RESUMO
Oxidative stress triggered by aging, radiation, or inflammation impairs ovarian function by inducing granulosa cell (GC) apoptosis. However, the mechanism inducing GC apoptosis has not been characterized. Here, we found that ovarian GCs from aging patients showed increased oxidative stress, enhanced reactive oxygen species activity, and significantly decreased expression of the known antiapoptotic factor sphingosine-1-phosphate/sphingosine kinase 1 (SPHK1) in GCs. Interestingly, the expression of Krüppel-like factor 12 (KLF12) was significantly increased in the ovarian GCs of aging patients. Furthermore, we determined that KLF12 was significantly upregulated in hydrogen peroxide-treated GCs and a 3-nitropropionic acid-induced in vivo model of ovarian oxidative stress. This phenotype was further confirmed to result from inhibition of SPHK1 by KLF12. Interestingly, when endogenous KLF12 was knocked down, it rescued oxidative stress-induced apoptosis. Meanwhile, supplementation with SPHK1 partially reversed oxidative stress-induced apoptosis. However, this function was lost in SPHK1 with deletion of the binding region to the KLF12 promoter. SPHK1 reversed apoptosis caused by hydrogen peroxide-KLF12 overexpression, a result further confirmed in an in vitro ovarian culture model and an in vivo 3-nitropropionic acid-induced ovarian oxidative stress model. Overall, our study reveals that KLF12 is involved in regulating apoptosis induced by oxidative stress in aging ovarian GCs and that sphingosine-1-phosphate/SPHK1 can rescue GC apoptosis by interacting with KLF12 in negative feedback.
Assuntos
Envelhecimento , Apoptose , Células da Granulosa , Peróxido de Hidrogênio , Fatores de Transcrição Kruppel-Like , Lisofosfolipídeos , Fosfotransferases (Aceptor do Grupo Álcool) , Esfingosina , Feminino , Humanos , Envelhecimento/metabolismo , Retroalimentação Fisiológica , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Peróxido de Hidrogênio/farmacologia , Técnicas In Vitro , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Lisofosfolipídeos/biossíntese , Lisofosfolipídeos/metabolismo , Técnicas de Cultura de Órgãos , Estresse Oxidativo/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Regiões Promotoras Genéticas , Esfingosina/biossíntese , Esfingosina/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: Circulating tumor cells (CTCs) with an epithelial-mesenchymal transition phenotype in peripheral blood may be a useful marker of carcinomas with poor prognosis. The aim of this study was to determine the prognostic significance of CTCs expressing Krüppel-like factor 8 (KLF8) and vimentin in pancreatic cancer (PC). METHODS: CTCs were isolated by immunomagnetic separation from the peripheral blood of 40 PC patients before undergoing surgical resection. Immunocytochemistry was performed to identify KLF8+ and vimentin+ CTCs. The associations between CTCs and time to recurrence (TTR), clinicopathologic factors, and survival were assessed. Univariate and multivariate analyzes were performed to identify risk factors. RESULTS: Patients with CTCs (n = 30) had a higher relapse rate compared to those without (n = 10) (70.0% vs 20.0%; P < 0.01). The proportion of KLF8+/vimentin+ CTCs to total CTCs was inversely related to TTR (r = -0.646; P < 0.01); TTR was reduced in patients with > 50% of CTCs identified as KLF8+/vimentin+ (P < 0.01). Independent risk factors for recurrence were perineural invasion and > 50% KLF8+/vimentin+ CTCs (both P < 0.05). CONCLUSION: Poor prognosis can be predicted in PC patients when > 50% of CTCs are positive for KLF8 and vimentin.
Assuntos
Fatores de Transcrição Kruppel-Like/biossíntese , Células Neoplásicas Circulantes/metabolismo , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Vimentina/biossíntese , Adulto , Biomarcadores Tumorais , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Recidiva Local de Neoplasia , Prognóstico , Fatores de RiscoRESUMO
Coronavirus disease 2019 (COVID-19) is regarded as an endothelial disease (endothelialitis) with its patho-mechanism being incompletely understood. Emerging evidence has demonstrated that endothelial dysfunction precipitates COVID-19 and its accompanying multi-organ injuries. Thus, pharmacotherapies targeting endothelial dysfunction have potential to ameliorate COVID-19 and its cardiovascular complications. The objective of the present study is to evaluate whether kruppel-like factor 2 (KLF2), a master regulator of vascular homeostasis, represents a therapeutic target for COVID-19-induced endothelial dysfunction. Here, we demonstrate that the expression of KLF2 was reduced and monocyte adhesion was increased in endothelial cells treated with COVID-19 patient serum due to elevated levels of pro-adhesive molecules, ICAM1 and VCAM1. IL-1ß and TNF-α, two cytokines elevated in cytokine release syndrome in COVID-19 patients, decreased KLF2 gene expression. Pharmacologic (atorvastatin and tannic acid) and genetic (adenoviral overexpression) approaches to augment KLF2 levels attenuated COVID-19-serum-induced increase in endothelial inflammation and monocyte adhesion. Next-generation RNA-sequencing data showed that atorvastatin treatment leads to a cardiovascular protective transcriptome associated with improved endothelial function (vasodilation, anti-inflammation, antioxidant status, anti-thrombosis/-coagulation, anti-fibrosis, and reduced angiogenesis). Finally, knockdown of KLF2 partially reversed the ameliorative effect of atorvastatin on COVID-19-serum-induced endothelial inflammation and monocyte adhesion. Collectively, the present study implicates loss of KLF2 as an important molecular event in the development of COVID-19-induced vascular disease and suggests that efforts to augment KLF2 levels may be therapeutically beneficial.
Assuntos
COVID-19 , Células Endoteliais da Veia Umbilical Humana , Fatores de Transcrição Kruppel-Like/biossíntese , SARS-CoV-2 , COVID-19/genética , COVID-19/metabolismo , COVID-19/patologia , COVID-19/prevenção & controle , Citocinas/biossíntese , Citocinas/genética , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Células Endoteliais da Veia Umbilical Humana/virologia , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Molécula 1 de Adesão Intercelular/genética , Fatores de Transcrição Kruppel-Like/genética , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Molécula 1 de Adesão de Célula Vascular/biossíntese , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
Insect metamorphosis into an adult occurs after the juvenile hormone (JH) titer decreases at the end of the juvenile stage. This generally coincides with decreased transcript levels of JH-response transcription factors Krüppel homolog 1 (Kr-h1) and broad (br), and increased transcript levels of the adult specifier E93. Thrips (Thysanoptera) develop through inactive and non-feeding stages referred to as "propupa" and "pupa", and this type of distinctive metamorphosis is called neometaboly. To understand the mechanisms of hormonal regulation in thrips metamorphosis, we previously analyzed the transcript levels of Kr-h1 and br in two thrips species, Frankliniella occidentalis (Thripidae) and Haplothrips brevitubus (Phlaeothripidae). In both species, the transcript levels of Kr-h1 and br decreased in the "propupal" and "pupal" stages, and their transcription was upregulated by exogenous JH mimic treatment. Here we analyzed the developmental profiles of E93 in these two thrips species. Quantitative RT-PCR revealed that E93 expression started to increase at the end of the larval stage in F. occidentalis and in the "propupal" stage of H. brevitubus, as Kr-h1 and br mRNA levels decreased. Treatment with an exogenous JH mimic at the onset of metamorphosis prevented pupal-adult transition and caused repression of E93. These results indicated that E93 is involved in adult differentiation after JH titer decreases at the end of the larval stage of thrips. By comparing the expression profiles of Kr-h1, br, and E93 among insect species, we propose that the "propupal" and "pupal" stages of thrips have some similarities with the holometabolous prepupal and pupal stages, respectively.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/biossíntese , Fatores de Transcrição Kruppel-Like/biossíntese , Tisanópteros/embriologia , Animais , Proteínas de Insetos/genética , Fatores de Transcrição Kruppel-Like/genética , Pupa/genética , Pupa/crescimento & desenvolvimento , Tisanópteros/genéticaRESUMO
Increasing the intrinsic regeneration potential of neurons is the key to promote axon regeneration and repair of nerve injury. Therefore, identifying the molecular switches that respond to nerve injury may play critical role in improving intrinsic regeneration ability. The mechanisms by which injury unlocks the intrinsic axonal growth competence of mature neurons are not well understood. The present study identified the key regulatory genes after sciatic nerve crush injury by RNA sequencing (RNA-Seq) and found that the hub gene Vav1 was highly expressed at both early response and regenerative stages of sciatic nerve injury. Furthermore, Vav1 was required for axon regeneration of dorsal root ganglia (DRG) neurons and functional recovery. Krüppel-like factor 2 (Klf2) was induced by retrograde Ca2+ signaling from injured axons and could directly promote Vav1 transcription in adult DRG neurons. The increased Vav1 then promoted axon regeneration by activating Rac1 GTPase independent of its tyrosine phosphorylation. Collectively, these findings break through previous limited cognition of Vav1, and first reveal a crucial role of Vav1 as a molecular switch in response to axonal injury for promoting axon regeneration, which might further serve as a novel molecular therapeutic target for clinical nerve injury repair.
Assuntos
Axônios/fisiologia , Fatores de Transcrição Kruppel-Like/biossíntese , Regeneração Nervosa/fisiologia , Traumatismos dos Nervos Periféricos/metabolismo , Proteínas Proto-Oncogênicas c-vav/biossíntese , Proteínas rac1 de Ligação ao GTP/biossíntese , Animais , Células Cultivadas , Masculino , Traumatismos dos Nervos Periféricos/patologia , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/fisiologia , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidoresRESUMO
Our previous study found that zinc finger protein 71 (ZNF71) mRNA expression was associated with chemosensitivity and its protein expression was prognostic of non-small-cell lung cancer (NSCLC). The Krüppel associated box (KRAB) transcriptional repression domain is commonly present in human zinc finger proteins, which are linked to imprinting, silencing of repetitive elements, proliferation, apoptosis, and cancer. This study revealed that ZNF71 KRAB had a significantly higher expression than the ZNF71 KRAB-less isoform in NSCLC tumors (n = 197) and cell lines (n = 117). Patients with higher ZNF71 KRAB expression had a significantly worse survival outcome than patients with lower ZNF71 KRAB expression (log-rank p = 0.04; hazard ratio (HR): 1.686 [1.026, 2.771]), whereas ZNF71 overall and KRAB-less expression levels were not prognostic in the same patient cohort. ZNF71 KRAB expression was associated with epithelial-to-mesenchymal transition (EMT) in both patient tumors and cell lines. ZNF71 KRAB was overexpressed in NSCLC cell lines resistant to docetaxel and paclitaxel treatment compared to chemo-sensitive cell lines, consistent with its association with poor prognosis in patients. Therefore, ZNF71 KRAB isoform is a more effective prognostic factor than ZNF71 overall and KRAB-less expression for NSCLC. Functional analysis using CRISPR-Cas9 and RNA interference (RNAi) screening data indicated that a knockdown/knockout of ZNF71 did not significantly affect NSCLC cell proliferation in vitro.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição Kruppel-Like/biossíntese , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/biossíntese , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Intervalo Livre de Doença , Docetaxel/farmacologia , Feminino , Humanos , Fatores de Transcrição Kruppel-Like/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Proteínas de Neoplasias/genética , Paclitaxel/farmacologia , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Taxa de SobrevidaRESUMO
Sepsis-induced acute lung injury is associated with dysregulated inflammatory reactions. MiR-19b-3p level was reported to be downregulated in patients with sepsis. To evaluate the role of miR-19b-3p in sepsis, cecum ligation and puncture-induced mouse sepsis model and lpopolysaccharide (LPS)-treated pulmonary microvascular endothelial cells (PMVECs) were used. For in vivo study, lung tissue was harvested for hematoxylin and eosin (H&E) staining, tumor necrosis factor-α, interleukin-6 (IL-6), IL-1ß, and p-p65, p-IκB measuring. Cell apoptosis was assessed by TUNEL assay. For in vitro study, cell proliferation and apoptosis were detected by CCK-8 and flow cytometry, respectively. Methylation of miR-19b-3p promoter was measured by methylation-specific PCR (MSP) assay. The target of miR-19b-3p was determined by dual-luciferase reporter gene assay. The level of miR-19b-3p was determined to be downregulated in vitro and in vivo. In addition, miR-19b-3p protected mice from inflammation injury through inhibiting NF-κB signaling pathway. Overexpression of miR-19b-3p increased cell viability, decreased apoptosis, and proinflammatory cytokines secretion in LPS-treated PMVECs. Besides these, Krüppel-like factor 7 (KLF7) was confirmed as the target of miR-19b-3p. And methylation of miR-19b-3p was the reason of decreased miR-19b-3p level. In conclusion, miR-19b-3p protected cells from sepsis-induced inflammation injury via inhibiting NF-κB signaling pathway, and KLF7 was a potential target.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Marcação de Genes/métodos , Mediadores da Inflamação/metabolismo , Fatores de Transcrição Kruppel-Like/biossíntese , MicroRNAs/biossíntese , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/genética , Animais , Fatores de Transcrição Kruppel-Like/genética , Lipopolissacarídeos/toxicidade , Masculino , Metilação , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , SepseRESUMO
Osteoporosis is a prevalent systemic skeletal disorder entailing bone fragility and increased fracture risk, often emerging in post-menopausal life. Emerging evidence implicates the dysregulation of microRNAs (miRNAs or miRs) in the progression of osteoporosis. This study investigated the effect of miR-199a-3p on osteoporosis and its underlying mechanism. We first examplished an ovariectomized (OVX)-induced rat osteoporosis model, and then isolated mesenchymal stem cells (MSCs) from bone marrow of the model rats. The overexpression and knock down of miR-199a-3p were conducted in OVX rats and MSCs to verify the role of miR-199a-3p on MSC differentiation. Calcium nodules were measured using alizarin red S (ARS) staining. RT-qPCR and Western blot assay were performed to measure the expression of miR-199a-3p, Kdm3a and osteogenic differentiation-related markers in rat tissues and cells. The correlation between miR-199a-3p and Kdm3a was confirmed using dual-luciferase reporter assay. The enrichment of Kdm3a at the Erk2 and Klf2 promoter was assessed using chromatin immunoprecipitation (ChIP) assay. Isolated MSCs were positive for CD29, CD44, CD90, and CD45, suggesting successful isolation of MSCs. There was increased expression of miR-199a-3p and inhibited osteogenic differentiation in OVX rats. Kdm3a was negatively targeted by miR-199a-3p. Our results also demonstrated that Kdm3a elevated the expression of Erk2 and Erk2 by promoting Erk2 and Klf2 demethylation, which further contributed to osteogenic differentiation. Overall, our results revealed a regulatory network of miR-199a-3p in osteogenic differentiation, highlighting miR-199a-3p as a potential target for therapeutic interventions in osteoporosis.
Assuntos
Histona Desmetilases/genética , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , Osteogênese/genética , Osteoporose/genética , Animais , Antígenos CD/biossíntese , Azepinas/farmacologia , Azepinas/uso terapêutico , Osso e Ossos/patologia , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Redes Reguladoras de Genes , Genes Reporter , Histona Desmetilases/antagonistas & inibidores , Histona Desmetilases/biossíntese , Humanos , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição Kruppel-Like/genética , MicroRNAs/biossíntese , Proteína Quinase 1 Ativada por Mitógeno/biossíntese , Proteína Quinase 1 Ativada por Mitógeno/genética , Osteoporose/metabolismo , Osteoporose/patologia , Osteoporose Pós-Menopausa/genética , Ovariectomia , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Ratos , Ratos Sprague-DawleyRESUMO
Acute kidney injury (AKI) occurs in 30%-70% of critically ill patients. Multiple organ failure (MOF), which is most often secondary to hypotension and septicemia, is a global public health problem. The prognosis of patients is poor. Currently, there is no specific therapeutic method. Finding new therapeutic targets is significant to improve the prognosis of AKI patients. This study explores expressions and related mechanisms of miR-212 and Kruppel-like factor 4 (KLF4) in rats with AKI. Sixty Wistar rats were randomly divided into 6 groups: Control group, sham operation group, model group, miR-212-agomir group, miR-212-antagomir group, miR-212-agomir+APTO-253 (joint group), n = 10. The expressions of miR-212, KLF4, inflammatory factors [tumor necrosis factor α (TNF-α), interleukin 6 (IL-6)], oxidative stress factors [superoxide dismutase (SOD), malondialdehyde (MDA)], and apoptosis-related proteins (bax, bcl-2) in renal tissue of rats were detected, and the relationship between miR-212 and KLF4 and the severity of AKI in rats were analyzed. The expression level of miR-212 increased (P < 0.05) and the expression level of KLF4 decreased (P < 0.05) in renal tissue of rats with AKI. miR-212 was negatively correlated with KLF4 expression (P < 0.05). MiR-212 was positively correlated with expressions of TNF-α, IL-6, MDA, and bax (P < 0.05), negatively correlated with expressions of SOD and bcl-2 (P < 0.05), KLF4 was negatively correlated with expressions of TNF-α, IL-6, MDA and bax (P < 0.05), and positively correlated with expressions of SOD and bcl-2 (P < 0.05). MiR-212 mimics can inhibit the luciferase activity of Wt-KLF4 (P < 0.05), and miR-212 inhibitor can promote the luciferase activity of Wt-KLF4 (P < 0.05). Down-regulation of miR-212 plays a protective role by targeting up-regulation of KLF4 to inhibit renal tissue inflammation, oxidative stress, and apoptosis in rats with AKI, which may be a potential target for clinical treatment of AKI in the future.
Assuntos
Injúria Renal Aguda/metabolismo , Regulação da Expressão Gênica , Fatores de Transcrição Kruppel-Like/biossíntese , MicroRNAs/biossíntese , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/tratamento farmacológico , Animais , Modelos Animais de Doenças , Fator 4 Semelhante a Kruppel , Ratos , Ratos WistarRESUMO
Cancer is one of the leading causes of mortality worldwide, ranked second after heart disease. Despite recent advancements in diagnosis and treatment, there are still numerous problems associated with cancer progression, disease recurrence, and therapeutic resistance that are partially explored. Several studies have recently revealed that Krüppel-like factor 8 (KLF8) regulates transcription of genes linked with diverse biological processes, including proliferation, epithelial to mesenchymal transition (EMT), migration, invasion, and inflammation. KLF8 is expressed ubiquitously in mammalian cells, and its aberrant expression has been manifested with several cancer types. Earlier studies demonstrated the crucial role of KLF8 in DNA repair and resistance to apoptosis in numerous cancer types. Hence, studying the function of KLF8 from the perspective of cancer progression and therapy resistance would help develop a new therapeutic avenue. In this review, we summarize the clinical relevance of KLF8 expression in various malignancies, focusing on recent updates in EMT, cellular signaling, and cancer stem cells. We also address the contribution of KLF8 in development, DNA repair, chemoresistance, and its clinical utility as a predictive biomarker.
Assuntos
Pesquisa Biomédica/tendências , Fatores de Transcrição Kruppel-Like/biossíntese , Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Animais , Reparo do DNA/fisiologia , Transição Epitelial-Mesenquimal/fisiologia , Humanos , Fatores de Transcrição Kruppel-Like/química , Fatores de Transcrição Kruppel-Like/genética , Neoplasias/genética , Neoplasias/patologia , Células-Tronco Neoplásicas/patologia , Estrutura Secundária de ProteínaRESUMO
Ligand-activated glucocorticoid receptor (GR) elicits variable glucocorticoid-modulated transcriptomes in different cell types. However, some genes, including Krüppel-like factor 9 (KLF9), a putative transcriptional repressor, demonstrate conserved responses. We show that glucocorticoids induce KLF9 expression in the human airways in vivo and in differentiated human bronchial epithelial (HBE) cells grown at air-liquid interface (ALI). In A549 and BEAS-2B pulmonary epithelial cells, glucocorticoids induce KLF9 expression with similar kinetics to primary HBE cells in submersion culture. A549 and BEAS-2B ChIP-seq data reveal four common glucocorticoid-induced GR binding sites (GBSs). Two GBSs mapped to the 5'-proximal region relative to KLF9 transcription start site (TSS) and two occurred at distal sites. These were all confirmed in primary HBE cells. Global run-on (GRO) sequencing indicated robust enhancer RNA (eRNA) production from three of these GBSs in BEAS-2B cells. This was confirmed in A549 cells, plus submersion, and ALI culture of HBE cells. Cloning each GBS into luciferase reporters revealed glucocorticoid-induced activity requiring a glucocorticoid response element (GRE) within each distal GBS. While the proximal GBSs drove modest reporter induction by glucocorticoids, this region exhibited basal eRNA production, RNA polymerase II enrichment, and looping to the TSS, plausibly underlying constitutive KLF9 expression. Post glucocorticoid treatment, interactions between distal and proximal GBSs and the TSS correlated with KLF9 induction. CBP/P300 silencing reduced proximal GBS activity, but negligibly affected KLF9 expression. Overall, a model for glucocorticoid-mediated regulation of KLF9 involving multiple GBSs is depicted. This work unequivocally demonstrates that mechanistic insights gained from cell lines can translate to physiologically relevant systems.
Assuntos
Dexametasona/farmacologia , Genômica , Glucocorticoides/farmacologia , Fatores de Transcrição Kruppel-Like/biossíntese , Pulmão/efeitos dos fármacos , Células A549 , Elementos Facilitadores Genéticos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Ligação Proteica , RNA Mensageiro/genética , Receptores de Glucocorticoides/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Krüppel-like factor 4 (KLF4), a zinc-finger transcription factor in klfs family, is known for its crucial role in regulating cell growth, proliferation, and differentiation. This research aimed to explore the prognostic significance of KLF4 in hepatocellular carcinoma's (HCC) patients after curative resection and the role of KLF4 in HCC progression. There were 185 HCC patients who had hepatectomy from July 2010 to July 2011 included in this study. KLF4 expression was detected by microarray immunohistochemical technique, western blot, and qRT-PCR. Then, the correlation between the prognosis of patients and KLF4 expression was evaluated based on patients' follow-up data. The research found KLF4 expression was significantly downregulated in HCC tissues compared to para-tumorous tissues. More importantly, the overall survival rate (OS) and recurrence-free survival rate (RFS) of HCC patients with low KLF4 expression were both significantly decreased compared to those with a high level of KLF4. Further function and mechanism analysis showed that KLF4 could inhibit the proliferation, migration, invasion and epithelial-mesenchymal transition of HCC cells. The study revealed that KLF4 was not only a tumor suppressor in HCC but also can be regarded as a valuable prognostic factor and potential biological target for diagnosis and treatment in HCC patients.
Assuntos
Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/cirurgia , Hepatectomia , Fatores de Transcrição Kruppel-Like/fisiologia , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/cirurgia , Carcinoma Hepatocelular/metabolismo , Feminino , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/biossíntese , Neoplasias Hepáticas/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
Hematopoietic stem and progenitor cells (HSPCs) have the ability to self-renew and differentiate into various blood cells, thus playing an important role in maintenance of lifelong hematopoiesis. Brahma-related gene 1 (BRG1), which acts as the ATP subunit of mammalian SWI-SNF-related chromatin remodeling complexes, is involved in human acute myeloid leukemia and highly expresses in short-term HSPCs. But its role and regulatory mechanism for HSPC development have not yet been well established. Here, we generated a brg1 knockout zebrafish model using TALEN technology. We found that in brg1-/- embryo, the primitive hematopoiesis remained well, while definitive hematopoiesis formation was significantly impaired. The number of hemogenic endothelial cells was decreased, further affecting definitive hematopoiesis with reduced myeloid and lymphoid cells. During embryogenesis, the nitric oxide (NO) microenvironment in brg1-/- embryo was seriously damaged and the reduction of HSPCs could be partially rescued by a NO donor. Chromatin immunoprecipitation (ChIP) assays showed that BRG1 could bind to the promoter of KLF2 and trigger its transcriptional activity of NO synthase. Our findings show that Brg1 promotes klf2a expression in hemogenic endothelium and highlight a novel mechanism for HSPC formation and maintenance.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Embrião não Mamífero/embriologia , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Nicho de Células-Tronco , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Células-Tronco Hematopoéticas/citologia , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição Kruppel-Like/genética , Óxido Nítrico/genética , Óxido Nítrico/metabolismo , Elementos de Resposta , Transcrição Gênica , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/genéticaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicinal herb Salvia miltiorrhiza Bunge(Danshen) and its components have been widely used to treat cardiovascular diseases for hundreds of years in China, including hypertension, diabetes, atherosclerosis, and chronic heart failure. Salvia miltiorrhiza injection (SMI), an aqueous extracts of Salvia miltiorrhiza Bunge, is one of most widely used traditional Chinese medicine injections. SMI is widely used in the treatment of diabetic vascular complications, However, the mechanisms remain to be defined. AIM OF THE STUDY: To investigate protective mechanism of Salvia miltiorrhiza Bunge against ROS generation in VSMCs of diabetic mice and patients. MATERIALS AND METHODS: Salvia miltiorrhiza injection (hereinafter referred to as SMI, 1.5 g mL-1), which was approved by the State Food and Drug Administration (approval number: Z32020161), was obtained from Shenlong Pharmaceutical Co., Ltd. (batch number: 11040314). SMI or vehicle were intraperitoneally administrated to the HFD-fed db/db mice, artery was harvested after 24weeks later. qRT-PCR and Western blot analysis were used to detect the expression of KLF6, KLF5, KLF4, KLF10, KLF12, and HO-1. DCFH-DA staining detected intracellular ROS production. Loss- and gain-of-function experiments of KLF10 were used to investigate the effect of KLF10 on the expression of HO-1. Dual-luciferase reporter assay evaluated the effect of KLF10 on the activity of the HO-1 promoter. RESULTS: KLF10 expression and ROS generation are significantly increased in the arteries of HFD-fed db/db mice, VSMCs of diabetic patients, as well as in high glucose-treated VSMCs. KLF10 overexpression suppresses, while its knockdown facilitates the expression of heme oxygenase (HO-1) mRNA and protein. Further, Salvia miltiorrhiza injection (SMI) abrogates KLF10 upregulation and reduces ROS generation induced by high glucose in VSMCs. Mechanistically, KLF10 negatively regulates the HO-1 gene transcription via directly binding to its promoter. Accordingly, SMI treatment of VSMCs reduces ROS generation through inhibiting KLF10 expression and thus relieving KLF10 repression of the expression of HO-1 gene, subsequently contributing to upregulation of HO-1. CONCLUSION: SMI exerts anti-oxidative effects on VSMCs exposed to high glucose through inhibiting KLF10 expression and thus upregulating HO-1.
Assuntos
Antioxidantes/uso terapêutico , Fatores de Transcrição de Resposta de Crescimento Precoce/antagonistas & inibidores , Glucose/toxicidade , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , Músculo Liso Vascular/efeitos dos fármacos , Extratos Vegetais/uso terapêutico , Salvia miltiorrhiza , Animais , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Linhagem Celular , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Angiopatias Diabéticas/tratamento farmacológico , Angiopatias Diabéticas/metabolismo , Dieta Hiperlipídica/efeitos adversos , Fatores de Transcrição de Resposta de Crescimento Precoce/biossíntese , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/biossíntese , Masculino , Camundongos , Músculo Liso Vascular/metabolismo , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologiaRESUMO
Coxsackie B3 virus (CVB3) is a member of small RNA viruses that belongs to the genus Enterovirus of the family Picornaviridae and CVB3 is the main pathogen of acute and chronic viral myocarditis. In this study RT-qPCR was used to determine the expression of miR-107 in CVB3-infected and uninfected HeLa cells. The experimental results show that the level of miR-107 began to rise at 4 h after the infection, and significantly boosted at 6 h. Based on the results of this experiment, we consider that miR-107 expression is related to CVB3 infection. In order to further clarify the effect of miR-107 in the process of CVB3 infection, we studied the effect of miR-107 upstream and downstream target genes on CVB3 replication. Levels of the target RNAs were detected by RT-qPCR after CVB3 infection, and the expression of CVB3 capsid protein VP1 by western blot analysis. Then the virus in the supernatant was quantitated via a viral plaque assay, reflecting the release of the virus. The experimental results showed that miRNA-107 expression is associated with CVB3 replication and proliferation, while KLF4 and BACE1 as the downstream of miR-107 weakened CVB3 replication. Overexpressions of KLF4 and BACE1 negatively regulated CVB3 replication, this effect on CVB3 was completely opposite to that of miR-107. Further experiments revealed that the upstream lncRNA004787, a new lncRNA that had not been reported, was located on chromosome 5, strand - from 37073250 to 37070908 (genome assembly: hg19). We sequenced and studied lncRNA004787 and found that it partially inhibited CVB3 replication. This prompted us to speculate that lncRNA004787 probably impacted the replication by other means. In conclusion, miR-107 interfered with CVB3 replication and release.
Assuntos
Enterovirus/genética , MicroRNAs/genética , Replicação Viral/genética , Secretases da Proteína Precursora do Amiloide/biossíntese , Secretases da Proteína Precursora do Amiloide/genética , Animais , Ácido Aspártico Endopeptidases/biossíntese , Ácido Aspártico Endopeptidases/genética , Cromossomos Humanos Par 5/genética , Células HeLa , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição Kruppel-Like/genética , RNA Longo não Codificante/genética , RNA Viral/biossíntese , RNA Viral/genética , Ensaio de Placa ViralRESUMO
OBJECTIVE: Long non-coding RNAs (lncRNAs) have been identified to exert an oncogenic or anti-tumor function in malignant tumors. LncRNA SNHG15 is verified to be an oncogene in hepatocellular carcinoma, colorectal cancer, and prostate cancer. In this paper, we mainly investigate the potential influence of SNHG15 on the progression of nasopharyngeal carcinoma (NPC). PATIENTS AND METHODS: SNHG15 levels in NPC tissues and cell lines were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Correlation between SNHG15 level and prognosis of NPC patients was evaluated by the Kaplan-Meier method. Regulatory effects of SNHG15 on proliferative, colony formation abilities, and apoptosis of SUNE1 and CNE1 cells were assessed through a series of functional experiments. Potential miRNAs binding SNHG15 and the downstream gene of the microRNA (miRNA) were predicted by bioinformatics method, which was confirmed by Dual-Luciferase reporter gene assay and Western blot. RESULTS: SNHG15 was upregulated in NPC tissues and cells. High level of SNHG15 indicated worse survival in NPC patients. Knockdown of SNHG15 markedly suppressed proliferative ability and induced apoptosis in SUNE1 and CNE1 cells. It is verified that miR-141-3p was the direct target binding SNHG15, and KLF9 was the downstream gene of miR-141-3p. SNHG15 was demonstrated to be a ceRNA to upregulate KLF9 by competitively binding miR-141-3p. CONCLUSIONS: SNHG15 is upregulated in NPC tissues, and this aggravates the progression of NPC by absorbing miR-141-3p to upregulate KLF9.
Assuntos
Proliferação de Células/fisiologia , Fatores de Transcrição Kruppel-Like/biossíntese , MicroRNAs/metabolismo , Carcinoma Nasofaríngeo/metabolismo , Neoplasias Nasofaríngeas/metabolismo , RNA Longo não Codificante/biossíntese , Linhagem Celular Transformada , Linhagem Celular Tumoral , Humanos , Carcinoma Nasofaríngeo/patologia , Neoplasias Nasofaríngeas/patologia , Regulação para Cima/fisiologiaRESUMO
Over the past few decades, long noncoding RNAs (lncRNAs) have been widely accepted to be involved in various diseases, and smooth muscle enriched long noncoding RNA (SMILR) was reported to participate in the proliferation of vascular smooth muscle cells (VSMCs). Nevertheless, the molecular mechanisms of SMILR in atherosclerosis (AS) have not been fully explored. In this study, VSMCs and human mononuclear cells (U937) treated with oxidized low-density lipoprotein (ox-LDL) were used as cell models of AS. We found that the expression of SMILR was upregulated in the serum of AS patients and ox-LDL-induced AS cell models. SMILR knockdown inhibited cell proliferation while increasing cell apoptosis in the AS cell models. In addition, SMILR acted as a sponge for miR-10b-3p, and miR-10b-3p counteracted SMILR-mediated regulation of AS. Moreover, we confirmed that miR-10b-3p could bind with KLF5, and SMILR regulated KLF5 expression by competitively binding miR-10b-3p. Furthermore, miR-10b-3p modulated cell proliferation and apoptosis in AS by targeting KLF5. Finally, miR-10b-3p regulated AS progression in vivo by targeting KLF5. Overall, our study demonstrated that SMILR participated in the progression of AS by targeting the miR-10b-3p/KLF5 axis, which may provide some clues for future studies of AS.
Assuntos
Aterosclerose/metabolismo , Progressão da Doença , Fatores de Transcrição Kruppel-Like/biossíntese , MicroRNAs/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Animais , Aterosclerose/patologia , Proliferação de Células/fisiologia , Expressão Gênica , Humanos , Fatores de Transcrição Kruppel-Like/genética , Camundongos , MicroRNAs/genética , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Células U937RESUMO
INTRODUCTION: Astrogliosis and glial scar formation following spinal cord injury (SCI) are viewed as major obstacles that hinder axonal regeneration and functional recovery. Regulating the glial scar and axonal regeneration in the lesion site is important for treating SCI. AIMS: Considering the important role of astrocyte in glial scar formation and subsequent axonal regeneration, we intended to investigate the effect of the transcription factors OCT4 and KLF4 on astrocyte and the underlying mechanism after spinal cord contusion injury in transgenic mice. RESULTS: Western blotting, q-PCR, immunofluorescence, and functional evaluation suggested that glial fibrillary acidic protein (GFAP) expression decreased in the lesion area, the porosity of the scar increased, and remyelination enhanced. Mice overexpressing the transcription factors OCT4 and KLF4 had higher Basso Mouse Scale scores than did the control mice. Moreover, using immunofluorescence and Western blotting, we discovered that some astrocytes expressed nestin and sox2 protein, suggesting that these astrocytes were reprogrammed into neural stem cell-like cells. Furthermore, a cell scratch assay showed that the migration ability of the astrocytes was significantly inhibited in the presence of the transcription factors OCT4 and KLF4. In addition, we demonstrated that the Hippo/Yap pathway was activated after these two transcription factors overexpressed in astrocytes. CONCLUSIONS: In summary, these results suggest that overexpression of the transcription factors OCT4 and KLF4 could induce astrocyte reprogramming, which subsequently improves remyelination and functional recovery after SCI.
Assuntos
Fatores de Transcrição Kruppel-Like/biossíntese , Fator 3 de Transcrição de Octâmero/biossíntese , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Astrócitos/patologia , Células Cultivadas , Expressão Gênica , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator 3 de Transcrição de Octâmero/genética , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/patologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
Aim: In this study, we aimed to develop a polycationic non-viral carrier for the delivery of the reprogramming factors to the L929 fibroblast cell.Methods: We have prepared (3-hydroxybutyrate-co-3-hydroxyhexanoate) PHBHHx-based nanoparticles with the solvent diffusion method. Cytotoxicity of PXNs was determined via MTT assay. Transfection efficiency was evaluated via screening GFP expression by fluorescence microscopy. The expression of reprogramming factors (Oct4, Klf4, and Sox2) was determined by RT-qPCR.Results: PXNs with 32.9 ± 0.41 mV zeta potential and 177.6 ± 0.80 nm size were used for transfection of L929 Fbroblast cells. The percentage of cell viability of PXN were between 91.8%(±2.9) and 42.1%(±1.3). The transfection efficiency was found as 71.6%(±3,5). According to RT-qPCR data, the rate of transfection factors was significantly increased after the 11th cycle compared to non-transfected cells. Based on these results, it can be concluded that newly developed PXN is thought to be an effective tool for reprogramming cells.
Assuntos
Caproatos/química , Nanopartículas/química , Reprogramação Celular , Expressão Gênica , Proteínas de Fluorescência Verde , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição Kruppel-Like/genética , Fator 3 de Transcrição de Octâmero/biossíntese , Fator 3 de Transcrição de Octâmero/genética , Tamanho da Partícula , Paxilina/genética , Fatores de Transcrição SOXB1/biossíntese , Fatores de Transcrição SOXB1/genética , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologia , Transfecção/métodosRESUMO
BACKGROUD: Osteoarthritis (OA) is a common disease caused by chondrocyte dysfunction. KLF10 is a member of the Sp1-like transcription factor family that is involved in regulating osteoblasts, but its expression and regulatory mechanism(s) in chondrocytes are unclear. In the present study, we aimed to investigate the regulatory role of KLF10 on the pathological process of OA. METHODS: KLF10 expression in the cartilaginous tissue of patients with osteoarthritis (OA) was evaluated by immunohistochemistry (IHC). Next, we generated an OA mouse model, and the histological changes in cartilage tissue were verified using H&E staining, Safranin O-Fast Green staining, and IHC assays. KLF10 expression in the articular chondrocytes of OA mice was determined by qRT-PCR and Western blotting. To investigate the role of KLF10 in regulating cell proliferation and migration, a KLF10 overexpression plasmid was constructed and transfected into primary mouse chondrocytes. Subsequently, we used RNA sequencing (RNA-seq) to screen differentially expressed genes in chondrocytes with or without KLF10 overexpression. qRT-PCR was used for verification purposes. RESULTS: We found that KLF10 was upregulated in the cartilaginous tissue of patients with OA as well as in cartilaginous tissue of the OA mouse model. KLF10 overexpression inhibited the proliferation and migration of chondrocytes. Furthermore, RNA sequencing results identified increased expression of Acvr1 and decreased expression of Inhbb in cells overexpressing KLF10. Changes in mRNA expression of Acvr1 and Inhbb were confirmed by qRT-PCR. CONCLUSIONS: KLF10 inhibits chondrocyte proliferation and migration by regulating the expression of Acvr1 and Inhbb in both human and mouse OA. These data suggest that KLF10 may be a potential therapeutic target for the treatment of OA.