caspase-3, LDH, and ROS expression, alongside reduced Sp1 and PI3K expression. In contrast, silencing miR-24-3p mitigated these detrimental effects and increased Sp1 and PI3K expression. Dual-luciferase assays confirmed that miR-24-3p directly targets Sp1. ConclusionDox induces cardiomyocyte damage, impairs cardiac function, and promotes cardiomyocyte apoptosis and oxidative stress. Silencing miR-24-3p offers a protective effect by activating the Sp1/PI3K signaling pathway in heart failure.

LINC01614 activated by SP1 promoted malignant behavior of triple-negative breast cancer cells via the WNT/b-Catenin signaling pathway.

Triple-negative breast cancer (TNBC) represents the most aggressive subtype of breast cancer (BC), characterized by a dismal prognosis. Dysregulated long non-coding RNA LINC01614 might be a potential biomarker for BC as previously reported. Nevertheless, its functions and mechanism in TNBC cells are unclear. The study aimed to study the effects of LINC01614 on TNBC cell migration, invasion, and epithelial-mesenchymal transition (EMT) process as well as the related mechanism. Reverse transcription quantitative polymerase chain reaction was performed to detect the expression of LINC01614 and SP1 in TNBC cells and tissues. The cellular localization of LINC01614 was determined by subcellular fraction assays. Cell counting kit-8 and Transwell invasion assays were conducted for measurement of TNBC cell viability and invasive ability. Cell migration was performed using wound healing assays and Transwell migration assays. Chromatin immunoprecipitation assays and luciferase reporter assays were used to explore the interaction between SP1 and LINC01614. Western blotting was used to assess protein levels of factors involved in EMT process and Wnt/ß-catenin signaling in TNBC cells. LINC01614 expression was elevated in TNBC tissues and cells. LINC01614 knockdown inhibited cell viability as well as migratory and invasive abilities of TNBC cells. LINC01614 knockdown also obstructed EMT process, as shown by E-cadherin upregulation and vimentin downregulation in TNBC cells. SP1 directly bound to the promoter of LINC01614 and activated LINC01614 expression. SP1 overexpression reversed the suppressive effect of LINC01614 knockdown on TNBC cell migration, invasion, and EMT process. Protein levels of Wnt and ß-catenin were diminished by LINC01614 knockdown, and the trend was partially rescued by SP1 overexpression. SP1-induced LINC01614 promoted malignant behavior of TNBC cells by activating the Wnt/ß-catenin signaling pathway.

Abstract P182: Antigen Presenting Cell-specific Keap1-Nrf2 pathway Mediates Salt-Sensitive Hypertension in Humans

Background: Hypertension remains the leading cause of mortality worldwide and is a primary risk factor for cardiovascular disease. Of the 1.3 billion people globally affected by hypertension, over 50% exhibit salt-sensitivity of blood pressure (SSBP), which leads to higher rates of mortality, morbidity, and kidney damage compared to those with salt-resistant hypertension. Previously our research has shown that high salt intake activates antigen-presenting cells (APCs), causing inflammation and hypertension. This pro-inflammatory response is partly driven by the epithelial sodium channel (ENaC), which facilitates sodium ion transport into APCs. While the Keap1-Nrf2 pathway is known for its role in antioxidant defense and inflammatory responses, its contribution to salt-sensitive hypertension remains unclear. We hypothesize that ENaC-dependent sodium entry triggers inflammation and activates the Sp1-MALAT1-Keap1-Nrf2 signaling axis in APCs, thereby contributing to SSBP. Method: We utilized a well-established inpatient protocol to phenotype participants for salt sensitivity of blood pressure. Additionally, we conducted RNA sequencing (RNAseq) on human monocytes exposed to elevated sodium levels in vitro and performed Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) analysis on peripheral blood mononuclear cells (PBMCs). Results: We found that whereas high salt did not significantly increases expression of Sp1 (5380.63 ± 359.77 vs 7414.54 ± 434.19, q=0.829), MALAT1 (86888.54 ±12200.12 vs 219429.90 ± 109994.16, q=0.717), Keap1 (1586.54 ± 132.94 vs 2203.72 ± 177.48, q=0.884), it significantly increased Nrf2 expression (5080.36 ± 416.64 vs 5443.54 ± 432.78, q=0.019) in RNAseq analysis of human high salt compared to normal salt treated monocyte. Furthermore, the results of the CITE-seq experiment indicated that changes in the expression of the Sp1 correlate with SSBP (r=0.5205, p=0.038). Conclusion: These preliminary findings reveal a physiological link between inflammation and salt-sensitive hypertension through the Sp1-MALAT1-Keap1-Nrf2 signaling axis in APCs. The activation of APCs mediated by Sp1 could potentially serve as a diagnostic marker for salt sensitivity in blood pressure.

Deciphering the Dysregulating IGF-1–SP1–CD248 Pathway in Fibroblast Functionality during Diabetic Wound Healing

Reduced fibroblast activity is a critical factor in the progression of diabetic ulcers. CD248, a transmembrane glycoprotein prominently expressed in activated fibroblasts, plays a pivotal role in wound healing. However, the role of CD248 in diabetic wound healing and the CD248 regulatory pathway remains largely unexplored. Our study shows that CD248 expression is significantly reduced in skin wounds from both diabetic patients and mice. Single-cell transcriptome data analyses reveal a marked reduction of CD248-enriched secretory-reticular fibroblasts in diabetic wounds. We identify insulin-like growth factor-1 (IGF-1) as a key regulator of CD248 expression through the Akt/mTOR signaling pathway and the Sp1 transcription factor. Overexpression of CD248 enhances fibroblast motility, elucidating the underrepresentation of CD248-enriched fibroblasts in diabetic wounds. Immunohistochemical staining of diabetic wound samples further confirm low SP1 expression and fewer CD248-positive secretory-reticular fibroblasts. Further investigation reveals that elevated tumor necrosis factor α (TNFα) levels in diabetic environment promotes IGF-1 resistance, and inhibiting IGF-1-induced CD248 expression. In summary, our findings underscore the critical role of the IGF1-SP1-CD248 axis in activating reticular fibroblasts during wound-healing processes. Targeting this axis in fibroblasts could help develop a therapeutic regimen for diabetic ulcers.

Role of post-translational modifications of Sp1 in cardiovascular diseases.

Specific protein 1 (Sp1) is pivotal in sustaining baseline transcription as well as modulating cell signaling pathways and transcription factors activity. Through interactions with various proteins, especially transcription factors, Sp1 controls the expression of target genes, influencing numerous biological processes. Numerous studies have confirmed Sp1's significant regulatory role in the pathogenesis of cardiovascular disorders. Post-translational modifications (PTMs) of Sp1, such as phosphorylation, ubiquitination, acetylation, glycosylation, SUMOylation, and S-sulfhydration, can enhance or modify its transcriptional activity and DNA-binding stability. These modifications also regulate Sp1 expression across different cell types. Sp1 is crucial in regulating non-coding gene expression and the activity of proteins in response to pathophysiological stimuli. Understanding Sp1 PTMs advances our knowledge of cell signaling pathways in controlling Sp1 stability during cardiovascular disease onset and progression. It also aids in identifying novel pharmaceutical targets and biomarkers essential for preventing and managing cardiovascular diseases.

The Anti-Metastatic Action of Oxyresveratrol via Suppression of Phosphoryl-ERK/-PKCα-Mediated Sp1/MMP1 Signaling in Human Renal Carcinoma Cells.

Oxyresveratrol (OxyR) exerts biological and pharmacological effects in a variety of tumor cells, including antioxidant action, antitumor activity, and proapoptotic effects. However, the regulation of targeted signaling pathways by OxyR and the mechanism underlying these effects in human renal cell carcinoma (RCC) have been less studied. We observed that OxyR at noncytotoxic doses did not affect the growth of human RCC cells or normal kidney HK2 cells. OxyR inhibited ACHN and Caki-1 cell migration and invasion through targeting matrix metalloproteinase 1 (MMP1) expression. Analysis of clinical databases showed that high MMP1 expression is associated with lower overall survival (OS) in these cancers (p < 0.01). OxyR significantly inhibited the mRNA and protein expression of Sp1. Furthermore, luciferase assay results showed that OxyR inhibited Sp1 transcriptional activity. Additionally, OxyR preferentially suppressed the activation of ERK and PKCα. Treatment with U0126 (MEK inhibitor) or G06976 (PKCα inhibitor) clearly decreased Sp1 and MMP1 expression and inhibited RCC cell migration and invasion. In conclusion, OxyR may be a potential antitumor therapy for the inhibition of migration and invasion by controlling p-ERK/Sp1 and p-PKCα/Sp1-mediated MMP1 expression in RCC.

MicroRNA-124-3p targets Sp1 transcription factor to regulate glioma progression in rats

Gliomas are the most prevalent malignancies of the central nervous system (CNS). Downregulation of microRNA‑124 (miR‑124) has been identified in glioma; however, its biological functions in glioma are not yet fully understood. Specificity protein 1 (SP1) is a type of transcription factor that is involved in cancer progression. In this study, we examined the targeting of Sp1 mRNA by miR-124-3p in a rat glioma model. After confirming and selecting the binding of Sp1 to miR-124 with the help of bioinformatics methods, adult male Wistar rats were used to induce glioma by microinjection of 1 × 106 C6 cells into the striatum area of brain. The rats were divided into 3 groups; intact, sham and glioma groups. The presence of glioma was confirmed 21 days after implantation through histological analysis. The expression levels of miR-124 and SP1 genes in the experimental groups were examined using quantitative real-time polymerase chain reaction (qRT-PCR). Our data showed that the expression of miR-124 was significantly downregulated in glioma group compared to the sham and intact group, while the expression of SP1 was significantly upregulated. We found that the expression levels of miR-124 and Sp1 were decreased and increased in C6 cell line compared to the normal brain tissue cell line, respectively. The results indicated that Sp1 was identified as a direct target of miR‑124 through luciferase reporter assays. In summary, this study demonstrated for the first time that miR-124 expression is downregulated and Sp1 expression is upregulated in an animal model of glioma, which, in turn, may be involved in the development of glioma brain cancer.

Core biomarkers analysis benefit for diagnosis on human intrahepatic cholestasis of pregnancy

BackgroundThe pregnant women with intrahepatic cholestasis were at high risk of fetal distress, preterm birth and unexpected stillbirth. Intrahepatic cholestasis of pregnancy (ICP) was mainly caused by disorder of bile acid metabolism, whereas the specific mechanism was obscure.MethodsWe performed proteomics analysis of 10 ICP specimens and 10 placenta specimens from patients without ICP through data-independent acquisition (DIA) technique to disclose differentially expressed proteins. We executed metabolomic analysis of 30 ICP specimens and 30 placenta specimens from patients without ICP through UPLC-MS/MS to identify differentially expressed metabolites. Enrichment and correlation analysis was used to obtain the direct molecular insights of ICP development. The ICP rat models were constructed to validate pathological features.ResultsThe heatmap of proteomics analysis showed the top 30 up-regulated and 30 down-regulated proteins. The metabolomic analysis revealed 20 richer and 4 less abundant metabolites in ICP samples compared with placenta specimens from patients without ICP, and enrichment pathways by these metabolites included primary bile acid biosynthesis, cholesterol metabolism, bile secretion, nicotinate and nicotinamide metabolism, purine metabolism and metabolic pathways. Combined analysis of multiple omics results demonstrated that bile acids such as Glycohyocholic acid, Glycine deoxycholic acid, beta-Muricholic acid, Noncholic acid, cholic acid, Gamma-Mercholic Acid, alpha-Muricholic acid and Glycochenodeoxycholic Aicd were significantly associated with the expression of GLRX3, MYL1, MYH7, PGGT1B, ACTG1, SP3, LACTB2, C2CD5, APBB2, IPO9, MYH2, PPP3CC, PIN1, BLOC1S1, DNAJC7, RASAL2 and ATCN3 etc. The core protein ACAT2 was involved in lipid metabolic process and animal model showed that ACAT2 was up-regulated in placenta and liver of pregnant rats and fetal rats. The neonates had low birth weight and Safranin O-Fast green FCF staining of animal models showed that poor osteogenic and chondrogenic differentiation of fetal rats.ConclusionMultiple metabolites-alpha-Muricholic acid, beta-Muricholic acid, Glycine deoxycholic acid and Glycochenodeoxycholic Acid etc. were perfect biomarkers to predict occurrence of ICP. Bile acids were significantly associated with varieties of protein expression and these proteins were differentially expressed in ICP samples. Our study provided several biomarkers for ICP detection and potential therapeutic targets for ICP development.

Identification of a Novel hsa_circ_0058058/miR-324-5p Axis and Prognostic/Predictive Molecules for Acute Myeloid Leukemia Outcome by Bioinformatics-Based Analysis.

Acute myeloid leukemia (LAML) is one of the most prevalent hematological malignancies. In recent years, while targeted approaches have shown promise in the fight against cancer, the treatability and prognosis of patients remain inadequate due to the shortage of drugs. Noncoding RNAs, especially circular RNA (circRNA) and microRNA (miRNA), have been shown to play a unique role in tumor development. This study aims to identify the disease-associated circRNA-miRNA-mRNA network by bioinformatic analysis and investigate the mechanisms in the development and progression of LAML. Additionally, it reveals the promising roles of these molecules as a diagnostic biomarker and therapeutic target for LAML treatment. Using various bioinformatics approaches, we identified the hsa_circ_0058058/miR-324-5p axis in LAML and its possible functions in LAML development. According to our results, hsa circ-0058058 can regulate the expression of AP1G1 and SP1 through miR-324-5p to support angiogenesis, the cell cycle, and DNA replication processes. Downregulation of hsa circ-0058058 may contribute to the anticancer functions of miR-324-5p on LAML tumorigenesis, and upregulation of miR-324-5p can abolish the oncogenic effects of AP1G1 and SP1 on LAML tumorigenesis. Additionally, highly enriched pathways indicated possible interactions between molecules underlying LAML pathology. Targeted molecules within this network may be able to function as therapeutic and diagnostic biomarkers for disease, while more research and clinical confirmation are needed.

Exposure to coal dust exacerbates cognitive impairment by activating the IL6/ERK1/2/SP1 signaling pathway

Coal dust (CD) is a common pollutant, and epidemiological surveys indicate that long-term exposure to coal dust not only leads to the occurrence of pulmonary diseases but also has certain impacts on cognitive abilities. However, there is little open-published literature on the effects and specific mechanisms of coal dust exposure on the cognition of patients with Mild Cognitive Impairment (MCI) and Alzheimer's Disease (AD). An animal model has been built in this study with clinical population samples to explore the changes in neuroinflammation and cognitive abilities with coal dust exposure. In the animal model, compared to C57BL/6 mice, APP/PS1 mice exposed to coal dust exhibited more severe cognitive impairment, accompanied by significantly elevated levels of neuroinflammatory factors Apolipoprotein E4 (AOPE4) and Interleukin-6 (IL6) in the hippocampus, and more severe neuronal damage. In clinical sample sequencing, it was found that there is significant upregulation of AOPE4, neutrophils, and IL6 expression in the peripheral blood of MCI patients compared to normal individuals. Mechanistically, cell experiments revealed that IL6 could promote the phosphorylation of ERK1/2 and enhance the expression of transcription factor SP1, thereby promoting AOPE4 expression. The results of this study suggest that coal dust can promote the upregulation of IL6 and AOPE4 in patients, exacerbating cognitive impairment.

Microglial Sp1 induced LRRK2 upregulation in response to manganese exposure, and 17β-estradiol afforded protection against this manganese toxicity

Chronic exposure to elevated levels of manganese (Mn) causes a neurological disorder referred to as manganism, presenting symptoms similar to those of Parkinson’s disease (PD), yet the mechanisms by which Mn induces its neurotoxicity are not completely understood. 17β-estradiol (E2) affords neuroprotection against Mn toxicity in various neural cell types including microglia. Our previous studies have shown that leucine-rich repeat kinase 2 (LRRK2) mediates Mn-induced inflammatory toxicity in microglia. The LRRK2 promoter sequences contain three putative binding sites of the transcription factor (TF), specificity protein 1 (Sp1), which increases LRRK2 promoter activity. In the present study, we tested if the Sp1-LRRK2 pathway plays a role in both Mn toxicity and the protection afforded by E2 against Mn toxicity in BV2 microglial cells. The results showed that Mn induced cytotoxicity, oxidative stress, and tumor necrosis factor-α production, which were attenuated by an LRRK2 inhibitor, GSK2578215A. The overexpression of Sp1 increased LRRK2 promoter activity, mRNA and protein levels, while inhibition of Sp1 with its pharmacological inhibitor, mithramycin A, attenuated the Mn-induced increases in LRRK2 expression. Furthermore, E2 attenuated the Mn-induced Sp1 expression by decreasing the expression of Sp1 via the promotion of the ubiquitin-dependent degradation pathway, which was accompanied by increased protein levels of RING finger protein 4, the E3-ligase of Sp1, Sp1 ubiquitination, and SUMOylation. Taken together, our novel findings suggest that Sp1 serves as a critical TF in Mn-induced LRRK2 expression as well as in the protection afforded by E2 against Mn toxicity through reduction of LRRK2 expression in microglia.

Evaluation the expression of Sp1 and Par3 in urinary bladder carcinoma

Evaluation the expression of Sp1 and Par3 in urinary bladder carcinoma

#1076 DNA hypomethylation of Syk induces oxidative stress and apoptosis via the PKCβ/ P66shc signaling pathway in diabetic kidney disease

Abstract Background and Aims Epigenetic alterations, especially DNA methylation, have been shown to play a role in the pathogenesis of diabetes mellitus (DM) and its complications, including diabetic kidney disease (DKD). Spleen tyrosine kinase (Syk) is known to be involved in immune and inflammatory disorders. We, therefore, investigated the possible involvement of Syk promoter methylation in DKD, and the mechanisms underlying this process. Method Kidney tissues were obtained from renal biopsies of patients with early and advanced DKD. A diabetic mouse model (ApoE−/−DM) was generated from ApoE knockout (ApoE−/−) mice using a high fat and high glucose diet combined with low-dose streptozocin (STZ) intraperitoneal injection. We also established an in vitro model using HK2 cells. Results A marked elevation in the expression levels of Syk, PKCβ, and P66shc in renal tubules was observed in patients with DKD. In ApoE−/− DM mice, Syk expression and the binding of Sp1 to the Syk gene promoter were both increased in the kidney. In addition, the promoter region of the Syk gene exhibited hypomethylation. Syk inhibitor (R788) intervention improved renal function and alleviated pathologic changes in ApoE−/− DM mice. Moreover, R788 intervention alleviated oxidative stress and apoptosis and down-regulated the expression of PKCβ/ P66shc signaling pathway proteins. In HK2 cells, oxLDL combined with high glucose stimulation upregulated Sp1 expression in the nucleus (compared with control and oxLDL groups), and this was accompanied by an increase in the binding of Sp1 to the Syk gene promoter. SP1 silencing down-regulated the expression of Syk and inhibited the production of reactive oxygen species and cell apoptosis. Finally, PKC agonist intervention reversed the oxidative stress and apoptosis induced by Syk inhibitor (R406). Conclusion In DKD, hypomethylation at the Syk gene promoter was accompanied by an increase in Sp1 binding at the promoter. As a consequence of this enhanced Sp1 binding, Syk gene expression was upregulated. Syk inhibitors could attenuate DKD-associated oxidative stress and apoptosis via down-regulation of PKCβ/ P66shc signaling pathway proteins. Together, our results identify Syk as a promising target for intervention in DKD.

Inhibition of Foxp3 expression in the placenta of mice infected intraperitoneally by toxoplasma gondii tachyzoites: insights into the PPARγ/miR-7b-5p/Sp1 signaling pathway

BackgroundToxoplasma gondii, an obligate intracellular parasitic protozoa, infects approximately 30% of the global population. Contracting T. gondii at the primary infection of the mother can result in neonatal microcephaly, chorioretinitis, hydrocephalus, or mortality. Our previous study indicated that pregnant mice infected with T. gondii displayed a decrease in both the number and the suppressive ability of regulatory T cells, accompanied by the reduced Forkhead box P3 (Foxp3). Numerous studies have proved that microRNAs (miRNAs) are implicated in T. gondii infection, but there is meager evidence on the relationship between alterations of miRNAs and downregulation of Foxp3 induced by T. gondii.MethodsQuantitative reverse transcription polymerase chain reaction was utilized to detect the transcriptions of miRNAs and Foxp3. Protein blotting and immunofluorescence were used to detect the expressions of Foxp3 and related transcription factors. The structure of mouse placenta was observed by hematoxylin and eosin (HE) staining.To examine the activity of miR-7b promoter and whether miR-7b-5p targets Sp1 to suppress Foxp3 expression, we constructed recombinant plasmids containing the full-length/truncated/mutant miR-7b promoter sequence or wildtype/mutant of Sp1 3' untranslated region (3' UTR) to detect the fluorescence activity in EL4 cells.ResultsIn T. gondii-infected mice, miR-7b transcription was significantly elevated, while Foxp3 expression was decreased in the placenta. In vitro, miR-7b mimics downregulated Foxp3 expression, whereas its inhibitors significantly upregulated Foxp3 expression. miR-7b promoter activity was elevated upon the stimulation of T. gondii antigens, which was mitigated by co-transfection of mutant miR-7b promoter lacking peroxisome proliferator-activated receptor γ (PPARγ) target sites. Additionally, miR-7b mimics diminished Sp1 expression, while miR-7b inhibitors elevated its expression. miR-7b mimics deceased the fluorescence activity of Sp1 3' untranslated region (3' UTR), but it failed to impact the fluorescence activity upon the co-transfection of mutant Sp1 3' UTR lacking miR-7b target site.ConclusionsT. gondii infection and antigens promote miR-7b transcription but inhibit Foxp3 protein and gene levels. T. gondii antigens promote miR-7b promoter activity by a PPARγ-dependent mechanism. miR-7b directly binds to Sp1 3' UTR to repress Sp1 expression. Understanding the regulatory functions by which T. gondii-induced miR-7b suppresses Foxp3 expression can provide new perspectives for the possible therapeutic avenue of T. gondii-induced adverse pregnancy outcomes.Graphical

SP1 transcriptionally activates HTR2B to aggravate traumatic spinal cord injury by shifting microglial M1/M2 polarization.

Spinal cord injury (SCI) can result in structural and functional damage to the spinal cord, which may lead to loss of limb movement and sensation, loss of bowel and bladder control, and other complications. Previous studies have revealed the critical influence of trans-acting transcription factor 1 (SP1) in neurological pathologies, however, its role and mechanism in SCI have not been fully studied. The study was performed using mouse microglia BV2 stimulated using lipopolysaccharide (LPS) and male adult mice subjected to spinal hitting. Western blotting was performed to detect protein expression of SP1, 5-hydroxytryptamine (serotonin) receptor 2B (HTR2B), BCL2-associated x protein (Bax), B-cell lymphoma-2 (Bcl-2), inducible nitric oxide synthase (iNOS), clusters of differentiation 86 (CD86), Arginase 1 (Arg-1) and clusters of differentiation 206 (CD206). Cell viability and apoptosis were analyzed by MTT assay and TUNEL assay. mRNA levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-4 (IL-4) and tumor necrosis factor-β (TNF-β) were quantified by quantitative real-time polymerase chain reaction. The association of SP1 and HTR2B was identified by chromatin immunoprecipitation assay and dual-luciferase reporter assay. HE staining assay was performed to analyze the pathological conditions of spinal cord tissues. LPS treatment induced cell apoptosis and inhibited microglia polarization from M1 to M2 phenotype, accompanied by an increase of Bax protein expression and a decrease of Bcl-2 protein expression, however, these effects were relieved after SP1 silencing. Mechanism assays revealed that SP1 transcriptionally activated HTR2B in BV2 cells, and HTR2B knockdown rescued LPS-induced effects on BV2 cell apoptosis and microglial M1/M2 polarization. Moreover, SP1 absence inhibited BV2 cell apoptosis and promoted microglia polarization from M1 to M2 phenotype by decreasing HTR2B expression. SCI mouse model assay further showed that SP1 downregulation could attenuate spinal hitting-induced promoting effects on cell apoptosis of spinal cord tissues and microglial M1 polarization. SP1 transcriptionally activated HTR2B to aggravate traumatic SCI by shifting microglial M1/M2 polarization.

Abstract LB296: Human CYP1B1 enhances cancer progression through induction of Sp1 via DNMT-mediated epigenetic regulation of miR-375

Abstract Human cytochrome P450 1B1 (CYP1B1) is a major catalytic enzyme for metabolism of 17β-estradiol (E2) to catechol estrogens, mainly carcinogenic metabolite 4-hydroxyestradiol (4-OHE2). To identify the unknown molecular mechanism for cancer progression induced by CYP1B1, we studied the role of CYP1B1 in human tumor cells, MCF-7, MDA-MB-231, and HeLa cells. Suppression of CYP1B1 by shRNA significantly reduced cancer cell viability and invasion. Clinical data from cancer patients showed that high level of CYP1B1 expression resulted in relatively lower survival rate. Interestingly, overexpression of CYP1B1 markedly activated Wnt/β-catenin signaling and EMT process, and suppression of specificity protein 1 (Sp1) by siRNA, an well-known transcription factor, demolished the promoting effects of CYP1B1 on cancer progression. To investigate the correlation between CYP1B1 and Sp1, we analyzed the clinical data from breast and cervical cancer patients and the results showed the positive correlation between two genes. Furthermore, TMS (tetramethoxystilbene), a specific CYP1B1 inhibitor, diminished the induction of Sp1 by CYP1B1, and 4-OHE2 exceedingly promoted the expression level of Sp1. Surprisingly, miR-375, known as an oncogenic microRNA, substantially decreased by 4-OHE2 and CYP1B1 overexpression in CYP1B1-transgenic (TG) mice. Methylation specific assay revealed that CYP1B1 suppressed miR-375 expression through induction of hyper-methylation on pre-miR-375. In addition, epigenetic regulation of miR-375 by CYP1B1 was executed via induction of DNA methyltransferases (DNMTs), DNMT1, 3a, and 3b. Enhanced expression level of Sp1 by 4-OHE2 was significantly deteriorated by 5-aza-dC, a DNMT inhibitor. Taken together, our data suggest that CYP1B1 enhances cancer progression through induction of cancer cell proliferation and invasion via DNMT-mediated epigenetic regulation of miR-375 and further promotion of Sp1 expression. Citation Format: Young-Jin Chun, Yeo-Jung Kwon, Tae-Uk Kwon. Human CYP1B1 enhances cancer progression through induction of Sp1 via DNMT-mediated epigenetic regulation of miR-375 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB296.

DNA hypomethylation of Syk induces oxidative stress and apoptosis via the PKCβ/P66shc signaling pathway in diabetic kidney disease.

Epigenetic alterations, especially DNA methylation, have been shown to play a role in the pathogenesis of diabetes mellitus (DM) and its complications, including diabetic kidney disease (DKD). Spleen tyrosine kinase (Syk) is known to be involved in immune and inflammatory disorders. We, therefore, investigated the possible involvement of Syk promoter methylation in DKD, and the mechanisms underlying this process. Kidney tissues were obtained from renal biopsies of patients with early and advanced DKD. A diabetic mouse model (ApoE-/- DM) was generated from ApoE knockout (ApoE-/-) mice using a high-fat and high-glucose diet combined with low-dose streptozocin intraperitoneal injection. We also established an invitro model using HK2 cells. A marked elevation in the expression levels of Syk, PKCβ, and P66shc in renal tubules was observed in patients with DKD. In ApoE-/- DM mice, Syk expression and the binding of Sp1 to the Syk gene promoter were both increased in the kidney. In addition, the promoter region of the Syk gene exhibited hypomethylation. Syk inhibitor (R788) intervention improved renal function and alleviated pathologic changes in ApoE-/- DM mice. Moreover, R788 intervention alleviated oxidative stress and apoptosis and downregulated the expression of PKCβ/P66shc signaling pathway proteins. In HK2 cells, oxLDL combined with high-glucose stimulation upregulated Sp1 expression in the nucleus (compared with control and oxLDL groups), and this was accompanied by an increase in the binding of Sp1 to the Syk gene promoter. SP1 silencing downregulated the expression of Syk and inhibited the production of reactive oxygen species and cell apoptosis. Finally, PKC agonist intervention reversed the oxidative stress and apoptosis induced by Syk inhibitor (R406). In DKD, hypomethylation at the Syk gene promoter was accompanied by an increase in Sp1 binding at the promoter. As a consequence of this enhanced Sp1 binding, Syk gene expression was upregulated. Syk inhibitors could attenuate DKD-associated oxidative stress and apoptosis via downregulation of PKCβ/P66shc signaling pathway proteins. Together, our results identify Syk as a promising target for intervention in DKD.

Changes in growth performance, gene expressions related to protein absorption and hepatic metabolism of triploid crucian carp (Carassius carassius triploid) caused by dietary protein level

The aim of this study was to investigate the effects of dietary protein inclusion level on growth, protein absorption pathway and liver metabolism-related pathway in triploid crucian carp (Carassius carassius triploid). Six experimental diets with different protein levels (26%, 29%, 32%, 35%, 38%, 41%) were formulated. A total of 540 fish with an initial weight of 11.79 ± 0.09 g were randomly assigned to 18 cages and six treatments with three replicates of 30 fish each for 8 weeks feeding. The results showed that the specific growth rate (SGR) and feed efficiency (FE) of triploid crucian carp were highly positively correlated with protein content (p < 0.05). The broken-line regression analysis showed that the optimal dietary inclusion level was determined to be 39% considering SGR of triploid crucian carp. In addition, genes related to protein absorption were influenced by the protein inclusion level of dietary. The mRNA expressions of pept1, lat2, cdx2, jak2 and sp1 exhibited similar change tendency and promoted to highest expression at 41% groups, 32% followed. Furthermore, the expressions of hepatic metabolism genes tor, s6k1, igf1, ampd1 and adsl reached peak at 38% dietary protein inclusion level, while 4e-bp2 gene showed the highest expression levels at 41% dietary protein inclusion level. Finally, igf1 expression in liver and cdx2 in intestine showed high pearson coefficients with dietary protein level suggesting their expressions were significant positively regulated by dietary protein level. In conclusion, this study evidenced the influence of dietary protein level on growth, protein absorption pathway, TOR and IGF pathways of hepatic metabolism related in triploid crucian carp. 39% dietary protein inclusion level would be optimum level of triploid crucian carp dietary, which will lead to a better growth and high feed efficiency of triploid crucian carp.