Effect of propofol on proliferation of neural stem cells in mice and the role of Sp1-EGFR-Akt signaling pathway / 中华麻醉学杂志

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.

Expression of specificity protein 1 and collagen I in primary pterygial tissues / Expressão da proteína de especificidade 1 e colágeno I em tecidos pterigiais primários

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.

Role of specificity protein 1 in transcription regulation of microRNA-92b in head and neck squamous cell carcinoma / 中华口腔医学杂志

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.

Transcription factor Sp1 in regulation of transcription of VEGF in hypoxia-induced hepatocellular cells / 军事医学

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.

Transcription factor Sp1 in regulation of transcription of VEGF in hypoxia-induced hepatocellular cells / 军事医学

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.

Expression of matrix metalloproteinase-11 in gastric cancer and its relation with transcription factor Sp1 / 肿瘤研究与临床

Objective To explore the expression of matrix metalloproteinase-11 (MMP-11) gene in gastric cancer and its correlation with transcription factor Sp1.Methods The expressions of the MMP-11 and Sp1 in level of RNA and protein in 11 specimens from the gastric cancer patients s were detected by RT-PCR,differential PCR and Western-blot,respectively.Results Electrophoresis illustration showed MMP-11 mRNA expression had significant differences in eight pairs of gastric cancer tissues and normal tissues,including 7 pairs of over expressed in gastric cancer tissues and low or no expression in normal tissues,and 1 pair of low expression in cancer tissues but high in normal tissues.The relative molecular weight of Sp1 protein was mainly 95×103 in cancer tissue and 106×103 in normal tissues.Activated MMP-11 protein mainly was over expressed in gastric cancer tissues,and low or no expression in normal tissues,but degradable MMP-1 1 protein was not observed.The enzyme prototype of and activated type of MMP-11 protein were also associated with Sp1 protein.Conclusions The MMP-11 mRNA expression in gastric tissue is higher than that in the adjacent tissues.Sp1 protein and MMP-11 protein are expressed in gastric cancer,but are low or no expression in normal tissue.There is relationship between Spl and MMP-11.

Esculetin, a Coumarin Derivative, Exhibits Anti-proliferative and Pro-apoptotic Activity in G361 Human Malignant Melanoma

BACKGROUND: Although esculetin, a coumarin compound, is known to induce apoptosis in human cancer cells, the effects and molecular mechanisms on the apoptosis in human malignant melanoma (HMM) cells are not well understood yet. In this study, we investigated the anti-proliferative effects of esculetin on the G361 HMM cells. METHODS: We analyzed the anti-proliferative effects and molecular mechanisms of esculetin on G361 cells by a 3-(4,5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, 4\',6-diamidino-2-phenylindole staining and Western blotting. RESULTS: Esculetin exhibited significant anti-proliferative effects on the HMM cells in a dose-dependent manner. Interestingly, we found that esculetin induced nuclear shrinkage and fragmentation, typical apoptosis markers, by suppression of Sp1 transcription factor (Sp1). Notably, esculetin modulated Sp1 downstream target genes including p27, p21 and cyclin D1, resulted in activation of apoptosis signaling molecules such as caspase-3 and PARP in G361 HMM cells. CONCLUSIONS: Our results clearly demonstrated that esculetin induced apoptosis in the HMM cells by downregulating Sp1 protein levels. Thus, we suggest that esculetin may be a potential anti-proliferative agent that induces apoptotic cell death in G361 HMM cells.

Role of miRNA-24 in regulation of endothelial nitric oxide synthase ex-pression and vascular endothelial cell proliferation / 中国病理生理杂志

[ABSTRACT]AIM:ToinvestigatewhethermiRNA-24isinvolvedintheregulationofendothelialnitricoxide synthase ( eNOS ) expression and vascular endothelial cell proliferation .METHODS: A plasmid that highly expressed miRNA-24 was constructed, and was transfected into the human umbilical vein endothelial cells (HUVECs) by liposome. The cell proliferation was detected by MTT assay .The expression of eNOS and Sp 1 at mRNA and protein levels was exa-mined by real-time PCR, immunohistochemistry and Western blotting .RESULTS:Compared with control group , the pro-liferation of endothelial cells in miRNA-24 group was significantly decreased by 41.97 % (0.47 ±0.04 vs 0.81 ±0.03, P<0.01), and the expression of eNOS at mRNA and protein levels was decreased by 44.8% (0.48 ±0.01 vs 0.87 ± 0.03, P<0.05) and 71.92%(0.16 ±0.06 vs 0.57 ±0.08, P<0.05), respectively.Meanwhile, the mRNA and pro-tein levels of Sp1 were significantly decreased by 53.00% (0.45 ±0.02 vs 0.93 ±0.01, P<0.05) and by 62.31%(0.13 ±0.07 vs 0.31 ±0.09, P<0.05), respectively.In miRNA-24 inhibitor group, the above indexes were decreased compared with control group , but significantly increased compared with miRNA-24 group.CONCLUSION: miRNA-24 significantly inhibits the proliferation of HUVECs and the eNOS expression .Sp1 possibly acts as one of the important factors in the regulation of eNOS expression by miRNA-24.

Progress of nuclear transcription factor Sp1 regulating angiogensis of tumor / 国际肿瘤学杂志

Sp1 is one of the nuclear transcription factors which are widely expressed in human body.It plays an important role in human growth and physiological activities,and is highly expressed in many kinds of tumors'tissue.Sp1 participates in regulating growth,invasion,angiogensis actions of tumor by regulating downstream gene(s) transcription.Some grow factors and tumor suppressor genes can regulate the expression of VEGF by Sp1,and then participate in angiogensis of tumor by two main mechanisms:kinases inducing Sp1 phosphorylation and the interact of Sp1 and other proteins.

The effect of silencing Sp1 gene on proliferation of colon carcinoma cell SW620 by RNA interference / 肿瘤研究与临床

Objective To inhibit the expression of transcription factor special protein 1(Sp1) through RNA interference (RNAi) technique and to investigate its impact on the proliferation ability of colorectal cancer cell line SW620. Methods The recombinant plasmid of Sp1 RNAi (pGenesil-1-Sp1) was constructed and transfected into SW620 cells by Lipofectamine. The transfcction efficiency was observed under fluorescence confocal microscopy. Expression levels of Sp1 mRNA and protein from SW620 after transfection were examined by real time PCR and Western blot respectively, after transduction of the recombinant plasmid into the SW620. The proliferation ability of SW620 cell line was evaluated by MTT assay. Results The expression plasmid (pGenesil-1-Sp1) against Sp1 was successfully constructed, recombinant vectors could reduce the expressions of Sp1 mRNA and protein in SW620, the ratio of inhibition of the expression of Sp1 mRNA and protein was 68.47 % and 73.82 % in 48th hour respectively. Compared with the control group, the difference was significant (P <0.05). MTT showed that the proliferation ability of SW620 cell was degraded. Conclusion Silencing Sp1 gene by the RNAi technology can actively inhibit the proliferation of SW620 cell. The successful application of Spl SiRNA extends the list of available therapeutic modalitics in the treatment of human colon cancer.

Transient downregulation of protein O-N-acetylglucosaminylation by treatment of high-dose nicotinamide in human cells

Nicotinamide at millimolar concentrations affects cell survival in various conditions, and is being utilized therapeutically in many human diseases. However, the effect of an acute treatment of nicotinamide at such high dose on gene expression and cellular metabolism has rarely been determined previously. In this study, we found that levels of O-N-acetylglucosamin(O- GlcNAc)ylated proteins including Sp1 acutely decreased upon treatment of 10 mM nicotinamide. Concomitantly, Sp1 protein level decreased rapidly through accelerated proteasome-mediated proteolysis. Cotreatment of glucosamine or 2-deoxyglucose, which inhibits protein deGlcNAcylation, effectively blocked the decrease induced by nicotinamide. Interestingly, the decline in the levels of Sp1 and protein O- GlcNAcylation was only transient lasting for two days post treatment, and this pattern matched closely the rapid fluctuation of the cellular [NAD(+)]. Our results suggest a possible link between cellular nicotinamide metabolism and protein O-GlcNAcylation, and an existence of cellular [NAD(+)] homeostasis.

Transcriptional induction of DLC-1 gene through Sp1 sites by histone deacetylase inhibitors in gastric cancer cells

We previously reported that trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, induced DLC-1 mRNA expression and accumulated acetylated histones H3 and H4 associated with the DLC-1 promoter in DLC-1 non-expressing gastric cancer cells. In this study, we demonstrated the molecular mechanisms by which TSA induced the DLC-1 gene expression. Treatment of the gastric cancer cells with TSA activates the DLC-1 promoter activity through Sp1 sites located at -219 and -174 relative to the transcription start site. Electrophoretic mobility-shift assay (EMSA) revealed that Sp1 and Sp3 specifically interact with these Sp1 sites and showed that TSA did not change their binding activities. The ectopic expression of Sp1, but not Sp3, enhances the DLC-1 promoter responsiveness by TSA. Furthermore, the TSA-induced DLC-1 promoter activity was increased by p300 expression and reduced by knockdown of p300. These results demonstrated the requirement of specific Sp1 sites and dependence of Sp1 and p300 for TSA-mediated activation of DLC-1 promoter.