Sp1 Expression Research Articles

ROS-Sp1 axis is involved in thermochemotherapy-enhanced sensitivity of pancreatic cancer cells to gemcitabine.

Gemcitabine (GEM)-based chemotherapy represents the first choice for locally unresectable advanced pancreatic cancer, while the benefit is limited due to acquired chemoresistance or drug delivery insufficiency. Hyperthermia treatment potentially improves the clinical efficacy of GEM. However, the underlying mechanism is incompletely revealed. Our study aims to investigate the effect and involved mechanism of thermochemotherapy on cell survival. Pancreatic cancer cells PANC-1 and ASPC-1 were either treated with GEM or thermochemotherapy, then cell viability, apoptosis, migration, invasion, reactive oxygen species (ROS) production, and Sp1 expression were evaluated. The results showed that GEM dose and time-dependently affected cell viability, and 30 μM GEM achieved favorable effect in suppressing cancer cell growth. Meanwhile, hyperthermia preconditioning (43°C for 60 min) 24 h before GEM treatment yielded superior effect than other treatment sequence. GEM caused significant cell apoptosis and proapoptotic genes of both cancer cells, and thermochemotherapy further promoted apoptosis and genes transcription, accompanied by excessive ROS production. Thermochemotherapy was superior to GEM in suppressing cell migration and invasion of pancreatic cancer cells. Meanwhile, GEM significantly reduced Sp1 mRNA and protein and its downstream gene Cox-2, and thermochemotherapy further suppressed their expressions. ROS elimination with N-acetyl-l-cysteine notably neutralizes the suppressive effect of GEM and thermochemotherapy on cell growth and expressions of Sp1 and Cox-2. In conclusion, thermochemotherapy inhibited pancreatic cell viability, migration and invasion, and promoted cell apoptosis by inducing excessive ROS production, which subsequently suppressed Sp1 expression and the downstream Cox-2.

Sp1 Upregulation Bolsters the Radioresistance of Glioblastoma Cells by Promoting Double Strand Breaks Repair.

Radioresistance remains a critical obstacle in the clinical management of glioblastoma (GBM) by radiotherapy. Therefore, it is necessary to explore the molecular mechanisms underlying radioresistance to improve patient response to radiotherapy and increase the treatment efficacy. The present study aimed to elucidate the role of specificity protein 1 (Sp1) in the radioresistance of GBM cells. Different human GBM cell lines and tumor-bearing mice were exposed to ionizing radiation (IR). Cell survival was determined by the colony formation assay. The expression of genes and proteins in the cells and tissues was analyzed by RT-PCR and western blotting, respectively. The γ-H2AX, p-Sp1 and dependent protein kinase catalytic subunit (DNA-PKcs phospho S2056) foci were analyzed by immunofluorescence. Apoptotic rates were measured by flow cytometry. Sp1 was upregulated after IR in vitro and in vivo and knocking down Sp1-sensitized GBM cells to IR. Sp1 activated the DNA-PKcs promoter and increased its expression and activity. Furthermore, the loss of Sp1 delayed double-strand breaks (DSB) repair and increased IR-induced apoptosis of GBM cells. Taken together, IR upregulates Sp1 expression in GBM cells, enhancing the activity of DNA-PKcs and promoting IR-induced DSB repair, thereby leading to increased radioresistance.

Astragalus polysaccharide ameliorates steroid-induced osteonecrosis of the femoral head by regulating miR-200b-3p-mediated Wnt/β-catenin signaling pathway via inhibiting SP1 expression

BackgroundSteroid-induced osteonecrosis of the femoral head (SONFH) is the necrosis of the femur bone caused by prolonged and massive use of corticosteroids. The present study probed into the significance of Astragalus polysaccharide (APS) in SONFH progression.MethodsSONFH cell model was constructed using murine long bone osteocyte Y4 (MLO-Y4) cells and then treated with APS. mRNA microarray analysis selected differentially expressed genes between control group and SONFH group. RT-qPCR determined SP1 and miR-200b-3p expression. Levels of SP1, β-catenin, autophagy-related proteins (LC3II/LC3I, Beclin1, p62) and apoptosis-related proteins (Bax, C-caspase3, C-caspase9, Bcl-2) were tested by Western blot. ChIP and luciferase reporter assays confirmed relationship between SP1 and miR-200b-3p. Fluorescence intensity of LC3 in cells was detected by immunofluorescence. Flow cytometry assessed cell apoptosis. Osteonecrosis tissues from SONFH mice were examined by HE and TRAP staining.ResultsAPS induced autophagy and suppressed apoptosis in SONFH cell model. APS inhibited SP1 expression and SP1 overexpression reversed effects of APS on SONFH cell model. Mechanistically, SP1 targeted miR-200b-3p to inhibit Wnt/β-catenin pathway. MiR-200b-3p depletion rescued the promoting effect of SP1 on SONFH cell model by activating Wnt/β-catenin pathway. HE staining showed that APS treatment reduced the empty lacunae and alleviated inflammation in trabecular bone of SONFH mice. TRAP staining revealed decreased osteoclasts number in SONFH mice after APS treatment.ConclusionAPS regulated osteocyte autophagy and apoptosis via SP1/miR-200b-3p axis and activated Wnt/β-catenin signaling, thereby alleviating SONFH, shedding new insights for therapy of SONFH.

Sp1 promotes tumour progression by remodelling the mitochondrial network in cervical cancer

BackgroundCervical cancer remains one of the most prevalent cancers worldwide. Accumulating evidence suggests that specificity protein 1 (Sp1) plays a pivotal role in tumour progression. The underlying role and mechanism of Sp1 in tumour progression remain unclear.MethodsThe protein level of Sp1 in tumour tissues was determined by immunohistochemistry. The effect of Sp1 expression on the biological characteristics of cervical cancer cells was assessed by colony, wound healing, transwell formation, EdU, and TUNEL assays. Finally, the underlying mechanisms and effects of Sp1 on the mitochondrial network and metabolism of cervical cancer were analysed both in vitro and in vivo.ResultsSp1 expression was upregulated in cervical cancer. Sp1 knockdown suppressed cell proliferation both in vitro and in vivo, while overexpression of Sp1 had the opposite effects. Mechanistically, Sp1 facilitated mitochondrial remodelling by regulating mitofusin 1/2 (Mfn1/2), OPA1 mitochondrial dynamin-like GTPase (Opa1), and dynamin 1-like (Drp1). Additionally, the Sp1-mediated reprogramming of glucose metabolism played a critical role in the progression of cervical cancer cells.ConclusionsOur study demonstrates that Sp1 plays a vital role in cervical tumorigenesis by regulating the mitochondrial network and reprogramming glucose metabolism. Targeting Sp1 could be an effective strategy for the treatment of cervical cancer.

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.

UVRAG Promotes Tumor Progression through Regulating SP1 in Colorectal Cancer.

Colorectal cancer (CRC) is the third most common type of cancer. The ultraviolet radiation resistance-associated gene (UVRAG) plays a role in autophagy and has been implicated in tumor progression and prognosis. However, the role of UVRAG expression in CRC has remained elusive. In this study, the prognosis was analyzed via immunohistochemistry, and the genetic changes were compared between the high UVRAG expression group and the low UVRAG expression group using RNA sequencing (RNA-seq) and single-cell RNA-seq (scRNA-seq) data, and genetic changes were then identified by in vitro experiments. It was found that UVRAG could enhance tumor migration, drug resistance, and CC motif chemokine ligand 2 (CCL2) expression to recruit macrophages by upregulating SP1 expression, resulting in poor prognosis of CRC patients. In addition, UVRAG could upregulate the expression of programmed death-ligand 1 (PD-L1). In summary, the relationship between UVRAG expression and the prognosis of CRC patients as well as the potential mechanisms in CRC were explored, providing evidence for the treatment of CRC.

Abstract 6733: Targeting Sp1 in Ewing sarcoma: a multi-approach method for the utilization of mithramycin

Abstract Purpose: Ewing Sarcoma (ES) is a bone and soft tissue cancer affecting young adults and children. Localized ES presents with a 5-year survival rate of 70%, while metastatic cases range from 15-30%. Our laboratory investigates combination treatments using less toxic agents to induce sensitization to chemotherapy in ES. The anti-cancer activity of an antineoplastic antibiotic, Mithramycin (Mit), against ES cells has been shown. Mit inhibits Specificity protein 1 (Sp1) a marker associated with aggressive cancer cell growth and resistance to chemo/radiation therapies. However, its mechanistic effects on other oncogenic proteins have yet to be elucidated in ES. The purpose of this study is to evaluate the effectiveness of Mit and combination with other chemotherapeutic agents, Etoposide and Vincristine, to inhibit ES cell growth and assess the effect on cancer related proteins regulated by Sp1. Future studies will expand upon Mithramycin's mechanism of action in Ewing Sarcoma utilizing RNA sequencing and computational methods. Methods: Anti-proliferative activity of Mit and/or Vincristine and Etoposide against ES cell lines, TC205 and CHLA10, was evaluated using CellTiterGlo kit. Dose curves and IC50 values were determined by Sigma-Plot software. The expression of Sp1 and survivin was determined by Western blot analysis. Cell lines were obtained from Children’s Oncology Group. The specific type of drug effect of the combination treatments were determined by analyzing the combination index obtained via Calcusyn software. Nude mice were injected with TC-205 cells and treated over two weeks with either Mit (1mg/kg per week) and/or Etoposide (5mg/kg per week) and tumor volume was compared. Protein models were obtained from RCSB PDB and homology tests were performed in the Swiss-model workspace. Results: Mit significantly decreased ES cell line viability and tumor volume in nude mice. Mit showed the ability to reduce the expression of Sp1 and offered differing effects on survivin expression, indicative of anti-apoptotic mechanisms being implemented in the ES cell lines. IC50 values of both chemotherapeutics and Mit were decreased by nearly 50% when used in combination and this effect was mirrored in further decreases in Sp1 expression. Synergistic drug responses were shown in the combination of Mit with both Vincristine and Etoposide. Sp1 and survivin protein models were established and homology verification using Ramachandran plots and QMEAN Z-scores indicated quality protein models for further computational studies. Conclusions: Mit may effectively sensitize ES cells and improve the response of chemotherapy while lowering necessary effective dosages. Studies to understand the mechanism of action of Mit on Sp1, survivin, and other proteins involved in Ewing Sarcomagenesis are underway. This study is partially supported by a grant from the Cancer Prevention and Research Institute of Texas (Award#: RP210046) Citation Format: Christoffer Lambring, Riyaz Basha, Umesh Sankpal. Targeting Sp1 in Ewing sarcoma: a multi-approach method for the utilization of mithramycin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6733.

Abstract 6255: Anti-proliferative effect of two copper complexes against medulloblastoma cells

Abstract Background and Purpose: Copper is a crucial structural component for many significant enzymes, as well as a key catalytic co-factor in redox processes. The flexible Cu(I/II) redox behavior facilitates the formation of copper complexes that are more potent and less toxic. The anticancer activity of several agents can be enhanced by forming copper complexes. The purpose of this study is to evaluate the anti-proliferative effects of two copper (II) complexes, copper-tolfenamic acid (Cu-TA) and copper thiosemicarbazone (Cu-acetylethTSC or CuTSC) against medulloblastoma (MB). MB is a cancer of the cerebellum which is associated with frequent relapse and drug resistance with the current treatments. It necessitates the development of alternative strategies. The anti-cancer activity of Cu-TA was evaluated in laboratory testing in some cancer models, but it has not been studied in MB. CuTSC against colorectal cancer, leukemia and breast cancer has been studied but it has not in MB. Methods: TA and TSC were complexed with Cu and Cu-TA and CuTSC complexes were characterized through colorimetric/mass spectrometric analyses. Cancer cells and cardiomyocytes were cultured using standard protocols and cell viability was measured using Cell Titer-Glo kit (Promega). Protein expression was determined by Western blot analysis. Experiments/Results: CuTA and CuTSC complexes showed proper characterization and stability during the testing period. Cardiomyocytes (H9C2) and human cancer cell lines representing breast, Ewing sarcoma and MB were screened for anti-proliferative activity of these two Cu complexes. While, cell viability was inhibited in all cancer cell lines, MB cell lines (DAOY and D283) showed higher efficacy while H9C2 cell viability was not affected. To understand the underlying mechanism of action, the effect of Cu-TA was assessed on the expression of c-PARP, Specificity protein 1 (Sp1) and an antiapoptotic protein, survivin. Western blot results revealed a clear decrease in the expression of both Sp1 and survivin and upregulation of c-PARP. Discussion/Conclusion: Both Cu-TA and CuTSC treatments result in a dose-time dependent anti-proliferative activity against cancer cells but not affecting non-malignant cardiomyocyte cells. Cu-TA can inhibit Sp1 and survivin and increase PARP cleavage suggesting the effect on Sp1-regulated oncogenes and an induction of apoptotic pathways. The studies to establish crossing of Blood Brain Barrier and further understand the underling mechanisms of these two Cu complexes in single and combination treatment alongside chemotherapeutic agents are currently under investigation. Citation Format: Hope Korshie Fiadjoe, Christoffer Lambring, Umesh T. Sankpal, Duaa Alajroush, Alvin A. Holder, Riyaz Basha. Anti-proliferative effect of two copper complexes against medulloblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6255.

SP1 transcriptionally regulates UBE2N expression to promote lung adenocarcinoma progression

Lung adenocarcinoma (LUAD) is the main cause of cancer-related death worldwide. Understanding the mechanisms of LUAD progression may provide insights into targeted therapy approaches for this malignancy. Ubiquitin-conjugating enzyme 2 N (UBE2N) has been demonstrated to play key roles in the progression of various cancers. However, the functions and mechanisms underlying UBE2N expression in LUAD are still unclear. In this study, we found that UBE2N is highly expressed in LUAD and patients with high UBE2N expression in their tumors have poor clinical outcomes. Moreover, we showed that UBE2N interference significantly inhibited LUAD progression in vitro and in vivo. At the molecular level, we demonstrated that the UBE2N is a bona fide target of transcription factor SP1. SP1 directly bound to the promoter of UBE2N and upregulated its expression in LUAD cells, which in turn contributed to the progression of LUAD. Furthermore, we found that there is a strong positive correlation between the expression of SP1 and UBE2N in LUAD samples. Importantly, LUAD patients with concomitantly high expression of SP1 and UBE2N were significantly associated with poor clinical outcomes. In conclusion, our study demonstrated that the SP1-UBE2N signaling axis might play a key role in the malignant progression of LUAD, which provides new targets and strategies for the treatment of LUAD.

LINC00115 regulates lung adenocarcinoma progression via sponging miR-154-3p to modulate Sp3 expression

The most commonly diagnosed and most lethal subtype of lung cancer is lung adenocarcinoma (LUAD). Therefore, more detailed understanding of the potential mechanism and identification of potential targets of lung adenocarcinoma is needed. A growing number of reports reveals that long non-coding RNAs (lncRNAs) play crucial roles in cancer progression. In present study, we found that lncRNA LINC00115 was upregulated in LUAD tissues and cells. Functional studies revealed that LINC00115 knockdown inhibits the proliferation, growth, invasion, and migration of LUAD cells. Mechanically, we indicated that miR-154-3p is target microRNA of LINC00115, and the effect of downregulated LINC00115 on LUAD cells was partially reversed by the miR-154-3p antisense oligonucleotide (ASO-miR-154-3p). Further investigation revealed that Specificity protein 3 (Sp3) directly interacted with miR-154-3p, and the Sp3 level was positively correlated with the LINC00115 expression. Rescue experiments further showed that Sp3 overexpression partially restored the effect of downregulated LINC00115 on LUAD cells. Similarly, in vivo experiments confirmed that downregulated LINC00115 inhibited xenograft growth and Sp3 expression. Our results demonstrated that LINC00115 knockdown inhibited LUAD progression via sponging miR-154-3p to modulate Sp3 expression. These data indicate that the LINC00115/miR-154-3p/Sp3 axis can be a potential therapeutic target of LUAD.

Tra2β Enhances Cell Proliferation by Inducing the Expression of Transcription Factor SP1 in Cervical Cancer

ObjectiveIn this study, the role and potential mechanism of transformer 2β (Tra2β) in cervical cancer were explored. MethodsThe transcriptional data of Tra2β in patients with cervical cancer from Gene Expression Profiling Interactive Analysis (GEPIA) and cBioPortal databases were investigated. The functions of Tra2β were evaluated by using Western blot, MTT, colony formation, Transwell assays, and nude mouse tumor formation experiments. Target genes regulated by Tra2β were studied by RNA-seq. Subsequently, representative genes were selected for RT-qPCR, confocal immunofluorescence, Western blot, and rescue experiments to verify their regulatory relationship. ResultsThe dysregulation of Tra2β in cervical cancer samples was observed. Tra2β overexpression in Siha and Hela cells enhanced cell viability and proliferation, whereas Tra2β knockdown showed the opposite effect. Alteration of Tra2β expression did not affect cell migration and invasion. Furthermore, tumor xenograft models verified that Tra2β promoted cervical cancer growth. Mechanically, Tra2β positively regulated the mRNA and protein level of SP1, which was critical for the proliferative capability of Tra2β. ConclusionThis study demonstrated the important role of the Tra2β/SP1 axis in the progression of cervical cancer in vitro and in vivo, which provides a comprehensive understanding of the pathogenesis of cervical cancer.

Givinostat Inhibition of Sp1-dependent MGMT Expression Sensitizes Glioma Stem Cells to Temozolomide.

Givinostat is a pan-histone deacetylase (HDAC) inhibitor that has demonstrated excellent tolerability as well as efficacy in patients with polycythemia vera. Accumulating in vitro and in vivo evidence suggests givinostat is also promising as a therapeutic agent targeting glioma stem cells (GSCs), the cancer stem cells of glioblastoma (GBM) considered responsible for its intractable nature. However, it remains to be shown how givinostat impacts the therapeutic effects of temozolomide, a DNA-alkylating agent and the key component of GBM treatment given not only during postoperative radiotherapy but also thereafter as maintenance chemotherapy. The effects of givinostat and knockdown of O6-methylguanine-DNA methyltransferase (MGMT) or Sp1 on the mRNA and protein expression of relevant genes in human GSC lines were examined by RT-PCR and western blot analyses. The dye exclusion method was used to evaluate cell viability. Givinostat enhanced the cytotoxic activity of temozolomide in GSC lines expressing MGMT, in which the MGMT expression was shown to contribute to their temozolomide resistance. Givinostat inhibited MGMT expression in GSCs and, in parallel, the expression of Sp1, a transcription factor involved in the control of MGMT promoter activity. Knockdown experiments demonstrated Sp1 expression was indeed required for MGMT expression in GSCs. Givinostat, in addition to its own cytotoxic activity, sensitizes GSCs to temozolomide by inhibiting Sp1-dependent MGMT expression in GSCs. Combining givinostat with temozolomide could therefore be a rational therapeutic strategy to effectively eliminate GSCs and thus help overcome the therapy resistance of GBM.

The downregulation of HSP90-controlled CRALBP expression is associated with age-related vision attenuation.

The dysfunction of CRALBP, a key regulator of the visual cycle, is associated with retinitis punctata albescens characterized by night vision loss and retinal degeneration. In this paper, we find that the expression of CRALBP is regulated by heat shock protein 90 (HSP90). Inhibition of HSP90α or HSP90β expression by using the CRISPR-Cas9 technology downregulates CRALBP's mRNA and protein expression in ARPE-19 cells by triggering the degradation of transcription factor SP1 in the ubiquitin-proteasome pathway. SP1 can bind to CRALBP's promoter, and inhibition of SP1 by its inhibitor plicamycin or siRNA downregulates CRALBP's mRNA expression. In the zebrafish, inhibition of HSP90 by the intraperitoneal injection of IPI504 reduces the thickness of the retinal outer nuclear layer and Rlbp1b mRNA expression. Interestingly, the expression of HSP90, SP1, and CRALBP is correlatedly downregulated in the senescent ARPE-19 and Pig primary RPE cells in vitro and in the aged zebrafish and mouse retinal tissues in vivo. The aged mice exhibit the low night adaption activity. Taken together, these data indicate that the HSP90-SP1 is a novel regulatory axis of CRALBP transcriptional expression in RPE cells. The age-mediated downregulation of the HSP90-SP1-CRALBP axis is a potential etiology for the night vision reduction in senior people.

Timing Expression of miR203a-3p during OA Disease: Preliminary In Vitro Evidence.

Osteoarthritis (OA) is a degenerative bone disease that involves the microenvironment and macroenvironment of joints. Progressive joint tissue degradation and loss of extracellular matrix elements, together with different grades of inflammation, are important hallmarks of OA disease. Therefore, the identification of specific biomarkers to distinguish the stages of disease becomes a primary necessity in clinical practice. To this aim, we investigated the role of miR203a-3p in OA progression starting from the evidence obtained by osteoblasts isolated from joint tissues of OA patients classified according to different Kellgren and Lawrence (KL) grading (KL ≤ 3 and KL > 3) and hMSCs treated with IL-1β. Through qRT-PCR analysis, it was found that osteoblasts (OBs) derived from the KL ≤ 3 group expressed high levels of miR203a-3p and low levels of ILs compared with those of OBs derived from the KL > 3 group. The stimulation with IL-1β improved the expression of miR203a-3p and the methylation of the IL-6 promoter gene, favoring an increase in relative protein expression. The gain and loss of function studies showed that the transfection with miR203a-3p inhibitor alone or in co-treatments with IL-1β was able to induce the expression of CX-43 and SP-1 and to modulate the expression of TAZ, in OBs derived from OA patients with KL ≤ 3 compared with KL > 3. These events, confirmed also by qRT-PCR analysis, Western blot, and ELISA assay performed on hMSCs stimulated with IL-1β, supported our hypothesis about the role of miR203a-3p in OA progression. The results suggested that during the early stage, miR203a-3p displayed a protective role reducing the inflammatory effects on CX-43, SP-1, and TAZ. During the OA progression the downregulation of miR203a-3p and consequently the upregulation of CX-43/SP-1 and TAZ expression improved the inflammatory response and the reorganization of the cytoskeleton. This role led to the subsequent stage of the disease, where the aberrant inflammatory and fibrotic responses determined the destruction of the joint.

Licochalcone A Suppresses Renal Cancer Cell Proliferation and Metastasis by Engagement of Sp1-Mediated LC3 Expression

Licochalcone A (LicA) is a strong anti-inflammatory, antioxidant, and anticarcinogenic substance that is useful against a variety of human malignancies. However, its precise mechanism in mediating the development of renal cell carcinoma (RCC) is not entirely understood. In this work, LicA was discovered to limit cell growth and survival, induce cell cycle arrest, promote autophagy and LC3B expression, and inhibit the migration and invasion of RCC cells. In addition, the proliferation, migration, and invasion inhibited by LicA were restored by the transfection of siRNA-LC3. The effects of LC3B on the metastatic phenotype of ACHN cells was enhanced with the overexpression of Sp1 or suppressed by inhibiting the phosphorylation of FAK and Src. Finally, LicA showed antitumor properties against RCC in an in vivo xenograft model. In conclusion, our study demonstrated the chemotherapeutic potential of LicA on proliferation, migration, invasion, and autophagy through the activation of LC3B expression, ultimately modulating FAK/Src signaling pathway-mediated Sp1 expression. These findings illustrate the novel role and molecular mechanisms of LicA against RCC cells.

Circ0005654 as a new biomarker of thyroid cancer interacting with SP1 to influence the prognosis: A case-control study.

Circular RNAs (CircRNAs) have been reported to play key roles in the progression of various cancers, including thyroid cancer (TC). Transcription factor 1 (SP1) promotes the development of thyroid cancer. This study aims at investigating the expression level of Circ0005654 in combination with Transcription factor1 (SP1) in patients with TC for diagnostic and therapeutic purposes. A total of 76 patients with thyroid cancer underwent radical surgery. Intraoperatively, thyroid cancer tissues and paired adjacent tissues and the corresponding clinicopathological data were collected. The expression of SP1 and β-catenin in thyroid cancer and adjacent tissues was determined by immunohistochemistry (IHC) while the Circ0005654 expression level was measured by semiquantitative real-time polymerase chain reaction (sqRT-PCR). Then, we compared the variability of Circ0005654, SP1, and Wnt/β-catenin expression in cancerous and adjacent tissues and determined the relationship between the correlation analysis and the clinicopathological features of the thyroid cancer patients. The diagnostic value of Circ0005654 in thyroid cancer tissues was analyzed with the help of the receiver operating characteristic (ROC) curve, counting the 3-year postoperative survival rate, and analyzing the effect of Circ0005654 and SP1 protein levels on the 3-year survival rate of the patients. sqRT-PCR showed that the expression level of Circ0005654 in thyroid cancer tissue was significantly higher than that of adjacent tissues. The area under the ROC of Circ0005654 was 0.9553, 95% confidence interval: (0.9211-0.9895) with a cutoff value of 0.7895, a sensitivity of 92.11%, and a specificity of 86.84%. The IHC results showed that the expression level of SP1, β-catenin, and Wnt was higher in cancer tissues than in adjacent tissues; Circ0005654, SP1, Wnt/β-catenin expression levels were associated with tumor diameter, lymph node metastasis, TNM stage, and envelope invasion (all P < .05). According to the Circ0005654 expression level in thyroid cancer tissue, the 3-year survival rate of the high expression group was 77.5% and 94.4% in the low expression group with a statistically significant difference; the 3-year survival rate of SP1 positive and negative patients was 78.6% and 100%, respectively, with the data being significantly different. Circ0005654 may serve as a potential biomarker for thyroid cancer diagnosis and may be involved in the development of thyroid cancer.

N-Acetyltransferase 9 Inhibits Porcine Reproductive and Respiratory Syndrome Virus Proliferation by N-Terminal Acetylation of the Structural Protein GP5.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a serious threat to the global swine industry. As a typical immunosuppressive virus, PRRSV has developed a variety of complex mechanisms to escape the host innate immunity. In this study, we uncovered a novel immune escape mechanism of PRRSV infection. Here, we demonstrate for the first time that the endoplasmic reticulum (ER)-resident N-acetyltransferase Nat9 is an important host restriction factor for PRRSV infection. Nat9 inhibited PRRSV proliferation in an acetyltransferase activity-dependent manner. Mechanistically, glycoprotein 5 (GP5) of PRRSV was identified as interacting with Nat9 and being N-terminally acetylated by it, which generates a GP5 degradation signal, promoting the K27-linked-ubiquitination degradation of GP5 to decrease virion assembly. Meanwhile, the expression of Nat9 was inhibited during PRRSV infection. In detail, two transcription factors, ETV5 and SP1, were screened out as the key transcription factors binding to the core promoter region of Nat9, and the PRRSV nonstructural protein 1β (Nsp1β), Nsp4, Nsp9, and nucleocapsid (N) proteins were found to interfere significantly with the expression of ETV5 and SP1, thereby regulating the transcription activity of Nat9 and inhibiting the expression of Nat9. The findings suggest that PRRSV decreases the N-terminal acetylation of GP5 to support virion assembly by inhibiting the expression of Nat9. Taken together, our findings showed that PRRSV has developed complex mechanisms to inhibit Nat9 expression and trigger virion assembly. IMPORTANCE To ensure efficient replication, a virus must hijack or regulate multiple host factors for its own benefit. Understanding virus-host interactions and the molecular mechanisms of host resistance to PRRSV infection is necessary to develop effective strategies to control PRRSV. The N-acetyltransferase Nat9 plays important roles during virus infection. Here, we demonstrate that Nat9 exhibits an antiviral effect on PRRSV proliferation. The GP5 protein of PRRSV is targeted specifically by Nat9, which mediates GP5 N-terminal acetylation and degradation via a ubiquitination-dependent proteasomal pathway. However, PRRSV manipulates the transcription factors ETV5 and SP1 to inhibit the expression of Nat9 and promote virion assembly. Thus, we report a novel function of Nat9 in PRRSV infection and elucidate a new mechanism by which PRRSV can escape the host innate immunity, which may provide novel insights for the development of antiviral drugs.

Modulatory Effects of Metformin on Farnesoid X receptor and Specificity Protein 1 in Human Pancreatic Cancer BxPC-3 Cells

Background: Pancreatic cancer is the fourth major cause of death, accounting for 7% of cancer-related deaths. Metformin (antihyperglycemic agent) users had a 62% lower risk of developing pancreatic cancer when compared to metformin non-users. Objective: This research aims to study the role of the Farnesoid X receptor (FXR) receptor in human pancreatic cancer cell line BxPC-3 and metformin’s modulatory effects on this receptor. Material and methods: Cell viability was assessed using MTT assay for INT747 (FXR agonist) and metformin, FXR and Specificity Protein 1 (Sp1) mRNA and protein levels were measured by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and western blot, respectively. Results: INT747 increased the growth and viability of BxPc-3 cells in addition to a significant increase in FXR and SP1 mRNA and protein levels. In contrast, BxPC-3 cells viability was significantly reduced by metformin in a dose- and time-dependent manner with downregulation of FXR and SP1 at mRNA and protein levels Conclusion: FXR may act as an oncogenic factor in pancreatic cancer BxPC-3 cells by increasing FXR and SP1 expression, while metformin significantly reduced FXR mRNA and protein levels in BxPC-3 cells, which may be due to a reduction in SP1 expression.

Sp1 Controls the Basal Level of Interleukin-34 Transcription

ABSTRACT Interleukin-34 (IL-34) is a cytokine that plays important roles at steady state and in diseases. The induced or inhibited expression of IL-34 by stimuli has been deeply investigated. However, the regulation of IL-34 basal expression is largely unknown. The aim of this study is to investigate whether IL-34 expression is regulated by a general transcription factor Specificity Protein 1 (Sp1) at transcription level. By using bioinformatic software, four putative Sp1-binding sites overlapping GC boxes were found in the core promoter region of IL-34. Alignment of the core promoter sequences of mammalian IL-34 showed GC box-C (−62/–57) and D (−11/–6) were conserved in some mammals. Luciferase assay results showed that only deletion of GC box-C (−62/–57) significantly reduced luciferase activities of IL-34 core promoter in SH-SY5Y cells. By using electrophoretic mobility shift assay (EMSA), it was found that Sp1 specifically interacted with GC box-C sequence CCCGCC (−62/–57) in the core promoter of IL-34. By using chromatin immunoprecipitation (ChIP), it was discovered that Sp1 bound to the core promoter of IL-34 in living cells. In addition, silencing of Sp1 expression by its specific siRNA reduced IL-34 mRNA and protein levels significantly in SH-SY5Y cells. Likewise, IL-34 expression was inhibited in a dose-dependent manner by a Sp1 inhibitor Plicamycin. Furthermore, silencing of Sp1 also downregulated mRNA and protein expression of IL-34 in GES-1 and 293T cell lines, suggesting that IL-34 transcription regulated by Sp1 was not cell-type specific. Taken together, these results indicate that Sp1 controls the basal level of IL-34 transcription.

Oncolytic HSV-1 suppresses cell invasion through downregulating Sp1 in experimental glioblastoma

Gliomas are highly aggressive intracranial tumors that are difficult to resect and have high lethality and recurrence rates. According to WHO grading criteria, glioblastoma with wild-type IDH1 has a poorer prognosis than WHO grade 4 IDH-mutant astrocytomas. To date, no effective therapeutic strategies have been developed to treat glioblastoma. Clinical trials have shown that herpes simplex virus (HSV)-1 is the safest and most efficacious oncolytic virus against glioblastoma, but the molecular antitumor mechanism of action of HSV-1 has not yet been determined. Deletion of the γ34.5 and ICP47 genes from a strain of HSV-1 yielded the oncolytic virus, oHSV-1, which reduced glioma cell viability, migration, and invasive capacity, as well as the growth of microvilli. Infected cell polypeptide 4 (ICP4) expressed by oHSV-1 was found to suppress the expression of the transcription factor Sp1, reducing the expression of host invasion-related genes. In vivo, oHSV-1 showed significant antitumor effects by suppressing the expression of Sp1 and invasion-associated genes, highly expressed in high-grade glioblastoma tissue specimens. These findings indicate that Sp1 may be a molecular marker predicting the antitumor effects of oHSV-1 in the treatment of glioma and that oHSV-1 suppresses host cell invasion through the ICP4-mediated downregulation of Sp1.