The SP1/SIRT1/ACLY signaling axis mediates fatty acid oxidation in renal ischemia-reperfusion-induced renal fibrosis.

BACKGROUND: Renal ischemia-reperfusion is the primary cause of acute kidney injury (AKI). Clinically, most patients who experience ischemia-reperfusion injury eventually progress gradually to renal fibrosis and chronic kidney disease (CKD). However, the underlying mechanism for AKI to CKD transition remain absent. Our study demonstrated that the downregulation of sirtuin 1 (Sirt1)-mediated fatty acid oxidation (FAO) facilitates IRI-induced renal fibrosis. METHODS: The IRI animal model was established, and ribonucleic acid (RNA) sequencing was used to explore potential differentially expressed genes (DEGs) and pathways. The SIRT1 knockout mice were generated, and a recombinant adeno-associated virus that overexpresses SIRT1 was injected into mice to explore the function of SIRT1 in renal fibrosis induced by renal IRI. In vitro, hypoxia/reoxygenation (H/R) was used to establish the classical model of renal IRI and overexpression or knockdown of SIRT1 to investigate the SIRT1 function through lentiviral plasmids. The underlying molecular mechanism was explored through RNA sequencing, bioinformatics analysis, and chromatin immunoprecipitation assay. RESULTS: RNA sequencing analysis and western blot demonstrated that the expression of SIRT1 was significantly decreased in IRI mice. Overexpression of SIRT1 improved renal function and reduced lipid deposition and renal fibrosis. On the contrary, knockout of SIRT1 aggravated kidney injury and renal fibrosis. RNA sequencing, bioinformatics analysis, and chromatin immunoprecipitation assay mechanistically revealed that SIRT1 impairs the acetylation of histone H3K27 on the promoter region of ACLY, thereby impeding FAO activity and promoting renal fibrosis. Additionally, SP1 regulated FAO by directly modulating SIRT1 expression. CONCLUSION: Our findings highlight that downregulation of SIRT1-modulated FAO facilitated by the SP1/SIRT1/ACLY axis in the kidney increases IRI, suggesting SIRT1 to be a potential therapeutic target for renal fibrosis induced by renal IRI.

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.

BACKGROUND: Toxoplasma 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. METHODS: Quantitative 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. RESULTS: In 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. CONCLUSIONS: T. 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.

KN-93 promotes HDAC4 nucleus translocation to promote fatty acid oxidation in myocardial infarction.

Myocardial infarction (MI) is a potentially fatal disease that causes a significant number of deaths worldwide. The strategy of increasing fatty acid oxidation in myocytes is considered a therapeutic avenue to accelerate metabolism to meet energy demands. We conducted the study aiming to investigate the effect of KN-93, which induces histone deacetylase (HDAC)4 shuttling to the nucleus, on fatty acid oxidation and the expression of related genes. A mouse model of myocardial infarction was induced by isoprenaline administration. Heart damage was assessed by the detection of cardiac injury markers. The level of fatty acid oxidation level was evaluated by testing the expression of related genes. Both immunofluorescence and immunoblotting in the cytosol or nucleus were utilized to observe the distribution of HDAC4. The interaction between HDAC4 and specificity protein (SP)1 was confirmed by co-immunoprecipitation. The acetylation level of SP1 was tested after KN-93 treatment and HDAC4 inhibitor. Oxygen consumption rate and immunoblotting experiments were used to determine whether the effect of KN-93 on increasing fatty acid oxidation is through HDAC4 and SP1. Administration of KN-93 significantly reduced cardiac injury in myocardial infarction and promoted fatty acid oxidation both in vitro and in vivo. KN-93 was shown to mediate nuclear translocation of HDAC4. HDAC4 was found to interact with SP1 and reduce SP1 acetylation. HDAC4 or SP1 inhibitors attenuated the effect of KN-93 on fatty acid oxidation. In conclusion, KN-93 promotes HDAC4 translocation to the nucleus, thereby potentially enhancing fatty acid oxidation by SP1.

Downregulation of UBB potentiates SP1/VEGFA-dependent angiogenesis in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) presents a unique profile characterized by high levels of angiogenesis and robust vascularization. Understanding the underlying mechanisms driving this heterogeneity is essential for developing effective therapeutic strategies. This study revealed that ubiquitin B (UBB) is downregulated in ccRCC, which adversely affects the survival of ccRCC patients. UBB exerts regulatory control over vascular endothelial growth factor A (VEGFA) by directly interacting with specificity protein 1 (SP1), consequently exerting significant influence on angiogenic processes. Subsequently, we validated that DNA methyltransferase 3 alpha (DNMT3A) is located in the promoter of UBB to epigenetically inhibit UBB transcription. Additionally, we found that an unharmonious UBB/VEGFA ratio mediates pazopanib resistance in ccRCC. These findings underscore the critical involvement of UBB in antiangiogenic therapy and unveil a novel therapeutic strategy for ccRCC.

Identification of TMZ resistance-associated histone post-translational modifications in glioblastoma using multi-omics data.

BACKGROUD: Glioblastoma multiforme (GBM) is among the most aggressive cancers, with current treatments limited in efficacy. A significant hurdle in the treatment of GBM is the resistance to the chemotherapeutic agent temozolomide (TMZ). The methylation status of the MGMT promoter has been implicated as a critical biomarker of response to TMZ. METHODS: To explore the mechanisms underlying resistance, we developed two TMZ-resistant GBM cell lines through a gradual increase in TMZ exposure. Transcriptome sequencing of TMZ-resistant cell lines revealed that alterations in histone post-translational modifications might be instrumental in conferring TMZ resistance. Subsequently, multi-omics analysis suggests a strong association between histone H3 lysine 9 acetylation (H3K9ac) levels and TMZ resistance. RESULTS: We observed a significant correlation between the expression of H3K9ac and MGMT, particularly in the unmethylated MGMT promoter samples. More importantly, our findings suggest that H3K9ac may enhance MGMT transcription by facilitating the recruitment of the SP1 transcription factor to the MGMT transcription factor binding site. Additionally, by analyzing single-cell transcriptomics data from matched primary and recurrent GBM tumors treated with TMZ, we modeled the molecular shifts occurring upon tumor recurrence. We also noted a reduction in tumor stem cell characteristics, accompanied by an increase in H3K9ac, SP1, and MGMT levels, underscoring the potential role of H3K9ac in tumor relapse following TMZ therapy. CONCLUSIONS: The increase in H3K9ac appears to enhance the recruitment of the transcription factor SP1 to its binding sites within the MGMT locus, consequently upregulating MGMT expression and driving TMZ resistance in GBM.

Transcriptional regulation of SARS-CoV-2 receptor ACE2 by SP1.

Angiotensin-converting enzyme 2 (ACE2) is a major cell entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The induction of ACE2 expression may serve as a strategy by SARS-CoV-2 to facilitate its propagation. However, the regulatory mechanisms of ACE2 expression after viral infection remain largely unknown. Using 45 different luciferase reporters, the transcription factors SP1 and HNF4α were found to positively and negatively regulate ACE2 expression, respectively, at the transcriptional level in human lung epithelial cells (HPAEpiCs). SARS-CoV-2 infection increased the transcriptional activity of SP1 while inhibiting that of HNF4α. The PI3K/AKT signaling pathway, activated by SARS-CoV-2 infection, served as a crucial regulatory node, inducing ACE2 expression by enhancing SP1 phosphorylation-a marker of its activity-and reducing the nuclear localization of HNF4α. However, colchicine treatment inhibited the PI3K/AKT signaling pathway, thereby suppressing ACE2 expression. In Syrian hamsters (Mesocricetus auratus) infected with SARS-CoV-2, inhibition of SP1 by either mithramycin A or colchicine resulted in reduced viral replication and tissue injury. In summary, our study uncovers a novel function of SP1 in the regulation of ACE2 expression and identifies SP1 as a potential target to reduce SARS-CoV-2 infection.

Sp1 Regulates the M1 Polarization of Microglia Through the HuR/NF-κB Axis after Spinal Cord Injury.

The M1 polarization of microglia, followed by the production of pro-inflammatory mediators, hinders functional recovery after spinal cord injury (SCI). Our previous study has illuminated that specificity protein 1 (Sp1) expression is increased following SCI, whereas the function and regulatory mechanism of Sp1 during M1 polarization of microglia following SCI remain unknown. RNA binding protein, HuR, has been shown to be up-regulated in the injured spinal cord through analysis of the GEO database. Further investigation using Chip-Atlas data suggests a binding between Sp1 and HuR. Emerging evidence indicates that HuR plays a pivotal role in neuroinflammation after SCI. In this research, Sp1 and HuR levels in mice with SCI and BV2 cells treated with lipopolysaccharide (LPS) was determined by using quantitative real-time polymerase chain reaction and Western blotting techniques. A series of in vitro assays were performed to investigate the function of Sp1 during M1 polarization of microglia. The association between Sp1 and its target gene HuR was confirmed through gene transfection and luciferase reporter assay. Enhanced expression of HuR was observed in both SCI mice and LPS-treated BV2 cells, while Sp1 knockdown restrained M1 polarization of microglia and its associated inflammation by inhibiting the NF-κB signaling pathway. Silencing Sp1 also suppressed microglia activation and its mediated inflammatory response, which could be reversed by overexpression of HuR. In conclusion, silencing Sp1 restrains M1 polarization of microglia through the HuR/NF-κB axis, leading to neuroprotection, and thus promotes functional restoration following SCI.

Translation regulatory long non-coding RNA 1 negatively regulates cell radiosensitivity via the miR-22-3p/SP1 axis in non-small cell lung cancer.

OBJECTIVE: Non-small cell lung cancer (NSCLC) occupies 85% of lung cancer. Long non-coding RNAs (LncRNAs) can regulate the radiosensitivity of cancers. This study explored the mechanism of lncRNA TRERNA1 in the radiosensitivity of NSCLC cells. METHODS: LncRNA TRERNA1 level in NSCLC cell lines was determined. NSCLC cell radiation tolerance was measured. TRERNA1 expression was silenced or overexpressed in A549/HCC827 cells with the highest/lowest radiation tolerance, respectively. The contents of γ-H2AX and SA-ß-gal in NSCLC cells after radiation induction were detected. The targeted binding of TRERNA1 to miR-22-3p and miR-22-3p to SP1 were verified by dual-luciferase assay. SP1 expression were detected. Functional rescue experiments were implemented to confirm the roles of miR-22-3p and SP1 in the regulatory mechanism of TRERNA1. RESULTS: TRERNA1 was upregulated in NSCLC cells. TRERNA1 silencing enhanced radiosensitivity of NSCLC cells. TRERNA1 silencing elevated the contents of γ-H2AX and SA-ß-gal in A549 cells after radiation induction, while TRERNA1 overexpression showed an opposite trend in HCC827 cells. There were targeting relationships between TRERNA1 and miR-22-3p, and miR-22-3p and SP1. miR-22-3p repression or SP1 overexpression abolished the effects of TRERNA1 silencing. CONCLUSION: TRERNA1 silencing enhanced radiosensitivity of NSCLC cells via the miR-22-3p/SP1 axis. This study may offer new targets for NSCLC treatment.

SP1-stimulated miR-208a-5p aggravates sepsis-induced myocardial injury via targeting XIAP.

The development of sepsis can lead to many organ dysfunction and even death. Myocardial injury is one of the serious complications of sepsis leading to death. New evidence suggests that microRNAs (miRNAs) play a critical role in infection myocardial injury. However, the mechanism which miR-208a-5p regulates sepsis-induced myocardial injury remains unclear. To mimic sepsis-induced myocardial injury in vitro, rat primary cardiomyocytes were treated with LPS. Cell viability and apoptosis were tested by CCK-8 and flow cytometry, respectively. The secretion of inflammatory factors was analyzed by ELISA. mRNA and protein levels were detected by RT-qPCR and Western blotting. The interaction among SP1, XIAP and miR-208a-5p was detected using dual luciferase report assay. Ultrasonic analysis and HE staining was performed to observe the effect of miR-208a-5p in sepsis-induced rats. Our findings indicated that miR-208a-5p expression in primary rat cardiomyocytes was increased by LPS. MiR-208a-5p inhibitor reversed LPS-induced cardiomyocytes injury through inhibiting the apoptosis. Furthermore, the inflammatory injury in cardiomyocytes was induced by LPS, which was rescued by miR-208a-5p inhibitor. In addition, downregulation of miR-208a-5p improved LPS-induced sepsis myocardial injury in vivo. Mechanistically, XIAP might be a target gene of miR-208a-5p. SP1 promoted transcription of miR-208a by binding to the miR-208a promoter region. Moreover, silencing of XIAP reversed the regulatory of miR-208a-5p inhibitor on cardiomyocytes injury. To sum up, those findings revealed silencing of miR-208a-5p could alleviate sepsis-induced myocardial injury, which would grant a new process for the treatment of sepsis.

Structure of a DNA G-quadruplex that Modulates SP1 Binding Sites Architecture in HIV-1 Promoter.

Nucleic acid sequences containing guanine tracts are able to form non-canonical DNA or RNA structures known as G-quadruplexes (or G4s). These structures, based on the stacking of G-tetrads, are involved in various biological processes such as gene expression regulation. Here, we investigated a G4 forming sequence, HIVpro2, derived from the HIV-1 promoter. This motif is located 60 nucleotides upstream of the proviral Transcription Starting Site (TSS) and overlaps with two SP1 transcription factor binding sites. Using NMR spectroscopy, we determined that HIVpro2 forms a hybrid type G4 structure with a core that is interrupted by a single nucleotide bulge. An additional reverse-Hoogsteen AT base pair is stacked on top of the tetrad. SP1 transcription factor is known to regulate transcription activity of many genes through the recognition of Guanine-rich duplex motifs. Here, the formation of HIVpro2 G4 may modulate SP1 binding sites architecture by competing with the formation of the canonical duplex structure. Such DNA structural switch potentially participates to the regulation of viral transcription and may also interfere with HIV-1 reactivation or viral latency.

Specificity protein 1 (Sp1) and glucocorticoid receptor (GR) stimulate bovine alphaherpesvirus 1 (BoHV-1) replication and cooperatively transactivate the immediate early transcription unit 1 promoter.

Bovine alphaherpesvirus 1 (BoHV-1) infections cause respiratory tract disorders and suppress immune responses, which can culminate in bacterial pneumonia. Following acute infection, BoHV-1 establishes lifelong latency in sensory neurons present in trigeminal ganglia (TG) and unknown cells in pharyngeal tonsil. Latently infected calves consistently reactivate from latency after an intravenous injection of the synthetic corticosteroid dexamethasone (DEX), which mimics the effects of stress. The immediate early transcription unit 1 (IEtu1) promoter drives expression of infected cell protein 0 (bICP0) and bICP4, two key viral transcriptional regulators. The IEtu1 promoter contains two functional glucocorticoid receptor (GR) response elements (GREs), and this promoter is transactivated by GR, DEX, and certain Krüppel transcription factors that interact with GC-rich motifs, including consensus specificity protein 1 (Sp1) binding sites. Based on these observations, we hypothesized that Sp1 stimulates productive infection and transactivates key BoHV-1 promoters. DEX treatment of latently infected calves increased the number of Sp1+ TG neurons and cells in pharyngeal tonsil indicating that Sp1 expression is induced by stress. Silencing Sp1 protein expression with siRNA or mithramycin A, a drug that preferentially binds GC-rich DNA, significantly reduced BoHV-1 replication. Moreover, BoHV-1 infection of permissive cells increased Sp1 steady-state protein levels. In transient transfection studies, GR and Sp1 cooperatively transactivate IEtu1 promoter activity unless both GREs are mutated. Co-immunoprecipitation studies revealed that GR and Sp1 interact in mouse neuroblastoma cells (Neuro-2A) suggesting this interaction stimulates IEtu1 promoter activity. Collectively, these studies suggested that the cellular transcription factor Sp1 enhances productive infection and stress-induced BoHV-1 reactivation from latency.IMPORTANCEFollowing acute infection, bovine alphaherpesvirus 1 (BoHV-1) establishes lifelong latency in sensory neurons in trigeminal ganglia (TG) and pharyngeal tonsil. The synthetic corticosteroid dexamethasone consistently induces BoHV-1 reactivation from latency. The number of TG neurons and cells in pharyngeal tonsil expressing the cellular transcription factor specificity protein 1 (Sp1) protein increases during early stages of dexamethasone-induced reactivation from latency. Silencing Sp1 expression impairs BoHV-1 replication in permissive cells. Interestingly, mithramycin A, a neuroprotective antibiotic that preferentially binds GC-rich DNA, impairs Sp1 functions and reduces BoHV-1 replication suggesting that it is a potential antiviral drug. The glucocorticoid receptor (GR) and Sp1 cooperatively transactivate the BoHV-1 immediate early transcript unit 1 (IEtu1) promoter, which drives expression of infected cell protein 0 (bICP0) and bICP4. Mithramycin A also reduced Sp1- and GR-mediated transactivation of the IEtu1 promoter. These studies revealed that GR and Sp1 trigger viral gene expression and replication following stressful stimuli.

Magnolin targeting of the JNK/Sp1/MMP15 signaling axis suppresses cervical cancer microenvironment and metastasis via microbiota modulation.

Magnolin (MGL), a compound derived from the magnolia plant, has inhibitory effects on tumor cell invasion and growth. His study aims to explore the antitumor effect and underlying molecular mechanism of MGL against human cervical cancer. We found that MGL inhibited the proliferation, migration, and invasiveness of cervical cancer cells in vitro and in vivo. The underlying mechanism was shown to involve MGL-induced inhibition of JNK/Sp1-mediated MMP15 transcription and translation. Overexpression of JNK/Sp1 resulted in significant restoration of MMP15 expression and the migration and invasion capabilities of MGL-treated cervical cancer cells. MGL modulated the cervical cancer microenvironment by inhibiting cell metastasis via targeting IL-10/IL-10 receptor B (IL-10RB) expression, thereby attenuating JNK/Sp1-mediated MMP15 expression. Analysis of the gut microbiota of mice fed MGL revealed a significant augmentation in Lachnospiraceae bacteria, known for their production of sodium butyrate. In vivo experiments also demonstrated synergistic inhibition of cervical cancer cell metastasis by MGL and sodium butyrate co-administration. Our study provides pioneering evidence of a novel mechanism by which MGL inhibits tumor growth and metastasis through the IL-10/IL-10RB targeting of the JNK/Sp1/MMP15 axis in human cervical cancer cells.

KLF6 activates Sp1-mediated prolidase transcription during TGF-ß1 signaling.

Prolidase (PEPD) is the only hydrolase that cleaves the dipeptides containing C-terminal proline or hydroxyproline-the rate-limiting step in collagen biosynthesis. However, the molecular regulation of prolidase expression remains largely unknown. In this study, we have identified overlapping binding sites for the transcription factors Krüppel-like factor 6 (KLF6) and Specificity protein 1 (Sp1) in the PEPD promoter and demonstrate that KLF6/Sp1 transcriptionally regulate prolidase expression. By cloning the PEPD promoter into a luciferase reporter and through site-directed deletion, we pinpointed the minimal sequences required for KLF6 and Sp1-mediated PEPD promoter-driven transcription. Interestingly, Sp1 inhibition abrogated KLF6-mediated PEPD promoter activity, suggesting that Sp1 is required for the basal expression of prolidase. We further studied the regulation of PEPD by KLF6 and Sp1 during transforming growth factor ß1 (TGF-ß1) signaling, since both KLF6 and Sp1 are key players in TGF-ß1 mediated collagen biosynthesis. Mouse and human fibroblasts exposed to TGF-ß1 resulted in the induction of PEPD transcription and prolidase expression. Inhibition of TGF-ß1 signaling abrogated PEPD promoter-driven transcriptional activity of KLF6 and Sp1. Knock-down of KLF6 as well as Sp1 inhibition also reduced prolidase expression. Chromatin immunoprecipitation assay supported direct binding of KLF6 and Sp1 to the PEPD promoter and this binding was enriched by TGF-ß1 treatment. Finally, immunofluorescence studies showed that KLF6 co-operates with Sp1 in the nucleus to activate prolidase expression and enhance collagen biosynthesis. Collectively, our results identify functional elements of the PEPD promoter for KLF6 and Sp1-mediated transcriptional activation and describe the molecular mechanism of prolidase expression.

MiR-29b Downregulation by p53/Sp1 Complex Plays a Critical Role in Bleb Scar Formation After Glaucoma Filtration Surgery.

Purpose: To investigate the function and mechanism of tumor protein p53 in pathological scarring after glaucoma filtration surgery (GFS) using human Tenon's fibroblasts (HTFs) and a rabbit GFS model. Methods: The expression of p53 in bleb scarring after GFS and transforming growth factor-ß (TGF-ß)-induced HTFs (myofibroblasts [MFs]) was examined by western blot and immunochemical analysis. The interaction between p53 and specificity protein 1 (Sp1) was investigated by immunoprecipitation. The role of p53 and Sp1 in the accumulation of collagen type I alpha 1 chain (COL1A1) and the migration of MFs was evaluated by western blot, quantitative real-time polymerase chain reaction (qRT-PCR), wound healing, and Transwell assay. The regulatory mechanisms among p53/Sp1 and miR-29b were detected via qRT-PCR, western blot, luciferase reporter assay, and chromatin immunoprecipitation assay. The therapeutic effect of mithramycin A, a specific inhibitor of Sp1, on scarring formation was evaluated in a rabbit GFS model. Results: p53 was upregulated in bleb scar tissue and MFs. p53 and Sp1 form a transcription factor complex that induces the accumulation of COL1A1 and promotes the migration of MFs through downregulation of miR-29b, a known suppressor of COL1A1. The p53/Sp1 axis inhibits miR-29b expression by the direct binding promoter of the miR-29b gene. Mithramycin A treatment attenuated bleb scar formation in vivo. Conclusions: The p53/Sp1/miR-29b signaling pathway plays a critical role in bleb scar formation after GFS. This pathway could be targeted for therapeutic intervention of pathological scarring after GFS. Translational Relevance: Our research indicates that inhibition of p53/Sp1/miR-29b is a promising therapeutic strategy for preventing post-GFS pathological scarring.

Over-expression of cFos by the Transcription Factors NFATc2 and Sp1 in Pancreatic Cancer Cells.

BACKGROUND/AIM: The transcription factors NFATc2 and Sp1 play a key role in the progression of pancreatic cancer because they interact inside the cells and exert their carcinogenic effect through transcriptional modification. Drugs can also induce a variety of oncogenic signalling cascades. The risk of tumour progression and metastasis seems to be significantly increased in the perioperative period. Our research group has previously demonstrated the function of the interaction between NFATc2 and Sp1 in pancreatic cancer and has identified the proto-oncogene cFos as a target gene. We also found that the anaesthetic drug propofol has anti-tumour properties. The aim of the present study was to investigate the effect of propofol on the expression of the transcription factors NFATc2, Sp1 and cFos in the pancreatic cancer cell lines PaTu 8988t and PANC-1 and to analyse the relevance of this effect for the cells. MATERIALS AND METHODS: Stimulation with propofol and its effects on the expression of NFATc2, Sp1 and cFos were assessed by immunoblot. Cell cycle distribution was analysed by flow cytometry, and cell proliferation was measured with the ELISA BrdU assay. Propofol and siRNA against cFos were used for stimulation. RESULTS: Propofol regulated the expression of NFATc2, Sp1 and cFos. Stimulation with 250 µM or 500 µM propofol decreased NFATc2, Sp1 and cFos signalling in the Western blot analysis. At the same time, propofol significantly inhibited proliferation and activated cell cycle. The same proliferation behaviour was observed after transient cFos inhibition. These effects were potentiated by simultaneous stimulation with propofol and transient inhibition of cFos, further inhibiting cell proliferation. Interestingly, the cell cycle activation observed after stimulation with propofol alone was reversed in both cell lines. CONCLUSION: Anaesthetists only see oncological patients in a short time window. However, the perioperative period is increasingly recognised as a very vulnerable time with a major impact on tumour progression. Further studies are needed to identify the underlying mechanisms and to verify their clinical relevance, especially in anaesthesia.

Effect of NFATc2- and Sp1-mediated TNFalpha Regulation on the Proliferation and Migration Behavior of Pancreatic Cancer Cells.

BACKGROUND/AIM: One in two people will develop a tumor during their lifetime. Adenocarcinoma of the pancreas is one of the most aggressive types of cancer in humans with very poor long-term survival. A central role in the carcinogenesis of pancreatic cancer has been attributed to NFAT transcription factors. Previous studies have identified the transcription factor Sp1 as a binding partner of NFATc2 in pancreatic cancer. Using expression profile analysis, our group was able to identify the tumor necrosis factor TNFalpha as a target gene of the interaction between NFATc2 and Sp1. The present study investigated the effect of TNFalpha over-expression via the transcription factors NFATc2 and Sp1 on the pancreatic cancer cell lines PaTu 8988t and PANC-1. MATERIALS AND METHODS: Transient transfection of NFATc2, Sp1, and TNFalpha siRNAs and their effects on the expression were investigated with immunoblot. Cell proliferation was measured with the ELISA BrdU assay. Cell migration was assayed with a Cell Migration Assay Kit using a Boyden chamber. RESULTS: Inhibition of the transfection factors NFATc2, Sp1, or TNFalpha by siRNA significantly inhibited proliferation, which was exacerbated when using the combination of NFATc2 and Sp1. TNFalpha was able to counterbalance this effect. In contrast to proliferation, migration of pancreatic cancer cells was increased by inhibiting these transfection factors. CONCLUSION: Tumor progression is strongly influenced by transcriptional changes in signaling cascades and oncogene mutations as well as by changes in tumor suppressor genes. Further studies are needed to understand the underlying mechanisms of these processes.

SP1 transcriptionally upregulates the expression of APOC3 in KGN cells to promote disease progression by regulating TLR2/NF-κB signalling pathway.

INTRODUCTION: Apolipoprotein C3 (APOC3) is known for its important functions in metabolism-related diseases. However, the function and molecular mechanism of APOC3 in polycystic ovarian syndrome (PCOS) have not been reported. MATERIAL AND METHODS: Quantitative polymerase chain reaction and western blot assays were used to detect the expression of APOC3 in KGN cells. Small interference APOC3 (siAPOC3) was applied to reduce APOC3 expression, and the proliferation ability of human granulosa cell line (KGN cells) was measured by cell counting kit-8 and colony formation assays. The protein levels of key genes related to apoptosis were detected by western blot assay. The transcriptional regulator of APOC3 was predicted by the UCSC and PROMO website, and verified by dual luciferase assay. siAPOC3 and pcDNA3.1-specific protein 1 (SP1) vector were co-transfected into KGN cells to detect the function of SP1 and APOC3 in KGN cells. RESULTS: APOC3 was overexpressed in KGN cells, and siAPOC3 transfection significantly reduced the growth ability of KGN cells and increased the apoptosis ability of KGN cells. SP1 directly bound to the promoter of APOC3 and transcriptional regulated APOC3 expression. Overexpression of SP1 increased the growth ability of KGN cells and decreased the apoptosis ability of KGN cells, which were reversed after siAPOC3 transfection. The increased levels of toll-like receptor 2 (TLR2) and p65 phosphorylation (p-P65) nuclear factor kappa B (NF-κB) caused by SP1 overexpression were inhibited by siAPOC3 transfection. APOC3, transcriptionally regulated by SP1, promoted the growth of KGN cells, and inhibited the apoptosis by regulating TLR2/NF-κB signalling pathway.

SGCE promotes breast cancer stemness by promoting the transcription of FGF-BP1 by Sp1.

Breast cancer stem cells are mainly responsible for poor prognosis, especially in triple-negative breast cancer (TNBC). In a previous study, we demonstrated that ε-Sarcoglycan (SGCE), a type Ⅰ single-transmembrane protein, is a potential oncogene that promotes TNBC stemness by stabilizing EGFR. Here, we further found that SGCE depletion reduces breast cancer stem cells, partially through inhibiting the transcription of FGF-BP1, a secreted oncoprotein. Mechanistically, we demonstrate that SGCE could interact with the specific protein 1 transcription factor and translocate into the nucleus, which leads to an increase in the transcription of FGF-BP1, and the secreted FBF-BP1 activates FGF-FGFR signaling to promote cancer cell stemness. The novel SGCE-Sp1-FGF-BP1 axis provides novel potential candidate diagnostic markers and therapeutic targets for TNBC.

N-terminal domain of classical swine fever virus Npro induces proteasomal degradation of specificity protein 1 with reduced HDAC1 expression to evade from innate immune responses.

IMPORTANCE: Of the flaviviruses, only CSFV and bovine viral diarrhea virus express Npro as the non-structural protein which is not essential for viral replication but functions to dampen host innate immunity. We have deciphered a novel mechanism with which CSFV uses to evade the host antiviral immunity by the N-terminal domain of its Npro to facilitate proteasomal degradation of Sp1 with subsequent reduction of HDAC1 and ISG15 expression. This is distinct from earlier findings involving Npro-mediated IRF3 degradation via the C-terminal domain. This study provides insights for further studies on how HDAC1 plays its role in antiviral immunity, and if and how other viral proteins, such as the core protein of CSFV, the nucleocapsid protein of porcine epidemic diarrhea virus, or even other coronaviruses, exert antiviral immune responses via the Sp1-HDAC1 axis. Such research may lead to a deeper understanding of viral immune evasion strategies as part of their pathogenetic mechanisms.