ABSTRACT
Tripartite motif containing protein 7 (TRIM7), as a member of the E3 ubiquitin ligase TRIM family, plays an important regulatory role in immune regulation, metabolism and other physiological processes. The aberrant expression of TRIM7 is closely related to the development and progression of hepatocellular carcinoma (HCC) and it shows a complex regulatory role. However, the regulatory mechanism for the expression of TRIM7 in HCC remains unknown. In this study, multiple online databases were used to analyze the expression of TRIM7 in HCC and data indicated that TRIM7 expression was upregulated in HCC and correlated to poor prognosis. Subsequently, the transcription factor binding sites in the TRIM7 promoter region were analyzed using UCSC and JASPAR databases, and the results showed that TRIM7 promoter contains four SP1 binding sites. In this work, we demonstrated that SP1 could directly bind to its binding sites in TRIM7 promoter and positively regulate the transcriptional activity driven by the TRIM7 promoter using dual luciferase reporter experiments and the ChIP-PCR method. Moreover, our results also showed SP1 overexpression upregulated the expression of TRIM7 at both mRNA and protein levels (P<0. 01),and SP1 inhibitor, mithramycin A, could reverse the activated effect of SP1 on TRIM7 expression (P<0. 01). In conclusion, this study preliminarily reveals the regulatory mechanism of TRIM7 upregulation in HCC, which provides an important theoretical basis for further study of the gene function, early diagnosis and targeted therapy.
ABSTRACT
OBJECTIVE@#To study the transcriptional regulation of SP1 on the scaffold protein ARRB1 and its influence on the progression of T-cell acute lymphoblastic leukemia (T-ALL).@*METHODS@#pGL3-ARRB1-luc, pCDNA3.1-SP1 and other transcription factor plasmids that might be combined were constructed, and the binding of transcription factors to the promoter of ARRB1 was identified by dual luciferase reporter gene assay. Stable cell lines with over-expressed SP1 (JK-SP1) was constructed by lentiviral transfection, and the expression correlation of SP1 with ARRB1 was demonstrated by RT-PCR and Western blot. Further, the apoptosis, cell cycle and reactive oxygen species (ROS) were detected by flow cytometry. The effect of SP1 on propagation of leukemic cells was observed on NCG leukemic mice.@*RESULTS@#The expression of fluorescein were enhanced by co-transfection with pCDNA3.1-SP1 and pGL3-ARRB1-luc plasmids in HEK293T cell line (P<0.001), meanwhile, compared with the control group, the expression of ARRB1 mRNA and protein were increased in JK-SP1 cells (both P<0.01). Further in vitro experiments showed that, compared with the control group, the apoptosis rate was higher (x=22.78%) , the cell cycle was mostly blocked in G1 phase (63.00%), and the content of reactive oxygen species increased in JK-SP1 cells. And in vivo experiments showed that the mice injected with JK-SP1 cells through tail vein had a favorable overall survival time (average 33.8 days), less infiltration in liver and spleen tissue.@*CONCLUSION@#Transcription factor SP1 promotes the transcription and expression of ARRB1 by binding the the promoter of ARRB1 directly, thus delays the progress of T-ALL in vitro and in vivo. The study improves the pathogenesis of ARRB1 regulating the initiation and development of T-ALL, and provides theoretical basis for the development of new possible targeted drugs.
Subject(s)
Humans , Animals , Mice , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , HEK293 Cells , Reactive Oxygen Species , Transcription Factors , T-Lymphocytes , Cell Line, Tumor , Sp1 Transcription Factor/metabolismABSTRACT
OBJECTIVE@#In this study, the role and potential mechanism of transformer 2β (Tra2β) in cervical cancer were explored.@*METHODS@#The 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.@*RESULTS@#The 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.@*CONCLUSION@#This 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.
Subject(s)
Humans , Animals , Mice , Female , Uterine Cervical Neoplasms/genetics , HeLa Cells , Cell Proliferation , Biological Assay , Transcription Factors , Sp1 Transcription Factor/geneticsABSTRACT
Objective:To evaluate the effect of propofol on proliferation of neural stem cells (NSCs) in mice and the role of specificity protein-1 (Sp-1)-epidermal growth factor receptor (EGFR)-protein kinase B (Akt) signaling pathway.Methods:Primary NSCs harvested from both the cortices and hippocampus of C57BL/6 mouse embryos were identified by immunofluorescent staining of Nestin.NSCs at passages 3-6 were divided into 3 groups ( n=21 each) using a random number table method: normal saline control group (C group), propofol group (P group) and propofol plus Sp1 inhibitor plicamycin group (PP group). Propofol at a final concentration of 10 μmol/L was added in group P. Propofol at a final concentration of 10 μmol/L and plicamycin at a final concentration of 100 nmol/L were added in group PP.The equal volume of normal saline was added in group C. The medium was replaced after 6 h of incubation and the cells were continuously incubated.The proliferation of NSCs was assessed by direct cell counting at 24, 36, 48, 60 and 72 h after the end of treatment with drugs.At 6 h after the end of treatment with drugs, the expression of Sp1 and EGFR mRNA was detected by real-time fluorescent quantitative polymerase chain reaction, and the expression of Sp1, Akt and phosphorylated Akt (p-Akt) by Western blot. Results:Compared with group C, the count of NSCs was significantly increased at 48, 60 and 72 h after treatment with drugs, and the expression of EGFR mRNA, Sp1 protein and mRNA and p-Akt was up-regulated in group P ( P<0.05 or 0.01), and no significant change was found in each parameter in group PP ( P>0.05). Compared with group P, the count of NSCs was significantly decreased at 48 and 60 h after treatment with drugs, and the expression of EGFR protein and mRNA and p-Akt was down-regulated in group PP ( P<0.05 or 0.01). Conclusions:Propofol can promote the proliferation of NSCs, and the mechanism may be related to activation of Sp1-EGFR-Akt signaling pathway in mice.
ABSTRACT
Objective:To evaluate the effect of down-regulation of Sp1 expression on the radiosensitivity of glioma cells.Methods:The oligonucleotide sequence encoding shRNA was designed and synthesized, and cloned into LV3 (H1/GFP & Puro) vector to construct the recombinant. U251 and U87 cells were infected with recombinant lentivirus, then the stably-transfected cell lines were obtained by puromycin screening. The expression levels of Sp1 mRNA and protein were detected by RT-PCR and Western blot. Cell survival was detected by clonal survival assay, cell cycle was determined by flow cytometry, and DNA damage was measured by immunofluorescence assay, respectively.Results:At 72 h after infection, high expression of Sp1 lentiviral vector was observed in two cell lines under fluorescence microscope. RT-PCR and Western blot confirmed that the expression levels of Sp1 mRNA and protein were significantly down-regulated in both transfected cells (both P<0.01) and the silencing rates of Sp1 were above 90%. The sensitization enhancement ratio (SER) of shRNA-U251 and shRNA-U87 cells at 10% cell survival level were 1.39 and 1.18, respectively. After irradiation, the G 2/M phase ratio and the number of γ-H2AX foci in two Sp1 knockout groups were significantly increased. Conclusion:shRNA silencing of Sp1 increases the G 2/M phase arrest induced by X-ray, aggravates the degree of DNA double-strand breaks, and improves the radiosensitivity of glioma cells.
ABSTRACT
Drug-induced long QT syndrome (LQTS) has become an important clinical research topic, and the occurrence of acquired long QT syndrome (acLQTS) is mainly caused by drug inhibition of the human ether-α-go-go related gene (hERG) channel. The hERG gene encodes the α subunit of the fast-activating delayed rectifying potassium ion channel (Ikr), which plays an important role in the process of action potential phase 3 repolarization and is also the target of most antiarrhythmic drugs. The purpose of this study was to investigate the effect of hydroxyrutaecarpine (HRU) on the hERG channel and to evaluate its cardiotoxicity. The whole cell patch clamp technique was used to detect the effects of HRU on the current and kinetics of the hERG channel, and to confirm the binding site on the hERG channel. PCR was used to determine the effect of HRU on hERG mRNA expression. Western blotting was used to detect the effects of HRU on the expression of hERG protein and transcription factor Sp1. Immunofluorescence was used to confirm the effects of HRU on localization and expression of hERG protein and transcription factor Sp1. Studies have shown that transient HRU can inhibit hERG current and shorten the inactivation time constant. Its binding sites to the hERG channel are F656 and Y652. After incubation for 24 h, HRU can reduce the expression of hERG protein, inhibit the hERG current, reduce the level of hERG mRNA, and reduce the expression of transcription factor Sp1 in the nucleus and hERG protein in the cytoplasm. Immunofluorescence experiments also showed the same results suggesting that the inhibition of Sp1 expression by HRU is the cause of the decreased expression of hERG mRNA. In conclusion, the acute inhibition of HRU accelerates the channel inactivation process and reduces the inactivation time constant by binding to the F656 and Y652 sites in the hERG channel, thus reducing the hERG current. In addition, HRU also inhibits the expression of hERG protein, mainly by inhibiting the expression of transcription factor Sp1, the transcription function of hERG channel protein is down-regulated, so that the hERG protein is reduced.
ABSTRACT
OBJECTIVE@#To compare the clinical effect between wheat-grain moxibustion at Yinbai (SP 1) and oral administration of dydrogesterone tablet for menstrual period prolongation after down-regulation treatment of in vitro fertilization embryo transfer (IVF-ET).@*METHODS@#A total of 54 patients with prolonged menstrual period after down-regulation treatment of IVF-ET were randomly divided into an observation group and a control group, 27 cases in each one. In the observation group, when the menstrual period delayed more than 7 days, the wheat-grain moxibustion at Yinbai (SP 1) was performed, once a day, with an interval of 1 day between two 3-day treatments; when the menstrual blood was cleaned, the ovulation was continued and the eggs were taken. In the control group, when the menstrual period delayed more than 7 days, the oral administration of dydrogesterone tablet was provided, 10 mg each time, twice a day; when the menstrual blood was cleaned, the ovulation was continued and the eggs were taken. The number of days for menstrual blood to be cleaned, the area change of uterine cavity hemorrhage, the morphology of endometrium, the blood supply of endometrium, the number of oocytes obtained, the grade of frozen embryo and the clinical effect were observed between the two groups after treatment.@*RESULTS@#Compared with the control group, the number of days for menstrual blood to be cleaned was shorter in the observation group after treatment (0.05). The cured rate in the observation group was 100.0% (27/27), higher than 33.3% (9/27) in the control group (<0.05).@*CONCLUSION@#The wheat-grain moxibustion at Yinbai (SP 1) could more effectively treat prolonged menstrual period after IVF-ET down-regulation treatment, which is beneficial to the preparation of the endometrium, and has no effect on the oocyte collection and embryo culture.
ABSTRACT
ABSTRACT Purpose: To determine the expression profiles of the transcription factor specificity protein 1 and collagen I in primary pterygial and normal conjunctival tissues, and to explore the role of specificity protein 1 and collagen I in pterygial development. Methods: The pterygial tissues of 20 patients who underwent resection of primary pterygial tissue in our hospital from June 2016 to December 2017 and the conjunctival tissues of 10 patients with enucleation due to trauma were collected. Reverse transcription quantitative-po lymerase chain reaction and western blot analyses were used to detect the relative expression levels of specificity protein 1 and type I collagen at the mRNA and protein levels. Results: The content of specificity protein 1 and collagen I mRNA and protein was significantly greater in primary pterygial tissue than it was in conjunctival tissue (p<0.05). There was a positive correlation between the mRNA and protein levels of specificity protein 1 and collagen I in primary pterygial tissues (protein: r=1, p<0.05; mRNA: r=1, p<0.05). Conclusion: Specificity protein 1 and collagen I are expressed in normal conjunctival and pterygial tissues, but expression is significantly greater in the latter. Specificity protein 1 and collagen I may be involved in the regulation of the development of primary pterygium.
RESUMO Objetivo: Determinar os perfis de expressão do fator de transcrição da proteína de especificidade 1 e do colágeno I em tecidos pterigiais primários e conjuntivais normais, e explorar o papel da proteína de especificidade 1 e colágeno I no desenvolvimento pterigial. Métodos: Foram coletados os tecidos pterigiais de 20 pacientes submetidos à ressecção de tecido de pterígio primário em nosso hospital no período de junho de 2016 a dezembro de 2017 e os tecidos conjuntivais de 10 pacientes com enucleação por trauma. A reação em cadeia da polimerase quantitativa de transcriptase reversa e a análise de Western blot foram utilizadas para detectar os níveis de expressão relativa da proteína de especificidade 1 e colágeno tipo I nos níveis de mRNA e proteína. Resultados: O conteúdo de especificidade da proteína 1 e do mRNA e proteína do colágeno I foi significativamente maior no tecido de pterígio primário do que no tecido conjuntival (p<0,05). Houve correlação positiva entre os níveis de mRNAs e proteína de especificidade 1 e colágeno I nos tecidos primários do pterígio (proteínas: r=1, p<0,05; mRNA: r=1, p<0,05). Conclusão: A proteína de especificidade 1 e do colágeno I é expressa nos tecidos conjuntivais e pterigiais normais, mas a expressão é significativamente maior no segundo. A especificidade da proteína 1 e do colágeno I pode ser envolvida na regulação do desenvolvimento do pterígio primário.
Subject(s)
Humans , Male , Female , Adult , Middle Aged , Aged , Pterygium/metabolism , RNA, Messenger/metabolism , Conjunctiva/abnormalities , Collagen Type I/metabolism , Pterygium/genetics , RNA, Messenger/genetics , Cells, Cultured , Blotting, Western , Conjunctiva/metabolism , Collagen Type I/geneticsABSTRACT
BACKGROUND: Emerging evidence showed that microRNAs (miRs) play critical roles in human cancers by functioning as either tumor suppressor or oncogene. MIR-382 was found to function as tumor suppressor in certain cancers. However, the role of MIR-382 in colorectal cancer (CRC) is largely unknown. Specificity protein 1 (SP1) is highly expressed in several cancers including CRC and is correlated with poor prognosis, but it is unclear whether or not MIR-382 can regulate the expression of SP1. METHODS: MIR-382 expression level was measured by reverse transcription-quantitative polymerase chain reaction. The connection between MIR-382 and SP1 was validated by luciferase activity reporter assay and western blot assay. Cell counting kit-8 assay and wound-healing assay were conducted to investigate the biological functions of MIR-382 in CRC. RESULTS: In this study, we found MIR-382 expression was downregulated in CRC tissues and cell lines, and the transfection of MIR-382 mimic decreased cell growth and migration. Furthermore, we identified SP1 was a direct target of MIR-382. Overexpression of MIR-382 decreased the expression of SP1, whereas MIR-382 knockdown promoted SP1 expression. We also observed an inversely correlation between MIR-382 and SP1 in CRC tissues. Additionally, we showed that knockdown of SP1 inhibited cell growth and migration and attenuated the effect of MIR-382 inhibitor on cell behaviors. CONCLUSIONS: In conclusion, the present study describes a potential mechanism underlying a MIR-382/SP1 link contributing to CRC development. Thus, MIR-382 may be able to be developed as a novel treatment target for CRC.
Subject(s)
Humans , Colorectal Neoplasms/metabolism , Gene Expression Regulation, Neoplastic/genetics , Sp1 Transcription Factor/metabolism , MicroRNAs/physiology , Transfection , Colorectal Neoplasms/pathology , Down-Regulation , Cell Movement , Sp1 Transcription Factor/genetics , MicroRNAs/genetics , Cell Line, Tumor , Cell Proliferation , Neoplasm Invasiveness/geneticsABSTRACT
Objective To discuss the mechanism of miR-24-3P in the process of acute respiratory distress syndrome (ARDS).Methods miR-24-3P mimics,miR-24-3P inhibitor and their negative controls were transfected in RAW264.7 cells respectively:The relative expression of miR-24-3P in each cell was detected.The relative expression of TNF-α,IL-6,SP1 and NF-κB were detected in each cell,and the regulation of miR-24-3P on the target gene SP1 was detected by dual luciferase reporter gene.Results The expression level of TNF-α mRNA and IL-6 mRNA in each group was statistically significant (P＜0.05).The relative expression level of SP1 mRNA and NF-κB mRNA in each group was not statistically significant (P＞0.05).The expression of SP1 in each group was statistically significant (P＜0.05).The gene analysis of dual luciferase reporter showed that the fluorescence activity was significantly inhibited after cotransfection of miR-24-3P and SP1 in HEK293 cells(P＜0.05).Conclusion miR-24-3P can play an important role in the process of ARDS by influencing the translation of SP1 gene and affecting the release of inflammatory mediators.
ABSTRACT
Objective@#To investigate the role of transcription factor specificity protein 1 (SP1) in proliferation, migration and invasion in head and neck squamous cell carcinoma (HNSCC), and the role of SP1 in transcription regulation of microRNA (miRNA)-92b.@*Methods@#Predicted the possible target miRNA of transcription factor SP1 by bioinformatic analysis. Furthermore, confirmed the binding sites of transcription factor SP1 and miRNA-92b promoter regions by chromatin immunoprecipitation. After transfecting SP1 siRNA and negative control siRNA, also performed quantitative real-time PCR (qPCR), cell proliferation assay and Transwell assay.@*Results@#The bioinformatic analysis shows SP1 is a possible transcription factor of miRNA-92b. Chromatin immunoprecipitation suggests there are three binding sites in miRNA-92b promoter regions that can be combined with SP1. qPCR suggests in PCI-4A and PCI-37A cells the expression of SP1 in experimental group (respectively was 0.064±0.020 and 0.639±0.008) were significantly lower than negative control group (both were 1)(P<0.05). In PCI-4A and PCI-37A cells the expression of miRNA-92b in experimental group (respectively was 0.215±0.033 and 0.497±0.104) were significantly lower than negative control group (both were 1)(P<0.05). In experimental group proliferation of SP1 in PCI-4A and PCI-37A cells value A were significantly lower than negative control group (P<0.05). In experimental group migration of SP1 in PCI-4A and PCI-37A cells (respectively was 37.0±4.6 and 40.7±2.1) were significantly lower than negative control group (101.0±5.3 and 82.7±5.7) (P<0.05). In experimental group invasion of SP1 in PCI-4A and PCI-37A cells (respectively was 31.3±10.8 and 37.0±4.6) were significantly lower than negative control group (92.3±3.1 and 70.3±3.1)(P<0.05).@*Conclusions@#SP1 promotes proliferation, migration and invasion abilities of HNSCC cells. SP1 is a transcription factor of miRNA-92b and can directly be involved in transcription regulation of miRNA-92b.
ABSTRACT
Objective:To investigate the expressions of miR23b and Sp1 in ovarian endometriosis and their clinic significance.Methods:qPCR was used to detect the expression of miR23b and Sp1 mRNA in paired ectopic/eutopic and normal endometrium.Immunohistochemistry and Western bolt were used to determine the expression and distribution of Sp1 in paired ectopic/eutopic and normal endometrium.The association ofmiR23b and Sp1 with the endometriosis was analyzed.Results:MiR23b mRNA expression in paired ectopic/eutopic and normal endometrium was gradually increased (P＜0.05).Sp1 protein mainly distributed in the nucleus of endometrial glandular epithelial and stromal cells,with a little or without expression in cytoplasm.Spl mRNA and protein expression in paired ectopic/eutopic and normal endometrium was gradually reduced (P＜0.05).Pearson correlation analysis showed that miR23b was negatively correlated with Sp 1 (r=-0.526,P＜0.05).Conclusion:MiR23b and Sp1 are involved in the pathogenesis of ovarian endometriosis,which may facilitate the formation of ectopic lesions.
ABSTRACT
Objective To investigate the role of Sp1 in the regulation of transcription of vascular endothelial growth factor ( VEGF ) in hypoxia-induced HepG2 cells.Methods Quantitative real-time polymerase chain reaction and immunoblot assay were used to examine the effect of hypoxia on Sp1 protein and VEGF mRNA expression .Mithramycin A, the selective inhibitor of Sp1 and knock-out Sp1 gene with siRNA were used to examine the effect on VEGF mRNA expression induced by hypoxia in HepG2 cells.Results Hypoxia induced Sp1 protein and VEGF mRNA expression in HepG2 cells.Mithramycin A produced a concentration-dependent decrease of hypoxia-induced VEGF mRNA expression . After inhibition of Sp1 RNAi, VEGF mRNA expression was significantly repressed in HepG 2 cells.Conclusion Hypoxia can increase the expression of Sp1 protein and VEGF mRNA in HepG2 cells induced by hypoxia.The transcription of VEGF is regulated by Sp1 in hypoxia-induced HepG2 cells.
ABSTRACT
Objective To investigate the role of Sp1 in the regulation of transcription of vascular endothelial growth factor ( VEGF ) in hypoxia-induced HepG2 cells.Methods Quantitative real-time polymerase chain reaction and immunoblot assay were used to examine the effect of hypoxia on Sp1 protein and VEGF mRNA expression .Mithramycin A, the selective inhibitor of Sp1 and knock-out Sp1 gene with siRNA were used to examine the effect on VEGF mRNA expression induced by hypoxia in HepG2 cells.Results Hypoxia induced Sp1 protein and VEGF mRNA expression in HepG2 cells.Mithramycin A produced a concentration-dependent decrease of hypoxia-induced VEGF mRNA expression . After inhibition of Sp1 RNAi, VEGF mRNA expression was significantly repressed in HepG 2 cells.Conclusion Hypoxia can increase the expression of Sp1 protein and VEGF mRNA in HepG2 cells induced by hypoxia.The transcription of VEGF is regulated by Sp1 in hypoxia-induced HepG2 cells.
ABSTRACT
Purpose To explore the difference of expression and prognostic significance of SP1,KLF4 and p21 in low grade ovarian serous carcinoma (LGSC) and high grade ovarian serous carcinoma (HGSC).Methods The expression of SP1,KLF4 and p21 protein was examined with immunohistochemistry EliVision method in cases with LGSC and HGSC.Kaplan-Meier analysis and Cox multivariate survival analysis were used to assess the impact of SP1,KLF4 and p21 expression on prognosis of LGSC and HGSC.Results SP1,KLF4 and p21 expression were detected respectively in 74.5％,17.0％ and 11.7％ HG-SC cases,and in 65.2％,34.8％ and 26.1％ LGSC cases.Compared to control group,the expression level of SP1 was significantly higher (P ＜ 0.05),but the expression level of KLF4 and p21 were significantly lower (P ＜0.05).There was no significant difference of SP1,KLF4 and p21 expression between HGSC and LGSC (P ＞ 0.05).The expression of SP1,KLF4 and p21 were associated with FIGO stage,meanwhile SP1 associated with residual tumor size in HGSC (P ＜ 0.05).There was a significant negative correlation between SP1 and KLF4,p21 proteins in HGSC (P ＜ 0.05).Kaplan-Meier analysis revealed that there were significantly poor overall survival (OS) of 5 years for patients with HGSC displaying high expression of SP1,or low expression of KLF4 and p21 (P ＜0.05),but no significantly improved OS for patients with LGSC (P ＞ 0.05).Cox analysis showed that SP1 overexpression is an independent prognosis factor for HGSC.Conclusion Overexpression of SP1 and low expresion of KLF4 and p21 contribute to carcinogenesis of HGSC and LGSC.They are associated with a poor prognosis of HGSC,but not LGSC,meanwhile SP1 is an independant prognosis factor for HGSC.
ABSTRACT
Objective The objective of this study was to investigate the expression and clinical significance of transcription factors Sp1 and Survivin in breast cancer and to further analyze their correlation.Methods Seventy cases of breast cancer patients were diagnosed as breast cancer from June 2013 to June 2015.The expression of Sp1 and Survivin in 70 cases of breast cancer and 20 cases of adjacent normal tissues were detected by immunohistochemical staining.The relationship was detected between the protein expression and the clinicopathological parameters of breast cancer.Results The positive rates of Sp1 and Survivin in breast cancer were 71.43% and 78.57%,respectively.The positive rates of Sp1 and Survivin were 30% and 10% in adjacent normal breast tissues.They showed a significant difference(P0.05).There were also no differences in the age,tumor size and histological grade of patients with breast cancer(P>0.05).In breast cancer tissues,there was a significant positive correlation between Sp1 and Survivin expression(r=0.517,P<0.01).Conclusion The transcription factors Sp1 and Survivin are closely related to the pathogenesis of breast cancer,which may serve as an adjunctive index for the diagnosis of breast cancer.It has certain guiding value for clinical diagnosis,treatment and prognosis of breast cancer.
ABSTRACT
SUMOylation is recently found to function as a targeting signal for the degradation of substrates through the ubiquitin-proteasome system. RNF4 is the most studied human SUMO-targeted ubiquitin E3 ligase. However, the relationship between SUMO proteases, SENPs, and RNF4 remains obscure. There are limited examples of the SENP regulation of SUMO2/3-targeted proteolysis mediated by RNF4. The present study investigated the role of SENP3 in the global protein turnover related to SUMO2/3-targeted ubiquitination and focused in particular on the SENP3 regulation of the stability of Sp1. Our data demonstrated that SENP3 impaired the global ubiquitination profile and promoted the accumulation of many proteins. Sp1, a cancer-associated transcription factor, was among these proteins. SENP3 increased the level of Sp1 protein via antagonizing the SUMO2/3-targeted ubiquitination and the consequent proteasome-dependent degradation that was mediated by RNF4. De-conjugation of SUMO2/3 by SENP3 attenuated the interaction of Sp1 with RNF4. In gastric cancer cell lines and specimens derived from patients and nude mice, the level of Sp1 was generally increased in parallel to the level of SENP3. These results provided a new explanation for the enrichment of the Sp1 protein in various cancers, and revealed a regulation of SUMO2/3 conjugated proteins whose levels may be tightly controlled by SENP3 and RNF4.
Subject(s)
Animals , Humans , Mice , Cysteine Endopeptidases , Genetics , Metabolism , Gene Expression Regulation, Neoplastic , Immunoenzyme Techniques , Immunoprecipitation , Mice, Inbred BALB C , Mice, Nude , Prognosis , Protein Processing, Post-Translational , Proteolysis , RNA, Messenger , Genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Small Ubiquitin-Related Modifier Proteins , Genetics , Metabolism , Sp1 Transcription Factor , Genetics , Metabolism , Stomach Neoplasms , Genetics , Metabolism , Pathology , Sumoylation , Tumor Cells, Cultured , Ubiquitination , Ubiquitins , Genetics , Metabolism , Xenograft Model Antitumor AssaysABSTRACT
Objective To investigate the influence of transcriptional factor Sp1 on expression of bile acids transporters MRP3 and MRP4 so as to perfect the regulatory mechanism of MRP3 and MRP4 expression.Methods Transformed Sp1-overexpression and Sp1 siRNA plas-mids to HepG2 cell and obtained the stably cells line.Then the expression levels of bile acids transporters MRP3 and MRP4 were measured by RT-qPCR,and the change of protein levels were detected by Western blot.Results The stably cells line Sp1-OE-HepG2 and Sp1siRNA-HepG2 were successfully transformed.The mRNA expression and protein levels of MRP3 and MRP4 were significantly increased in Sp1-OE-HepG2 cells,among which the mRNA expression of MRP3 mRNA increased 2.8 times,the protein levels of MRP3 increased 3.0 times,and the mRNA expression and protein levels of MRP4 increased 3.2 times and 2.5 times respectively.Conversely,the mRNA expression and protein levels of MRP3 and MRP4 were decreased in Sp1 siRNA-HepG2 cells,among which the mRNA expression of MRP3 mRNA de-creased 52%,the mRNA expression of MRP4 mRNA decreased 58%,the protein levels of MRP3 decreased 57%,and the protein levels of MRP4 decreased 60%.Conclusion Transcriptional factor Sp1 could regulate the expression of bile acids transporters MRP3 and MRP4 in HepG2 cells.
ABSTRACT
Objective To identify the expression of transcription factor Sp1 in NK/T-cell lymphoma (NK/TCL) cell lines and to investigate the role of Sp1 in regulation of cell invasion. Methods Real-time PCR, immunofluorescence and Western blot were performed to detect the expression of Sp1 in NK/TCL cell lines SNK-1 and SNK-6 and normal NK cells. Expression levels of IGF-1R and MMP-2 were measured by real-time PCR and Western blot, respectively. Transwell assay was applied to observe the effects of mythramycin A(MIT) on cell invasion. Results Sp1 expression in mRNA and protein were over-expression in NK/TCL cell lines SNK-1 and SNK-6 when compared with normal NK cells. Inhibition of Sp1 by MIT remarkably reduced expression of IGF-1R and MMP-2 in SNK-1, SNK-6 and as a result, or significantly suppressed cell invasion. Expression levels of Sp1 mRNA in SNK-1 and SNK-6 were (9.4±0.3) and (10.6±0.3) foldsincrease as compared with that of control group, respectively (P=0.005 2, P=0.003 7). Levels of Sp1 protein were (5.4±0.3) and (8.6±0.5) foldsincrease times than control groups, respectively (P=0.008 3, P=0.006 9). Inhibition of Sp1 by MIT (100 nmol/L) remarkably reduced expression levels of IGF-1R mRNA by (83.9±3.7) % and (65.8±4.2) % (P = 0.008 2, P = 0.009 7) as compared with controls. Meanwhile, levels of IGF-1R protein were reduced by (51.5±7.1) % and (49.6±9.1) % (P = 0.017 8, P = 0.015 5) as compared with control group. Inhibition of Sp1 by MIT (100 nmol/L) significantly reduced cell invasion and MMP-2 expression in the two cell lines,the cell invasion rates were reduced by (29.6±6.4) % and (37.2±7.6) % (P =0.041 8, P = 0.037 2) in SNK-1 and SNK-6 as compared with control group. The MMP-2 protein levels were found to be (52.7±4.7) % and (29.7±5.6) % (P = 0.028 6, P = 0.020 2) of control group. Conclusion Sp1 is over-expressed in NK/TCL cell lines, and it promotes NK/TCL cell invasion by up-regulating IGF-1R and further increasing MMP-2 expression.
ABSTRACT
AIM: To identify the potential elements within the survivin promoter indispensable for the upregu-lation of survivin by Kaposi’ s sarcoma-associated herpesvirus encoded replication and transcription activator ( KSHV RTA).METHODS: A series of truncated survivin promoter luciferase constructs were generated.Reporter assay and chromatin immunoprecipitation were performed to detect the interaction between RTA and the important cis-acting elements. RESULTS: Deletion of the GC/Sp1 and p53 binding sites within the survivin promoter almost completely shut down the survivin promoter activity and the p53 cis-acting element synergistically contributes to survivin promoter activation by RTA. CONCLUSION: KSHV RTA interacts with the GC/Sp1 and p53 cis-acting elements and regulates the expression of cellu-lar survivin by specifically increasing the activity of survivin promoter.