Sp1 Protein Expression Research Articles

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.

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.

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.

Sp1 mediated the inhibitory effect of glutamate on pulmonary surfactant synthesis.

Studies have shown that the release of endogenous glutamate (Glu) participates in lung injury by activating N-methyl-D-aspartate receptor (NMDAR), but the mechanism is still unclear. This study was to investigate the effects and related mechanisms of Glu on the lipid synthesis of pulmonary surfactant (PS) in isolated rat lung tissues. The cultured lung tissues of adult SD rats were treated with Glu. The amount of [3H]-choline incorporation into phosphatidylcholine (PC) was detected. RT-PCR and Western blot were used to detect the changes of mRNA and protein expression of cytidine triphosphate: phosphocholine cytidylyltransferase alpha (CCTα), a key regulatory enzyme in PC biosynthesis. Western blot was used to detect the expression of NMDAR1, which is a functional subunit of NMDAR. Specific protein 1 (Sp1) expression plasmids were used. After transfected with Sp1 expression plasmids, the mRNA and protein levels of CCTα were detected by RT-PCR and Western blot in A549 cells. After treated with NMDA and MK-801, the mRNA and protein levels of Sp1 were detected by RT-PCR and Western blot in A549 cells. Glu decreased the incorporation of [3H]-choline into PC in a concentration- and time- dependent manner. Glu treatment significantly reduced the mRNA and protein levels of CCTα in lungs. Glu treatment up-regulated NMDAR1 protein expression, and the NMDAR blocker MK-801 could partially reverse the reduction of [3H]-choline incorporation induced by Glu (10-4 mol/L) in lungs. After transfected with Sp1 plasmid for 30 h, the mRNA and protein expression levels of CCTα were increased and the protein expression of Sp1 was also up-regulated. After A549 cells were treated with NMDA, the level of Sp1 mRNA did not change significantly, but the expression of nucleus protein in Sp1 was significantly decreased, while the expression of cytoplasmic protein was significantly increased. However, MK-801could reverse these changes. Glu reduced the biosynthesis of the main lipid PC in PS and inhibited CCTα expression by activating NMDAR, which were mediated by the inhibition of the nuclear translocation of Sp1 and the promoter activity of CCTα. In conclusion, NMDAR-mediated Glu toxicity leading to impaired PS synthesis may be a potential pathogenesis of lung injury.

Solamargine enhanced gefitinib antitumor effect via regulating MALAT1/miR-141-3p/Sp1/IGFBP1 signaling pathway in non-small cell lung cancer.

Lung cancer is the leading cause of cancer-related deaths worldwide. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) showed great therapeutic efficacy for non-small cell lung cancer (NSCLC) patients. However, acquired resistance severely limits the clinical application and efficacy of EGFR-TKIs. In the current study, we found that solamargine (SM), a natural alkaloid derived from the fruit of Lycium tomato lobelia, has been found to inhibit the progression of NSCLC and enhance the anticancer effect of EGFR-TKIs. In brief, SM significantly inhibited the cell viability of NSCLC cells and enhanced the anticancer effect of gefitinib (GFTN) and erlotinib (ERL). Mechanistically, SM decreased the expression of MALAT1 and induced miR-141-3p, whereas reduced SP1 protein levels. Interestingly, both MALAT1 and Sp1 have classical and conservative binding sites of miR-141-3p in their 3'-UTR regions. Silence of MALAT1 and overexpression of miR-141-3p both decreased the protein expression of Sp1. Subsequently, promoter activity and protein expression of IGFBP1 were upregulated by SM, which was not observed in cells with SP1 overexpression. Moreover, the inhibitory effect of SM on cell growth was significantly blocked by knockdown of IGFBP1 expression. More importantly, the combination of SM and GFTN synergistically inhibited the progression of lung cancer. Similar results were observed in experiments in vivo. Finally, the clinical relevance of MALAT1, Sp1 and IGFBP1 was further validated using bioinformatics analysis. Taken together, we confirmed that SM significantly enhanced the anticancer effect of EGFR-TKIs by regulating the MALAT1/miR-141-3p/Sp1/IGFBP1 signaling pathway. This study unravels a novel mechanism and suggests a new potential NSCLC-associated therapy.

The Potent G-Quadruplex-Binding Compound QN-302 Downregulates S100P Gene Expression in Cells and in an In Vivo Model of Pancreatic Cancer

The naphthalene diimide compound QN-302, designed to bind to G-quadruplex DNA sequences within the promoter regions of cancer-related genes, has high anti-proliferative activity in pancreatic cancer cell lines and anti-tumor activity in several experimental models for the disease. We show here that QN-302 also causes downregulation of the expression of the S100P gene and the S100P protein in cells and in vivo. This protein is well established as being involved in key proliferation and motility pathways in several human cancers and has been identified as a potential biomarker in pancreatic cancer. The S100P gene contains 60 putative quadruplex-forming sequences, one of which is in the promoter region, 48 nucleotides upstream from the transcription start site. We report biophysical and molecular modeling studies showing that this sequence forms a highly stable G-quadruplex in vitro, which is further stabilized by QN-302. We also report transcriptome analyses showing that S100P expression is highly upregulated in tissues from human pancreatic cancer tumors, compared to normal pancreas material. The extent of upregulation is dependent on the degree of differentiation of tumor cells, with the most poorly differentiated, from more advanced disease, having the highest level of S100P expression. The experimental drug QN-302 is currently in pre-IND development (as of Q1 2023), and its ability to downregulate S100P protein expression supports a role for this protein as a marker of therapeutic response in pancreatic cancer. These results are also consistent with the hypothesis that the S100P promoter G-quadruplex is a potential therapeutic target in pancreatic cancer at the transcriptional level for QN-302.

Transforming growth factor-β receptor 1: An intervention target for genetic poor cartilage quality induced by prenatal dexamethasone exposure

IntroductionFetal-originated osteoarthritis is relative to poor cartilage quality and may exhibit transgenerational genetic effects. Previous findings revealed prenatal dexamethasone exposure (PDE) induced poor cartilage quality in offspring. ObjectivesThis study focused on further exploring molecular mechanism, heritability, and early intervention of fetal-originated osteoarthritis. MethodsPregnant rats (F0) were segregated into control and PDE groups depending upon whether dexamethasone was administered on gestational days (GDs) 9–20. Some female offspring were bred with healthy males during postnatal week (PW) 8 to attain the F2 and F3 generations. The F3-generation rats were administrated with glucosamine intragastrically at PW12 for 6 weeks. The knee cartilages of male and female rats at different time points were harvested to assay their morphologies and functions. Furthermore, primary chondrocytes from the F3-generation rats were isolated to confirm the mechanism and intervention target of glucosamine. ResultsCompared with the control, female and male rats in each generation of PDE group showed thinner cartilage thicknesses; shallower and uneven staining; fewer chondrocytes; higher Osteoarthritis Research Society International scores; and lower mRNA and protein expression of SP1, TGFβR1, Smad2, SOX9, ACAN and COL2A1. After F3-generation rats were treated with glucosamine, all of the above changes could be reversed. In primary chondrocytes isolated from the F3-generation rats of PDE group, glucosamine promoted SP1 expression and binding to TGFβR1 promoter to increase the expression of TGFβR1, p-Smad2, SOX9, ACAN and COL2A1, but these were prevented by SB431542 (a potent and selective inhibitor of TGFβR1). ConclusionsPDE induced chondrodysplasia in offspring and stably inherited in F3-generation rats, which was related to decreased expression of SP1/TGFβR1/Smad2/SOX9 pathway to reduce the cartilage matrix synthesis, without major sex-based variations. Glucosamine could alleviate the poor genetic cartilage quality in offspring induced by PDE by up-regulating SP1/TGFβR1 signaling, which was prevented by a TGFβR1 inhibitor. This study elucidated the molecular mechanism and therapeutic target (TGFβR1) of genetic chondrodysplasia caused by PDE, which provides a research basis for precisely treating fetal-originated osteoarthritis.

The suppressive role of phytochemical-induced glutathione S-transferase Mu 2 in human urothelial carcinoma cells

Glutathione S-transferases (GSTs) belong to one class of phase 2 detoxification enzymes which are important in metabolism and/or detoxification of various electrophilic endogenous metabolites and xenobiotics. From the available database, we found that GSTM2 gene expression is lower in high stages of bladder urothelial carcinoma than in stage 1 and normal bladder tissue. GSTM2 overexpression retards invasion, migration and tumor sphere formation of bladder cancer cells. Analysis of GSTM2 promoter activity shows that one SP1 site located at − 48 to − 40 bp is important for GSTM2 gene expression in BFTC 905 cells. An SP1 inhibitor, mithramycin A, inhibits GSTM2 promoter activity and protein expression. SP1 overexpression also increases GSTM2 expression in BFTC 905 and 5637 cells. Eight potential phytochemicals were analyzed for GSTM2 promoter activation, and results indicated that baicalein, berberrubine, chalcone, curcumin, resveratrol, and wogonin can increase promoter activity. In endogenous GSTM2 expression, berberrubine and resveratrol activated GSTM2 mRNA and protein expression the most. A DNA methylation inhibitor, 5-aza-deoxycytidine, can decrease GSTM2 gene methylation level and then increase its gene expression; 50 μM berberrubine decreased the GSTM2 gene methylation level, providing a mechanism for activating GSTM2 gene expression. Berberrubine and resveratrol also increased SP1 protein expression as one of the mechanisms for GSTM2 gene expression. In summary, berberrubine and resveratrol activates GSTM2 expression which inhibits cell proliferation, migration, and invasion of bladder cancer cells. The GSTM2 expression mechanism is partially via SP1 activation, and the effect of berberrubine is also partly via DNA CpG demethylation.

Construction and application of lentivirus overexpression vector with two labeling genes fused with CopGFP and PuroR

Objective: To construct the lentivirus overexpression vector with two label genes fused with CopGFP and PuroR and to detect the emission of green fluorescence as well as resistance to puromycin in liver cancer cells infected with lentivirus packaged with the above vector. Methods: Firstly, two fragments containing copGFP and PuroR coding sequences were amplified from pCDH-CMV-MCS-copGFP and pLKO.1 respectively; secondly, the two amplified regions were fused with each other by recombinant PCR; thirdly, the fusion DNA fragment was cut and inserted into pCDH-CMV-MCS-copGFP vector, which was linearized with the same restriction endonuclease as used to digest fusion DNA fragment: BamH Ⅰ and Sal Ⅰ. The fusion region in the constructed vector was confirmed by DNA sequencing. The checked vector was co-transfected with package assistant plasmids, namely PLP1, PLP2 and VSVG into in 293T cells and the culture supernatant was subjected to centrifuge and infect liver cancer MHCC97H cells, which were then used to detect their resistance to puromycin (infected cells were treated with 1 mg/ml puromycin for 7 days after infection) and to observe green fluorescence emission in microscope. To determine its efficiency in expressing foreign target protein, the Sp1 coding region was inserted into the MCS sites of the vector, and Sp1 mRNA and protein expression levels were compared with the vehicle vector by RT-qPCR and Western blot. Results: The lentivirus overexpression vector with two label genes fused with CopGFP and PuroR was successfully constructed, and the liver cancer cells infected with lentivirus packaged with the vector expressing two labeling genes fused with CopGFP and PuroRshowed both emission of green fluorescence and resistance to puromycin simultaneously, while cells containing with the vector inserted with Sp1 coding region improved Sp1 mRNA level with 3.3 fold and protein level with 2.2 fold higher in comparison with cells containing the vehicle vector (P＜0.01). Conclusion: The fused label genes consisting of copGFP and PuroR are correctly cloned into the lentivirus vector and confer cells with the ability to emission of green fluorescence and resistance to puromycin, besides, the vector may promote the expression of the target gene with long coding sequence.

Cardiac Expression of Esophageal Cancer-Related Gene-4 is Regulated by Sp1 and is a Potential Early Target of Doxorubicin-Induced Cardiotoxicity.

Esophageal Cancer-Related Gene 4 (Ecrg4) expressed in cardiomyocytes and the cardiac conduction system is downregulated during cardiac ischemia and atrial fibrillation. To explore whether Ecrg4 plays any role in doxorubicin (DOX)-induced cardiotoxicity. Rats and neonatal rat cardiomyocytes (NRCMs) were employed to study the effect of DOX on Ecrg4 transcription. Bioinformatics combined with promoter analysis were used to map the rat Ecrg4 promoter. ChIP assay was used to evaluate the binding of Sp1 to the Ecrg4 promoter. Transient transfection was used to study the effect of Sp1 on the expression of endogenous Ecrg4. DOX decreased endogenous Ecrg4 gene expression in the heart and cultured NRCMs. In silico analysis showed that the 5'UTR immediately upstream of the start codon ATG, harbors a putative promoter that is GC-rich, and contains CpG islands, multiple overlapping Sp1sites. Transcription is initiated mainly on the 'C' at - 15. Serial 5'-deletion combined with dual-luciferase assays showed that the rat Ecrg4 core promoter resides at - 1/- 800. Sp1 transactivated Ecrg4 gene, which was almost abolished by DOX. Furthermore, ChIP assay showed that Sp1 specifically bound to the Ecrg4 promoter was interrupted by DOX. Finally, DOX suppressed Sp1 protein expression, and restoration of Sp1 increased Ecrg4 expression that was resistant to DOX-induced Ecrg4 downregulation. Importantly, cardiomyocyte-specific loss of Ecrg4 significantly enriched the differentially expressed proteins in the signaling pathways commonly involved in DOX-induced cardiotoxicity. Our results indicate that Sp1 mediates DOX-induced suppression of Ecrg4, which may contribute indirectly to its cardiotoxicity.

The Acetylation Modification of SP1 Regulates the Protein Stability in Silkworm.

Acetylation is a highly conservative and reversible post-translational modification. Acetylation modification can regulate gene expression by altering protein function and is widely identified in an increasing number of species. Previously, the acetylated proteome of silkworm was identified by combining acetylated polypeptide enrichment with nano-HPLC/MS/MS; the identification revealed that the SP proteins (SPs) were high acetylated. In this study, the acetylation of SP1, one of the SPs, was further confirmed using immunoprecipitation (IP) and Western blotting. Then, we found the acetylation could upregulate SP1 protein expression by enhancing the protein stability. Further research found that the acetylation of SP1 protein can competitively inhibit its ubiquitination and thus improve the stability and cell accumulation of SP1 protein by inhibiting the ubiquitin-mediated proteasome degradation pathway. This result provides a basis for acetylation to regulate the nutrient storage and utilization of silkworm.

Human Telomerase Reverse Transcriptase as a Therapeutic Target of Dihydroartemisinin for Esophageal Squamous Cancer

Objective: To elucidate the oncogenic role of human telomerase reverse transcriptase (hTERT) in esophageal squamous cancer and unravel the therapeutic role and molecular mechanism of dihydroartemisinin (DHA) by targeting hTERT. Methods: The expression of hTERT in esophageal squamous cancer and the patients prognosis were analyzed by bioinformatic analysis from TCGA database, and further validated with esophageal squamous cancer tissues in our cohort. The Cell Counting Kit-8 (CCK8) and colony formation assay were used to evaluate the proliferation of esophageal squamous cancer cell lines (Eca109, KYSE150, and TE1) after hTERT overexpression or treated with indicated concentrations of DHA. Transwell migration assay and scratch assay were employed to determine the migration abilities of cancer cells. Fluorescence microscopy and flow cytometry were conducted to measure the intracellular reactive oxygen species (ROS) levels in cancer cells after treated with DHA. Moreover, RT-PCR and Western blot were performed to test the alteration of associated genes on mRNA and protein level in DHA treated esophageal squamous cancer cell lines, respectively. Furthermore, tumor-bearing nude mice were employed to evaluate the anticancer effect of DHA in vivo. Results: We found that hTERT was significantly upregulated in esophageal squamous cancer both from TCGA database and our cohort also. Overexpression of hTERT evidently promoted the proliferation and migration of esophageal squamous cancer cells in vitro. Moreover, DHA could significantly inhibit the proliferation and migration of esophageal cancer cell lines Eca109, KYSE150, and TE1 in vitro, and significantly down-regulate the expression of hTERT on both mRNA and protein level in a time- and dose-dependent manner as well. Further studies showed that DHA could induce intracellular ROS production in esophageal cancer cells and down-regulate SP1 expression, a transcription factor that bound to the promoter region of hTERT gene. Moreover, overexpression of SP1 evidently promoted the proliferation and migration of Eca109 and TE1 cells. Intriguingly, rescue experiments showed that inhibiting ROS by NAC alleviated the downregulation of SP1 and hTERT in cells treated with DHA. Furthermore, overexpression of SP1 or hTERT could attenuate the inhibition effect of DHA on the proliferation and migration of Eca109 cells. In tumor-bearing nude mice model, DHA significantly inhibited the growth of esophageal squamous cancer xenografts, and downregulated the expression of SP1 and hTERT protein, while no side effects were observed from heart, kidney, liver, and lung tissues by HE stain. Conclusion: hTERT plays an oncogenic role in esophageal squamous cancer and might be a therapeutic target of DHA through regulating ROS/SP1 pathway.

Enhanced Sp1/YY1 Expression Directs CBS Transcription to Mediate VEGF-Stimulated Pregnancy-Dependent H2S Production in Human Uterine Artery Endothelial Cells.

[Figure: see text].

Radiosensitizing effect of gold nanoparticle loaded with small interfering RNA-SP1 on lung cancer: AuNPs-si-SP1 regulates GZMB for radiosensitivity

Radioresistance is a major challenge that largely limits the efficacy of radiotherapy in lung cancer. Gold nanoparticles (AuNPs) are emerging as novel radiosensitizers for cancer patients. Therefore, this study was designed to explore the radiosensitizing effect and mechanism of AuNPs loaded with small interfering RNA (siRNA)-SP1 (AuNPs-si-SP1) on lung cancer. AuNPs-si-SP1 was prepared by the noncovalent binding between AuNPs and siRNA-SP1. The adsorption capacity of AuNPs to siRNA-SP1 was analyzed by gel electrophoresis. The cell uptake of AuNPs-si-SP1 was observed under a laser confocal microscopy. Silencing efficacy of AuNPs-si-SP1 was validated by RT-qPCR and Western blot analysis. Cell viability was determined by CCK-8 assay, radiosensitization by plate colony formation assay, cell apoptosis and cell cycle by flow cytometry, and DNA double strand breaks by immunofluorescence in the presence or absence of AuNPs-si-SP1 or GZMB. The downstream mechanism of SP1 was predicted by bioinformatics analysis, followed by verification by Western blot analysis. Subcutaneous tumorigenesis in nude mice was established to verify the radiosensitization of AuNPs-si-SP1 and GZMB in vivo. AuNPs-si-SP1 effectively absorbed SP1 siRNA and was highly internalized by A549 cells to reduce SP1 protein expression. AuNPs-si-SP1 or GZMB overexpression promoted cells to G2/M phase, DNA double strand breaks, and enhanced radiosensitivity. SP1 could repress GZMB expression in lung cancer cells. In vivo experiments manifested that AuNPs-si-SP1 could inhibit the growth of solid tumor in nude mice to achieve radiosensitization by inhibiting SP1 to upregulate GZMB. AuNPs-si-SP1 might increase the radiosensitivity of lung cancer by inhibiting SP1 to upregulate GZMB.

Uremic Toxin Indoxyl Sulfate Impairs Hydrogen Sulfide Formation in Renal Tubular Cells

Hydrogen sulfide (H2S) is the third gasotransmitter that recognized as a cytoprotectant. Recent study demonstrated that exogenous supplement of H2S ameliorates functional insufficiency in chronic kidney disease (CKD). However, how H2S system is impaired by CKD has not been elucidated. The uremic toxin indoxyl sulfate (IS) is known to accumulate in CKD patients and harm the renal tubular cells. This study therefore treated proximal tubular cells LLC-PK1 with IS to see how IS affects H2S formation. Our results showed that H2S release from LLC-PK1 cells was markedly attenuated by IS when compared to that in control cells. The H2S donors NaHS and GYY-4137 significantly attenuated IS-induced tubular damage by enhancing cell survival in dose-dependent manners. Inhibition of remnant H2S production in the IS-treated cells by AOAA exacerbated tubulotoxicity. These results clearly indicated IS impairs H2S formation. We then examined protein and mRNA expression of H2S-producing enzymes cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST). IS downregulated both CBS and 3-MST protein and mRNA expressions after 24-72 h of treatment. CSE protein and mRNA levels were lowered at 24 h and 24-48 h, respectively, by IS. The low expression of CBS and 3-MST at both protein and mRNA levels could be reversed by AhR inhibition. As transcription factor specificity protein 1 (Sp1) regulates gene expression of H2S-producing enzymes, we further showed that IS significantly decreased the DNA binding activity of Sp1, this however did not affect Sp1 protein expression. AhR inhibition reversed low Sp1 activity caused by IS. Together these results, we conclude that IS activates AhR which then attenuates Sp1 function on regulation of H2S-producing enzyme expression. Attenuation of H2S formation contributed to uremic toxin-mediated renal tubular cell damage.

Down\u2010regulation of the Sp1 transcription factor by an increase of microRNA-4497\xa0in human placenta is associated with early recurrent miscarriage

BackgroundThe pathophysiological mechanism of recurrent miscarriage (RM) is unclear. The goals of this study were to determine the role of microRNA-4497 overexpression in placental villus tissues in early RM; To identify the potential target mRNAs of miRNA-4497; And to investigate the microRNA-4497-mediated regulatory mechanisms in placental trophoblasts.MethodsBioinformatics analysis was performed to identify the candidate target genes of miRNA-4497. The protein expression of Sp1 transcription factor (SP1), chemokine (C-X-C motif) receptor 5 (CXCR5) and bone morphogenetic protein 8a (BMP8A) were determined in the villus tissues of the RM and normal groups by Western blotting and immunohistochemistry. Cultured 293T cells were co-transfected with the miRNA-4497 agomir or luciferase reporter vectors containing the wild-type or mutant 3’-UTRs of the target mRNAs to verify the regulatory role of miRNA-4497.ResultsBioinformatics analysis suggested that SP1, CXCR5 and BMP8A mRNAs are potential targets of miRNA-4497. The expression of SP1, CXCR5 and BMP8A proteins in the chorionic villus tissues of RM placentas were significantly decreased compared to those in the normal controls. Moreover, SP1 protein levels were inversely correlated with the levels of miRNA-4497 in the placentas of RM patients and normal controls. The expression of SP1 mRNA and protein were down-regulated in HTR-8/SVneo cells after forced overexpression of the miRNA-4497 agomir. The results of the co-transfection assay showed that mutation of the miRNA-4497-binding sites in the 3’-untranslated region (3’-UTR) of SP1 led to a recovery of luciferase activity upon overexpression of miRNA-4497, suggesting that SP1 could be a direct target of miRNA-4497.ConclusionsAn increased miRNA-4497 level in the placental villus tissues associated with recurrent miscarriage may down-regulate SP1 expression. The negative regulation of SP1 by miRNA-4497 may potentially contribute to the pathogenesis of recurrent miscarriage through promotion of trophoblast apoptosis. These findings provide novel information on the regulation of placental trophoblast apoptosis, and could be useful for the development of new therapeutic strategies for better management of recurrent miscarriage.

G-Quadruplex Modulation of SP1 Functional Binding Sites at the KIT Proximal Promoter.

The regulation of conformational arrangements of gene promoters is a physiological mechanism that has been associated with the fine control of gene expression. Indeed, it can drive the time and the location for the selective recruitment of proteins of the transcriptional machinery. Here, we address this issue at the KIT proximal promoter where three G-quadruplex forming sites are present (kit1, kit2 and kit*). On this model, we focused on the interplay between G-quadruplex (G4) formation and SP1 recruitment. By site directed mutagenesis, we prepared a library of plasmids containing mutated sequences of the WT KIT promoter that systematically exploited different G4 formation attitudes and SP1 binding properties. Our transfection data showed that the three different G4 sites of the KIT promoter impact on SP1 binding and protein expression at different levels. Notably, kit2 and kit* structural features represent an on-off system for KIT expression through the recruitment of transcription factors. The use of two G4 binders further helps to address kit2-kit* as a reliable target for pharmacological intervention.

Curcumin inhibits proliferation, migration and neointimal formation of vascular smooth muscle via activating miR-22

Context Curcumin has antitumor, antioxidative, anti-inflammatory, and anti-proliferative properties. Objective To investigate the role of miR-22 during curcumin-induced changes in vascular smooth muscle cells (VSMC) and neointima formation in balloon-injured rat abdominal aorta. Materials and methods Sprague-Dawley rats were randomised to the sham-operated (n = 10), operated control (injured, n = 10), and curcumin treatment (n = 10) groups. miR-22 expression was determined by real-time PCR. SP1 was assessed by western blot and real-time PCR. Rat aortic smooth muscle A7r5 cells were used to determine VSMC proliferation and migration, which were measured by the MTS, EdU staining, Transwell, and wound healing assays. Results miR-22 levels declined following arterial balloon injury in vivo (48% at 3d, p < 0.05) and serum stimulation in vitro (45% at 24 h, p < 0.01). Functional studies revealed that miR-22 negatively regulated the proliferation and migration of VSMCs by directly targeting the SP1 transcription factor in VSMCs. Curcumin increased the expression of miR-22 (81%, p < 0.05) and decreased the protein expression of SP1 in VSMCs (25%, p < 0.05). miR-22 inhibition was found to attenuate the effects of curcumin on VSMC functions. Curcumin increased miR-22 (46%, p < 0.01), decreased the SP1 protein (19%, p < 0.05), and inhibited vascular neointimal area (48%, p < 0.01) in vivo. Discussion The miR-22/SP1 pathway is involved in the protective role of curcumin during arterial balloon injury, but the mechanisms remain unclear. Conclusion miR-22 is involved in the inhibitory effects of curcumin on VSMCs’ proliferation, migration and neointima hyperplasia after arterial balloon injury in rats. Curcumin could be used to prevent neointimal hyperplasia after angioplasty.

The regulation and interaction of colon cancer-associated transcript-1 and miR7-5p contribute to the inhibition of SP1 expression by solamargine in human nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) is a common head and neck malignancy with higher incidence in Southern China and Southeast Asia. Solamargine (SM), a steroidal alkaloid glycoside, has been shown to have anticancer properties. However, the underlying mechanism involved remains undetermined. In this study, we showed that SM inhibited the growth of NPC cells. Mechanistically, we found that solamargine decreased lncRNA colon cancer‐associated transcript‐1 (CCAT1) and increased miR7‐5p expression. There was a reciprocal interaction of CCAT1 and miR7‐5p. In addition, SM inhibited the expression of SP1 protein and promoter activity, which was strengthened by miR7‐5p mimics and inhibited by overexpressed CCAT1. MiR7‐5p could bind to 3'‐UTR of SP1 and attenuated SP1 gene expression. Exogenously expressed SP1 feedback resisted SM‐increased miR7‐5p expression and more importantly reversed SM‐inhibited growth of NPC cells. Finally, SM inhibited NPC tumor growth in vivo. Collectively, our results show that SM inhibits the growth of NPC cells through reciprocal regulation of CCAT1 and miR7‐5p, followed by inhibition of SP1 gene expression in vitro and in vivo. The interregulation and correlation among CCAT1, miR7‐5p and SP1, and the feedback regulatory loop unveil the novel molecular mechanism underlying the overall responses of SM in anti‐NPC.

Abstract B126: A small molecule inhibitor of the RNA m6A writer METTL3 inhibits the development of acute myeloid leukemia (AML) in vivo

Abstract METTL3 is an RNA methyltransferase which is responsible for the deposition of N-6-methyladenosine (m6A) on mRNA targets such as SP1, to modulate their stability and expression. METTL3 was identified as an essential gene for the growth of AML cells and proposed as a novel target for cancer therapy (Barbieri 2017). We present the in vitro and in vivo characterization of novel small molecule inhibitors of METTL3, which recapitulate the genetic validation of METTL3 as a novel cancer target using a pharmacological audit trail. Small molecule inhibitors from 2 distinct chemical series were identified and optimised using a structure-guided medicinal chemistry platform. Compounds 1 and 2 are from different series, and both showed biochemical inhibition of METTL3 enzyme with single digit nanomolar potency. Direct binding to METTL3 was confirmed by SPR with comparable potency. Compound 3 is an inactive analog which was confirmed inactive in the enzyme assay (IC50 &amp;gt;50microM). Compounds 1-2 are selective for METTL3 and did not inhibit a panel of other RNA, DNA or protein methyltransferases tested (&amp;gt;10microM IC50). Cellular target engagement was confirmed by the demonstration that compounds 1 and 2 inhibited SP1 and Brd4 protein expression with submicromolar potency, whereas the inactive analog compound 3 had no effect. Compounds 1 and 2 treatment of MOLM13 cells inhibited their proliferation which correlated with SP1 inhibition, and compound 3 had no effect, demonstrating that their activity was METTL3-dependent. Compound 1 has excellent oral bioavailability with good dose-proportional exposure in mice and a half-life of 3.5 hrs, and was well-tolerated with no body weight loss or clinical signs. Compound 1 was evaluated for anti-tumor effects in an MLL-AF9 driven primary murine AML model. 30 mg/kg daily oral dosing of compound 1 gave a significant reduction in AML expansion and a reduction in spleen weight compared to vehicle control, indicating a pronounced anti-tumor effect in vivo. Target engagement was confirmed in bone marrow and spleen as measured by reduction of METTL3-dependent m6A targets. We have described the comprehensive characterization of potent and selective inhibitors of the METTL3 RNA methyltransferase, and demonstrated their activity and utility using biochemical, cellular and in vivo systems. We have demonstrated that inhibition of METTL3 by small molecules in vivo leads to a pronounced anti-tumor effect in a physiologically relevant model of acute myeloid leukemia. To our knowledge, this is the first demonstration of in vivo activity of inhibitors of an RNA methyltransferase and providing proof of concept that RNA modifying enzymes are a new target class for the development of novel cancer therapeutics. Citation Format: Mark Albertella, Wesley Blackaby, Richard Fosbeary, Alan Hendrick, Dan Leggate, Yaara Ofir-Rosenfeld, Alexandra Sapetschnig, Konstantinos Tzelepis, Eliza Yankova, Tony Kouzarides, Oliver Rausch. A small molecule inhibitor of the RNA m6A writer METTL3 inhibits the development of acute myeloid leukemia (AML) in vivo [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B126. doi:10.1158/1535-7163.TARG-19-B126