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1.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 38(2): 117-122, 2021 Feb 10.
Artigo em Chinês | MEDLINE | ID: mdl-33565061

RESUMO

OBJECTIVE: To compare the mRNA level of cell proliferation-related genes Twist1, SIRT1, FGF2 and TGF-ß3 in placenta mesenchymal stem cells (PA-MSCs), umbilical cord mensenchymals (UC-MSCs) and dental pulp mesenchymal stem cells (DP-MSCs). METHODS: The morphology of various passages of PA-MSCs, UC-MSCs and DP-MSCs were observed by microscopy. Proliferation and promoting ability of the three cell lines were detected with the MTT method. Real-time PCR (RT-PCR) was used to determine the mRNA levels of Twist1, SIRT1, FGF2, TGF-ß3. RESULTS: The morphology of UC-MSCs and DP-MSCs was different from that of PA-MSCs. Proliferation ability and promoting ability of the PA-MSCs was superior to that of UC-MSCs and DP-MSCs. In PA-MSCs, expression level of Twist1 and TGF-ß3 was the highest and FGF2 was the lowest. SIRT1 was highly expressed in UC-MSCs. With the cell subcultured, different expression levels of Twist1, SIRT1, FGF2, TGF-ß3 was observed in PA-MSCs, UC-MSCs and DP-MSCs. CONCLUSION: Up-regulated expression of the Twist1, SIRT1 and TGF-ß3 genes can promote proliferation of PA-MSCs, UC-MSCs and DP-MSCs, whilst TGF-ß3 may inhibit these. The regulatory effect of Twist1, SIRT1, FGF2 and TGF-ß3 genes on PA-MSCs, UC-MSCs and DP-MSCs are different.


Assuntos
Proliferação de Células/genética , Fator 2 de Crescimento de Fibroblastos/genética , Células-Tronco Mesenquimais/citologia , Proteínas Nucleares/genética , Sirtuína 1/genética , Fator de Crescimento Transformador beta3/genética , Proteína 1 Relacionada a Twist/genética , Diferenciação Celular , Células Cultivadas , Polpa Dentária/citologia , Feminino , Humanos , Placenta/citologia , Gravidez , Cordão Umbilical/citologia
2.
Zhongguo Zhong Yao Za Zhi ; 45(19): 4699-4704, 2020 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-33164435

RESUMO

To explore the effect of slient mating type information regulation 2 homolog 1(SIRT1) on the delay of D-galactose(D-gal) induced premature ovarian failure in mice with ginsenoside Rg_1(Rg_1). Fifty-four female SPF BALB/c mice were randomly divided into PBS group, D-gal group, and Rg_1 group. In the D-gal group, D-galactose(200 mg·kg~(-1)·d~(-1)) was injected subcutaneously into the neck and back for 42 days. In the PBS group, the equal amount of phosphate buffered saline(PBS) was injected into the neck and back for 42 days. In addition to the therapy of D-gal group, Rg_1 group was given Rg_1(20 mg·kg~(-1)·d~(-1)) through the intraperitoneal injection on the 15 th day for 28 days. At the same time, the D-gal group and the PBS group were also given an equal amount of PBS through hintraperitoneal injection on the 15 th day for 28 days. The changes in body weight and ovarian weight coefficient of mice were detected. Expressions of estradiol 2(E_2), luteinizing hormone(LH), superoxide dismutase(SOD), catalase(CAT) and follicle-stimulating hormone(FSH) in peripheral blood were detected. Morphological changes of ovaries were detected by HE staining. Changes in expression of aging regulator SIRT1 were detected by fluorescent quantitative PCR and Western blot. The results showed that compared with the PBS group, the body weight growth rate of the D-gal group was significantly slowed, the ovarian weight coefficient was decreased, the serum levels of E_2, LH, SOD, CAT were significantly reduced, and FSH was significantly increased. After the administration with Rg_1, the body weight growth rate, ovarian weight coefficient, serum levels of E_2, LH, SOD, and CAT in the mice were higher than those in the D-gal group, while FSH was lower than those in the D-gal group. HE staining showed that the follicular morphology and structure of the PBS group were normal; the number of follicles in the D-gal group was reduced, the corpus luteum was vacuolated, and the number of atretic follicles was increased. Compared with the D-gal group, the number of follicles in the Rg_1 group was increased, whereas the number of corpus luteum was decreased. Compared with the PBS group, SIRT1 expression was down-regulated in the D-gal group, while SIRT1 expression was up-regulated in the Rg_1 group compared with the D-gal group. The results suggested that Rg_1 could delay D-gal-induced premature ovarian failure in a mouse model of premature ovarian failure, and SIRT1 played an important role in this.


Assuntos
Ginsenosídeos , Insuficiência Ovariana Primária , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Insuficiência Ovariana Primária/induzido quimicamente , Insuficiência Ovariana Primária/tratamento farmacológico , Insuficiência Ovariana Primária/genética , Sirtuína 1/genética
3.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 36(9): 788-793, 2020 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-32967762

RESUMO

Objective To investigate SIRT1-PGC-1α signaling pathway-mediated effect of hyperoxia on mitochondrial function in A549 human alveolar epithelial cells and its possible mechanism. Methods Human alveolar epithelial cells in logarithmic growth phase were randomly divided into control group and hyperoxia group. The control group was cultured in a 37DegreesCelsius, 50 mL/L CO2 saturated humidity incubator, and the hyperoxia group was treated with 950 mL/L O2. Following 24-hour culture, Mito SOXTM staining was used to detect the level of mitochondrial reactive oxygen species (Mito-ROS) and JC-1 staining to detect the mitochondrial membrane potential. Real-time quantitative PCR was performed to detect the mitochondrial DNA content and the mRNA levels of SIRT1, PGC-1α, nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM), and Western blotting to detect the protein levels of SIRT1, PGC-1α, NRF1 and TFAM. Results Compared with the control group, the Mito-ROS of the hyperoxia group increased significantly, while the membrane potential decreased obviously; the mitochondrial DNA content of the hyperoxia group went down, and the mRNA and protein expression of SIRT1, PGC-1α, NRF1 and TFAM dropped. Conclusion Hyperoxia induces mitochondrial dysfunction in human alveolar epithelial cells by inhibiting the expression of SIRT1 and PGC-1α.


Assuntos
Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Sirtuína 1/genética , Células Epiteliais Alveolares/metabolismo , Hipóxia Celular , Linhagem Celular , Regulação para Baixo , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Sirtuína 1/metabolismo
4.
Life Sci ; 257: 118055, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32634429

RESUMO

AIMS: Human adipose derived mesenchymal stem cells (hAD-MSCs) as the most promising target for cell therapy and regenerative medicine, face senescence as a major drawback resulting in their limited proliferation and differentiation potentials. To evaluate the efficacy of miR-34a silencing as an anti-senescence strategy in hAD-MSCs, in this study common hallmarks of senescence were assessed after transient inhibition of miR-34a in hAD-MSCs. MATERIALS AND METHODS: The expression levels of miR-34a in hAD-MSCs at different passages were evaluated by real-time quantitative PCR. hAD-MSCs at passage 2 and passage 7 were transfected with miR-34a inhibitor. Doubling time assay, colony forming assay, and cell cycle analysis were performed to evaluate cell proliferation rate. The activity of senescence associated ß-galactosidase (SA-ß-gal) was assessed by histochemical staining. Moreover, the senescence associated molecular alterations including that of pro-senescence (P53, P21 and P16) and anti-senescence (SIRT1, HTERT and CD44) genes were examined by quantitative RT-PCR and western blot assays. To evaluate the differentiation potentials of MSCs, following adipogenic and osteogenic induction, the expression levels of lineage specific markers were analyzed by qPCR. KEY FINDINGS: Our results showed that inhibition of miR-34a enhances the proliferation, promotes the adipogenic and osteogenic differentiation potency, reduces the senescence associated-ß gal activity, and reverses the senescence associated molecular alterations in hAD-MSCs. SIGNIFICANCE: In this study, we showed that inhibition of miR-34a reduces the cellular senescence through the activation of SIRT1. Our findings support the silencing of miR-34a as an anti-senescence approach to improve the therapeutic potentials of hAD-MSCs.


Assuntos
Diferenciação Celular/fisiologia , Senescência Celular/fisiologia , Células-Tronco Mesenquimais/citologia , MicroRNAs/genética , Sirtuína 1/genética , Adipogenia/fisiologia , Tecido Adiposo/citologia , Inativação Gênica , Humanos , Receptores de Hialuronatos/genética , Osteogênese/fisiologia , Telomerase/genética
5.
Life Sci ; 256: 117898, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32522566

RESUMO

BACKGROUND: Atherosclerosis as a progressive inflammatory disease is the main cause of Coronary Artery Disease (CAD). Multiple genetic and environmental factors are involved in susceptibility to atherosclerotic vascular diseases. FOXO1 gene acts as a key molecular proinflammatory transcription factor and the FBOX32 gene as an F-box protein plays pivotal roles in regulation of muscle atrophy and inhibition of the pathologic cardiac hypertrophy. MiR-27a has been reported to contribute to atherosclerosis prevention and the inflammatory processes of atherosclerosis. MicroRNA-23a has been found to promote atherosclerotic plaque progression and vulnerability. Hence, given the importance of these subjects, the present study was carried out to investigate the expression levels of the desired genes. METHODOLOGY: In this case-control study, 82 patients with CAD and 80 healthy controls were investigated. Expression levels of miRNAs -27a and 23a, FOXO1, Sirtuin 1 (SIRT1) in the Peripheral Blood Mononuclear Cells (PBMCs), serum concentration of IL6 and TNF-α of the studied subjects were evaluated using the real-time Polymerase Chain Reaction (PCR) technique. The correlation between the variables was also investigated. RESULTS: Results of the study demonstrated that expression of FOXO1, IL-6, TNF-α, miR-27a, and miR-23a increased in the PBMCs of the patients with CAD and their expression levels were significantly correlated with the severity of stenosis. A significant decrease was observed in the expression of SIRT1 in the patients with CAD compared to the healthy controls. Furthermore, the Receiver Operating Characteristic (ROC) curve was plotted to find the effectiveness of FOXO1 and miRNA-27a gene expression as a diagnostic marker for CAD. CONCLUSIONS: Findings of the study suggested that miRs-27a and FOXO1 genes have a potential role in the progression of atherosclerosis and mediate the molecular and genetic disturbances of the intracellular communication in the atherosclerosis.


Assuntos
Doença da Artéria Coronariana/sangue , Citocinas/sangue , Proteína Forkhead Box O1/metabolismo , Regulação da Expressão Gênica , Mediadores da Inflamação/sangue , Leucócitos Mononucleares/metabolismo , MicroRNAs/sangue , Estudos de Casos e Controles , Doença da Artéria Coronariana/genética , Feminino , Proteína Forkhead Box O1/genética , Humanos , Modelos Lineares , Masculino , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Sirtuína 1/genética , Sirtuína 1/metabolismo
6.
J Biol Regul Homeost Agents ; 34(2): 379-391, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32517436

RESUMO

Chondrocyte apoptosis is linked to cartilage degeneration, and considered as a crucial event during the development of osteoarthritis (OA). X inactive specific transcript (XIST) is an oncogenic long non-coding RNA (lncRNA). However, its role in the pathophysiological process of OA remains largely unknown. In this work, quantitative real-time reverse transcriptase PCR (qRT-PCR) was employed to measure the expression of XIST, miR-653-5p and sirtuin1 (SIRT1) mRNA in OA and normal cartilage tissues. Chondrocyte cell lines, CHON-001 and ATDC5, were treated with different doses of interleukin- 1ß (IL-1ß) to mimic the inflammatory environment of OA in vitro. Overexpression plasmids, microRNA (miRNA) mimics, miRNA inhibitors and small interfering RNAs (siRNAs) were constructed and transfected into CHON-001 and ATDC5 cells. 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay was adopted to determine the cell viability. Western blot was used to detect the expression of apoptosis-related proteins. Enzyme-linked immunosorbent assay (ELISA) was employed to probe the expression levels of inflammatory factors. Flow cytometry was used to analyze the cell apoptosis. StarBase and TargetScan databases were used to predict the binding sites between XIST and miR-653-5p, miR-653-5p and 3'UTR of SIRT1, respectively, which were then verified by dual luciferase reporter assay. The data in the present study demonstrated that XIST and SIRT1 were down-regulated while miR-653-5p was up-regulated in OA tissues and cell models. The up-regulation of XIST increased the viability of CHON-001 and ATDC5 cells, while it impeded their apoptosis and inflammatory response induced by IL-1ß. Conversely, miR-653-5p had opposite effects. It was proved that miR-653-5p could be sponged and suppressed by XIST. Additionally, SIRT1 was identified as a target of miR-653-5p, and SIRT1 could be suppressed by XIST indirectly. In conclusion, down-regulated XIST was involved in the injury of chondrocytes during the pathophysiological process of OA, and XIST up-regulation protected chondrocytes from inflammatory injury via regulating miR-653-5p/SIRT1 axis.


Assuntos
Apoptose , Condrócitos/citologia , MicroRNAs/genética , RNA Longo não Codificante/genética , Sirtuína 1/genética , Animais , Linhagem Celular , Humanos , Interleucina-1beta/farmacologia , Camundongos , Osteoartrite
7.
Oncogene ; 39(27): 5056-5067, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32518374

RESUMO

Williams syndrome transcription factor (WSTF) is a transcription factor and tyrosine kinase. WSTF overexpression promotes migration and proliferation of various cancers, and Ser158 (WSTFS158) phosphorylation plays an important role in this process. However, the role of the other posttranslational modifications of WSTF is unknown. Here, we report that lysine (K) 426 on WSTF is acetylated by MOF and deacetylated by SIRT1. Mechanistically, male-specific lethal (MSL) 1v1 interaction with WSTF facilitates its interaction with MOF for WSTF acetylation, which in turn promotes WSTFS158 phosphorylation. The kinase and transcriptional regulatory activity of WSTF were enhanced by acetylation. WSTFK426ac levels positively and significantly correlated with tumor size, histological grade, and age. Moreover, we demonstrated that acetylated WSTF promotes cancer cell proliferation, migration, invasion, and tumor formation. In conclusion, we identified the enzymes regulating WSTF K426 acetylation, and demonstrated an acetylation-dependent mechanism that modulates the activities of WSTF and contributes to tumorigenesis. Our findings provide new clues to study WSTF-mediated normal development and disease.


Assuntos
Carcinogênese/patologia , Histona Acetiltransferases/metabolismo , Neoplasias/patologia , Fatores de Transcrição/metabolismo , Acetilação , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Feminino , Regulação da Expressão Gênica , Células HEK293 , Histona Acetiltransferases/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica/genética , Transplante de Neoplasias , Fosforilação , Processamento de Proteína Pós-Traducional , Interferência de RNA , RNA Interferente Pequeno/genética , Sirtuína 1/genética , Sirtuína 1/metabolismo , Transplante Heterólogo
8.
Anticancer Res ; 40(6): 3155-3161, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32487610

RESUMO

BACKGROUND/AIM: The deacetylase sirtuin1 (SIRT1) inhibits tumor suppressor p53 and may promote tumorigenesis; however, SIRT1 effects on leukemia cells are controversial. The aim of this study was to clarify the activity of SIRT1 in leukemia cells. MATERIALS AND METHODS: The effects of SIRT1 inhibition or activation and SIRT1 knockdown or overexpression were examined in two T cell acute lymphoblastic leukemia (T-ALL) cell lines carrying NOTCH1 mutations and three acute myeloid leukemia (AML) cell lines. RESULTS: The growth of T-ALL cells was promoted by SIRT1 inhibition and SIRT1 knockdown but was reduced by SIRT1 activation and overexpression; however, no effects were observed in AML cells. SIRT1 activation decreased NOTCH, NF-κB, and mTOR signaling and inhibited p53, suggesting that the possible mechanisms of T-ALL growth suppression by SIRT1 are independent of p53. CONCLUSION: SIRT1 activators acting through the down-regulation of NOTCH, NF-κB, and mTOR pathways can be novel targeted drugs for NOTCH1-mutated T-ALLs.


Assuntos
NF-kappa B/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Receptores Notch/metabolismo , Sirtuína 1/metabolismo , Carbazóis/farmacologia , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , Mutação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Receptor Notch1/genética , Receptor Notch1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/biossíntese , Sirtuína 1/genética , Serina-Treonina Quinases TOR/metabolismo , Transfecção
9.
Mol Pharmacol ; 98(2): 88-95, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32487734

RESUMO

Arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic-metabolizing enzyme that also has a role in cancer cell growth and metabolism. Recently, it was reported that NAT1 undergoes lysine acetylation, an important post-translational modification that can regulate protein function. In the current study, we use site-directed mutagenesis to identify K100 and K188 as major sites of lysine acetylation in the NAT1 protein. Acetylation of ectopically expressed NAT1 in HeLa cells was decreased by C646, an inhibitor of the protein acetyltransferases p300/CREB-binding protein (CBP). Recombinant p300 directly acetylated NAT1 in vitro. Acetylation of NAT1 was enhanced by the sirtuin (SIRT) inhibitor nicotinamide but not by the histone deacetylase inhibitor trichostatin A. Cotransfection of cells with NAT1 and either SIRT 1 or 2, but not SIRT3, significantly decreased NAT1 acetylation. NAT1 activity was evaluated in cells after nicotinamide treatment to enhance acetylation or cotransfection with SIRT1 to inhibit acetylation. The results indicated that NAT1 acetylation impaired its enzyme kinetics, suggesting decreased acetyl coenzyme A binding. In addition, acetylation attenuated the allosteric effects of ATP on NAT1. Taken together, this study shows that NAT1 is acetylated by p300/CBP in situ and is deacetylated by the sirtuins SIRT1 and 2. It is hypothesized that post-translational modification of NAT1 by acetylation at K100 and K188 may modulate NAT1 effects in cells. SIGNIFICANCE STATEMENT: There is growing evidence that arylamine N-acetyltransferase 1 has an important cellular role in addition to xenobiotic metabolism. Here, we show that NAT1 is acetylated at K100 and K188 and that changes in protein acetylation equilibrium can modulate its activity in cells.


Assuntos
Arilamina N-Acetiltransferase/química , Arilamina N-Acetiltransferase/metabolismo , Proteína de Ligação a CREB/genética , Proteína p300 Associada a E1A/genética , Isoenzimas/química , Isoenzimas/metabolismo , Sirtuína 1/genética , Sirtuína 2/genética , Acetilcoenzima A/metabolismo , Acetilação/efeitos dos fármacos , Arilamina N-Acetiltransferase/genética , Benzoatos/farmacologia , Proteína de Ligação a CREB/metabolismo , Cristalografia por Raios X , Proteína p300 Associada a E1A/metabolismo , Células HeLa , Humanos , Ácidos Hidroxâmicos/farmacologia , Isoenzimas/genética , Lisina/química , Lisina/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Niacinamida/farmacologia , Conformação Proteica , Pirazóis/farmacologia , Sirtuína 1/metabolismo , Sirtuína 2/metabolismo , Transfecção
10.
J Biol Regul Homeost Agents ; 34(3): 435-443, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32529818

RESUMO

This study explored the effects of propofol on the cognitive function and expressions of superoxide dismutase (SOD) and malondialdehyde (MDA) through the silent information regulator 1 (SIRT1) signaling pathway during cerebral ischemia-reperfusion (I/R) injury. C57BL/6J mice were used and divided into Sham group, I/R group (I/R model established via ligation of artery) and Treated group (peritoneal injection of propofol) according to different treatments. The memory ability of mice was evaluated using Morris water maze test, and the motor coordination was assessed using Rota rod test and oblique beam walking test. The brain tissues were prepared into embedded sections, and then the pathological changes in brain neurons were detected via hematoxylin-eosin (HE) staining, and the changes in apoptosis of brain tissues were detected via flow cytometry. Moreover, after the mice were anesthetized and sacrificed, the brain tissues were isolated and whole blood was collected. Then the changes in SIRT1 protein were determined using Western blotting, and the changes in MDA and SOD activity were determined through biochemical assays. The results of Morris water maze test and elevated plus-maze test revealed that transfer latency time (TLT) was significantly prolonged, and escape latency time (ELT) was significantly shortened in the I/R group compared with those in Sham group (*P<0.05), indicating memory impairment after cerebral I/R injury. TLT was shortened, and ELT was significantly prolonged in the Treated group compared with those in I/R group (#P<0.05). In Rota rod test, the falling down time was obviously shorter in the I/R group than in the Sham group (*P<0.05), while it was obviously longer in the Treated group than that in the I/R group (#P<0.05). Compared with the Sham group, the I/R group had neurological impairment, manifested as the evident increase in motor performance score (*P<0.05), and the motor performance score in the Treated group was evidently lower than that in the I/R group (#P<0.05). The apoptosis was markedly enhanced in the I/R group (*P<0.05), while it was markedly weakened in the Treated group (#P<0.05) compared with that in the Sham group. In addition, the results of Western blotting showed that the expression of SIRT1 was evidently higher in I the /R group than that in the Sham group, while it evidently declined after treatment with propofol (#P<0.05).


Assuntos
Anestésicos Intravenosos , Propofol , Traumatismo por Reperfusão , Sirtuína 1 , Anestésicos Intravenosos/farmacologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Propofol/farmacologia , Ratos Sprague-Dawley , Traumatismo por Reperfusão/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/genética , Sirtuína 1/metabolismo
11.
Cardiovasc Drugs Ther ; 34(5): 641-650, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32564302

RESUMO

PURPOSE: Advancing age is the major risk factor for thoracic aortic aneurysm/dissection (TAAD). However, the causative link between age-related molecules and TAAD remains elusive. Here, we investigated the role of Sirtuin 1 (SIRT1, also known as class III histone deacetylase), the best studied member of the longevity-related Sirtuin family, in TAAD development in vivo. METHODS: We used male smooth muscle-specific SIRT1 transgenic (ST-Tg) mice, smooth muscle-specific SIRT1 knockout (ST-KO) mice, and their wild-type (WT) littermates on a C57BL/6J background to establish a TAAD model induced by oral administration of 3-aminopropionitrile fumarate (BAPN). We analyzed the incidence and fatality rates of TAAD in the groups. We examined matrix metallopeptidase 2 (MMP2) and MMP9 expression in aortas or cultured A7r5 cells via western blotting and real-time polymerase chain reaction (PCR). We performed chromatin immunoprecipitation (ChIP) to clarify the epigenetic mechanism of SIRT1-regulated MMP2 expression in vascular smooth muscle cells (VSMCs). RESULTS: BAPN treatment markedly increased the incidence of TAAD in WT mice but caused less disease in ST-Tg mice. Moreover, ST-KO mice had the highest BAPN-induced TAAD fatality rate of all the groups. Mechanistically, SIRT1 overexpression resulted in lower MMP2 and MMP9 expression after BAPN treatment in both mouse aortas and cultured A7r5 cells. The downregulation of BAPN-induced MMP2 expression by SIRT1 was mediated by deacetylation of histone H3 lysine 9 (H3K9) on the Mmp2 promoter in the A7r5 cells. CONCLUSION: Our findings suggest that SIRT1 expression in SMCs protects against TAAD and could be a novel therapeutic target for TAAD management.


Assuntos
Aneurisma Dissecante/prevenção & controle , Aneurisma da Aorta Torácica/prevenção & controle , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Sirtuína 1/metabolismo , Acetilação , Aneurisma Dissecante/enzimologia , Aneurisma Dissecante/genética , Aneurisma Dissecante/patologia , Animais , Aorta Torácica/enzimologia , Aorta Torácica/patologia , Aneurisma da Aorta Torácica/enzimologia , Aneurisma da Aorta Torácica/genética , Aneurisma da Aorta Torácica/patologia , Linhagem Celular , Modelos Animais de Doenças , Histonas/metabolismo , Masculino , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Transdução de Sinais , Sirtuína 1/genética
12.
Mol Cell Endocrinol ; 515: 110917, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32593740

RESUMO

Obesity patients are more susceptible to develop COVID-19 severe outcome due to the role of angiotensin-converting enzyme 2 (ACE2) in the viral infection. ACE2 is regulated in the human cells by different genes associated with increased (TLR3, HAT1, HDAC2, KDM5B, SIRT1, RAB1A, FURIN and ADAM10) or decreased (TRIB3) virus replication. RNA-seq data revealed 14857 genes expressed in human subcutaneous adipocytes, including genes mentioned above. Irisin treatment increased by 3-fold the levels of TRIB3 transcript and decreased the levels of other genes. The decrease in FURIN and ADAM10 expression enriched diverse biological processes, including extracellular structure organization. Our results, in human subcutaneous adipocytes cell culture, indicate a positive effect of irisin on the expression of multiple genes related to viral infection by SARS-CoV-2; furthermore, translatable for other tissues and organs targeted by the novel coronavirus and present, thus, promising approaches for the treatment of COVID-19 infection as therapeutic strategy to decrease ACE2 regulatory genes.


Assuntos
Adipócitos/efeitos dos fármacos , Fibronectinas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína ADAM10/genética , Proteína ADAM10/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Betacoronavirus/genética , Betacoronavirus/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Infecções por Coronavirus/virologia , Fibronectinas/genética , Fibronectinas/metabolismo , Furina/genética , Furina/metabolismo , Ontologia Genética , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histona Desacetilase 2/genética , Histona Desacetilase 2/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Biológicos , Anotação de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Obesidade/virologia , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Sirtuína 1/genética , Sirtuína 1/metabolismo , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismo , Proteínas rab1 de Ligação ao GTP/genética , Proteínas rab1 de Ligação ao GTP/metabolismo
13.
BMC Bioinformatics ; 21(1): 186, 2020 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-32410570

RESUMO

BACKGROUND: Continuous enzyme kinetic assays are often used in high-throughput applications, as they allow rapid acquisition of large amounts of kinetic data and increased confidence compared to discontinuous assays. However, data analysis is often rate-limiting in high-throughput enzyme assays, as manual inspection and selection of a linear range from individual kinetic traces is cumbersome and prone to user error and bias. Currently available software programs are specialized and designed for the analysis of complex enzymatic models. Despite the widespread use of initial rate determination for processing kinetic data sets, no simple and automated program existed for rapid analysis of initial rates from continuous enzyme kinetic traces. RESULTS: An Interactive Continuous Enzyme Kinetics Analysis Tool (ICEKAT) was developed for semi-automated calculation of initial rates from continuous enzyme kinetic traces with particular application to the evaluation of Michaelis-Menten and EC50/IC50 kinetic parameters, as well as the results of high-throughput screening assays. ICEKAT allows users to interactively fit kinetic traces using convenient browser-based selection tools, ameliorating tedious steps involved in defining ranges to fit in general purpose programs like Microsoft Excel and Graphpad Prism, while still maintaining simplicity in determining initial rates. As a test case, we quickly analyzed over 500 continuous enzyme kinetic traces resulting from experimental data on the response of the protein lysine deacetylase SIRT1 to small-molecule activators. CONCLUSIONS: ICEKAT allows simultaneous visualization of individual initial rate fits and the resulting Michaelis-Menten or EC50/IC50 kinetic model fits, as well as hits from high-throughput screening assays. In addition to serving as a convenient program for practicing enzymologists, ICEKAT is also a useful teaching aid to visually demonstrate in real-time how incorrect initial rate fits can affect calculated Michaelis-Menten or EC50/IC50 kinetic parameters. For the convenience of the research community, we have made ICEKAT freely available online at https://icekat.herokuapp.com/icekat.


Assuntos
Enzimas/metabolismo , Sistemas On-Line , Software , Algoritmos , Concentração Inibidora 50 , Cinética , Sirtuína 1/genética
14.
J Biomed Sci ; 27(1): 61, 2020 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-32381096

RESUMO

BACKGROUND: The disruption of the blood-brain barrier (BBB) plays a critical event in the pathogenesis of ischemia stroke. TGR5 is recognized as a potential target for the treatment for neurologic disorders. METHODS: This study investigated the roles of TGR5 activation in attenuating BBB damage and underlying mechanisms after middle cerebral artery occlusion (MCAO). Sprague-Dawley rats were subjected to model of MCAO and TGR5 agonist, INT777, was administered intranasally. Small interfering RNA (siRNA) for TGR5 and BRCA1 were administered through intracerebroventricular injection 48 h before MCAO. Infarct volumes, brain water content, BBB permeability, neurological scores, Western blot, immunofluorescence staining and co- immunoprecipitation were evaluated. RESULTS: Endogenous TGR5 and BRCA1 were upregulated in the injured hemisphere after MCAO and TGR5 expressed in endothelial cells. Treatment with INT777 alleviated brain water content and BBB permeability, reduced infarction volume and improved neurological scores at 24 h and 72 h after ischemia. INT777 administration increased BRCA1 and Sirt1 expression, as well as upregulated expressions of tight junction proteins. Ischemic damage induced interaction of TGR5 with BRCA1. TGR5 siRNA and BRCA1 siRNA significantly inhibited expressions of BRCA1 and Sirt1, aggravated BBB permeability and exacerbated stroke outcomes after MCAO. The protective effects of INT777 at 24 h after MCAO were also abolished by TGR5 siRNA or BRCA1 siRNA. CONCLUSIONS: Our findings demonstrate that activating TGR5 could reduce BBB breakdown and improve neurological functions through BRCA1/Sirt1 signaling pathway after MCAO. TGR5 may serve as a potential new candidate to relieve brain injury after MCAO.


Assuntos
Barreira Hematoencefálica/fisiologia , Infarto da Artéria Cerebral Média/patologia , Receptores Acoplados a Proteínas-G/genética , Transdução de Sinais/genética , Animais , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas-G/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo
15.
Lab Invest ; 100(9): 1223-1237, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32461588

RESUMO

MicroRNAs regulate gene expression at the posttranscriptional level, and this process has been shown to be implicated in the pathological processes of temporal lobe epilepsy. At present, studies about the impact of microRNA-181a (miR-181a) on epilepsy have focused on hippocampal neurons, and the effect of miR-181a on other cells in the hippocampus remains poorly understood. Herein, we explored the role of miR-181a-5p in a lithium-pilocarpine model of epilepticus in immature rats. We found that the hippocampal expression level of miR-181a-5p was increased. Inhibition of miR-181a-5p protected the hippocampus against epilepsy, including hippocampal insults, neuronal apoptosis, astrocyte and microglia activation, neuroinflammation, oxidative stress, mitochondrial function, and cognitive dysfunction. Moreover, miR-181a-5p inhibition exerted a seizure-suppressing effect via SIRT1 upregulation. Overall, our findings reveal the potential role of the miR-181a-5p/SIRT1 pathway in the development of temporal lobe epilepsy, and this pathway may represent a novel target for ameliorating epilepsy and its sequelae.


Assuntos
Astrócitos/metabolismo , Epilepsia do Lobo Temporal/genética , MicroRNAs/genética , Estresse Oxidativo , Sirtuína 1/genética , Fatores Etários , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Epilepsia do Lobo Temporal/induzido quimicamente , Epilepsia do Lobo Temporal/metabolismo , Regulação da Expressão Gênica , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Masculino , Microglia/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Pilocarpina , Ratos Sprague-Dawley , Sirtuína 1/metabolismo
16.
Nature ; 583(7814): 139-144, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32461691

RESUMO

MicroRNAs (miRNAs) regulate the levels of translation of messenger RNAs (mRNAs). At present, the major parameter that can explain the selection of the target mRNA and the efficiency of translation repression is the base pairing between the 'seed' region of the miRNA and its counterpart mRNA1. Here we use R1ρ relaxation-dispersion nuclear magnetic resonance2 and molecular simulations3 to reveal a dynamic switch-based on the rearrangement of a single base pair in the miRNA-mRNA duplex-that elongates a weak five-base-pair seed to a complete seven-base-pair seed. This switch also causes coaxial stacking of the seed and supplementary helix fitting into human Argonaute 2 protein (Ago2), reminiscent of an active state in prokaryotic Ago4,5. Stabilizing this transient state leads to enhanced repression of the target mRNA in cells, revealing the importance of this miRNA-mRNA structure. Our observations tie together previous findings regarding the stepwise miRNA targeting process from an initial 'screening' state to an 'active' state, and unveil the role of the RNA duplex beyond the seed in Ago2.


Assuntos
Pareamento de Bases , MicroRNAs/genética , RNA Mensageiro/genética , Sirtuína 1/genética , Proteínas Argonauta/metabolismo , Sítios de Ligação , Células HEK293 , Humanos , Modelos Moleculares , Complexo de Inativação Induzido por RNA/metabolismo
18.
Life Sci ; 255: 117849, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32473249

RESUMO

AIMS: The associations between colorectal neoplasia differentially expressed (CRNDE), a novel long non-coding RNA (lncRNA), and inflammation and cell apoptosis have been underscored recently. However, its function in sepsis-induced myocardial injury remains undetermined. The current study sets to examine the putative mechanism of CRNDE in myocardial injury evoked by sepsis. MATERIALS AND METHODS: Firstly, the rat model of sepsis was developed and verified. Subsequently, differentially expressed lncRNAs in myocardial tissues of septic rats were determined. Afterwards, CRNDE overexpression or knockdown was introduced into the myocardial tissues of rats. Then, H9c2 cells were induced by lipopolysaccharide (LPS) and transfected with overexpression of CRNDE and sirtuin 1 (SIRT1), si-CRNDE or microRNA (miR)-29a mimic. Apoptosis, Caspase-3 activity, secretion of inflammatory factors, and intracellular reactive oxygen species (ROS) content were subsequently measured in rat tissues and transfected cells. Finally, the NF-κB/PARP-1 signaling activity in rat myocardial tissues and cells was detected. KEY FINDINGS: CRNDE expression was reduced in the myocardial tissues of rats with sepsis, and CRNDE restoration alleviated following myocardial injury. Additionally, overexpression of CRNDE inhibited cardiomyocyte apoptosis, ROS content, Caspase-3 activity, nuclear NF-κB p65 phosphorylation and PARP-1 expression. Dual-luciferase assays showed that the CRNDE/miR-29a/SIRT1 network regulated sepsis-induced myocardial injury. Moreover, miR-29a mimic attenuated the protective effect of CRNDE overexpression on LPS-induced cardiomyocytes. SIGNIFICANCE: CRNDE protects the myocardial tissues against sepsis-induced cardiomyocyte apoptosis and oxidative damage by inhibiting the post-transcriptional regulatory function of miR-29a on SIRT1.


Assuntos
MicroRNAs/genética , Miocárdio/patologia , RNA Longo não Codificante/genética , Sepse/fisiopatologia , Sirtuína 1/genética , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Inflamação/genética , Inflamação/patologia , Lipopolissacarídeos , Masculino , Miócitos Cardíacos/patologia , NF-kappa B/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Sepse/genética
19.
BMC Cardiovasc Disord ; 20(1): 240, 2020 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-32434515

RESUMO

BACKGROUND: This study aimed to investigate the regulatory effect of rno-microRNA-30c-5p (rno-miR-30c-5p) on myocardial ischemia reperfusion (IR) injury in rats and the underlying molecular mechanisms. METHODS: A rat model of myocardial IR injury was established. The infarct size was detected by 2,3,5-triphenyltetrazolium chloride staining. The pathologic changes of myocardial tissues were detected by hematoxylin-eosin staining. The apoptosis of myocardial cells was measured by TUNEL staining and flow cytometry. The mRNA expression of rno-miR-30c-5p and Sirtuin 1 (SIRT1) was detected by quantitative real-time PCR. The levels of IL-1ß, IL-6 and TNF-α were detected by enzyme linked immunosorbent assay. The protein expression of Bax, Bcl-2, caspase-3, p-IκBα, IκBα, p-NF-κB p65, NF-κB p65 and SIRT1 was detected by Western blot. The interaction between rno-miR-30c-5p and SIRT1 was predicted by TargetScan, and further identified by dual luciferase reporter gene and RNA immunoprecipitation assay. RESULTS: The myocardial IR injury model was successfully established in rats. IR induced the myocardial injury in rats and increased the expression of rno-miR-30c-5p. Overexpression of rno-miR-30c-5p enhanced the inflammation, promoted the apoptosis, and activated NF-κB pathway in IR myocardial cells. SIRT1 was the target gene of rno-miR-30c-5p. Silencing of SIRT1 reversed the effects of rno-miR-30c-5p inhibitor on the apoptosis and NF-κB pathway in IR myocardial cells. CONCLUSIONS: Rno-miR-30c-5p promoted the myocardial IR injury in rats through activating NF-κB pathway and down-regulating SIRT1.


Assuntos
MicroRNAs/metabolismo , Infarto do Miocárdio/enzimologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Miocárdio/enzimologia , NF-kappa B/metabolismo , Sirtuína 1/metabolismo , Animais , Apoptose , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Mediadores da Inflamação/metabolismo , Masculino , MicroRNAs/genética , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , Ratos Sprague-Dawley , Transdução de Sinais , Sirtuína 1/genética
20.
J Biomed Sci ; 27(1): 68, 2020 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-32446297

RESUMO

BACKGROUND: Tissue oxidative stress, sympathetic activation and nutrient sensing signals are closely related to adult hypertension of fetal origin, although their interactions in hypertension programming remain unclear. Based on a maternal high-fructose diet (HFD) model of programmed hypertension, we tested the hypothesis that dysfunction of AMP-activated protein kinase (AMPK)-regulated angiotensin type 1 receptor (AT1R) expression and sirtuin1 (SIRT1)-dependent mitochondrial biogenesis contribute to tissue oxidative stress and sympathoexcitation in programmed hypertension of young offspring. METHODS: Pregnant female rats were randomly assigned to receive normal diet (ND) or HFD (60% fructose) chow during pregnancy and lactation. Both ND and HFD offspring returned to ND chow after weaning, and blood pressure (BP) was monitored from age 6 to 12 weeks. At age of 8 weeks, ND and HFD offspring received oral administration of simvastatin or metformin; or brain microinfusion of losartan. BP was monitored under conscious condition by the tail-cuff method. Nutrient sensing molecules, AT1R, subunits of NADPH oxidase, mitochondrial biogenesis markers in rostral ventrolateral medulla (RVLM) were measured by Western blot analyses. RVLM oxidative stress was measured by fluorescent probe dihydroethidium and lipid peroxidation by malondialdehyde assay. Mitochondrial DNA copy number was determined by quantitative real-time polymerase chain reaction. RESULTS: Increased systolic BP, plasma norepinephrine level and sympathetic vasomotor activity were exhibited by young HFD offspring. Reactive oxygen species (ROS) level was also elevated in RVLM where sympathetic premotor neurons reside, alongside augmented protein expressions of AT1R and pg91phox subunit of NADPH oxidase, decrease in superoxide dismutase 2; and suppression of transcription factors for mitochondrial biogenesis, peroxisome proliferator-activated receptor γ co-activator α (PGC-1α) and mitochondrial transcription factor A (TFAM). Maternal HFD also attenuated AMPK phosphorylation and protein expression of SIRT1 in RVLM of young offspring. Oral administration of a HMG-CoA reductase inhibitor, simvastatin, or an AMPK activator, metformin, to young HFD offspring reversed maternal HFD-programmed increase in AT1R and decreases in SIRT1, PGC-1α and TFAM; alleviated ROS production in RVLM, and attenuated sympathoexcitation and hypertension. CONCLUSION: Dysfunction of AMPK-regulated AT1R expression and SIRT1-mediated mitochondrial biogenesis may contribute to tissue oxidative stress in RVLM, which in turn primes increases of sympathetic vasomotor activity and BP in young offspring programmed by excessive maternal fructose consumption.


Assuntos
Proteínas Quinases Ativadas por AMP/genética , Frutose/administração & dosagem , Regulação da Expressão Gênica , Mitocôndrias/fisiologia , Receptor Tipo 1 de Angiotensina/genética , Sirtuína 1/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Feminino , Hipertensão/genética , Exposição Materna , Biogênese de Organelas , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina/metabolismo , Sirtuína 1/metabolismo
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