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1.
Proc Natl Acad Sci U S A ; 117(27): 15846-15851, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32561648

RESUMO

Combination use of BRAF V600E inhibitor dabrafenib and MEK inhibitor trametinib has become a standard treatment for human cancers harboring BRAF V600E. Its anticancer efficacies vary, however, with dramatic efficacy in some patients and drug resistance/tumor recurrence in others, which is poorly understood. Using thyroid cancer, melanoma, and colon cancer cell models, we showed that dabrafenib and trametinib induced robust apoptosis of cancer cells harboring both BRAF V600E and TERT promoter mutations but had little proapoptotic effect in cells harboring only BRAF V600E. Correspondingly, the inhibitors nearly completely abolished the growth of in vivo tumors harboring both mutations but had little effect on tumors harboring only BRAF V600E. Upon drug withdrawal, tumors harboring both mutations remained hardly measurable but tumors harboring only BRAF V600E regrew rapidly. BRAF V600E/MAP kinase pathway is known to robustly activate mutant promoter of TERT, a strong apoptosis suppressor. Thus, for survival, cancer cells harboring both mutations may have evolved to rely on BRAF V600E-promoted and high-TERT expression-mediated suppression of apoptosis. As such, inhibition of BRAF/MEK can trigger strong apoptosis-induced cell death and hence tumor abolishment. This does not happen in cells harboring only BRAF V600E as they have not developed reliance on TERT-mediated suppression of apoptosis due to the lack of mutant promoter-driven high-TERT expression. TERT promoter mutation governs BRAF-mutant cancer cells' apoptotic and hence therapeutic responses to BRAF/MEK inhibitors. Thus, the genetic duet of BRAF V600E and TERT promoter mutation represents an Achilles Heel for effective therapeutic targeting and response prediction in cancer.


Assuntos
Apoptose/efeitos dos fármacos , Mutação , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/efeitos dos fármacos , Proteínas Proto-Oncogênicas B-raf/genética , Telomerase/genética , Animais , Morte Celular , Linhagem Celular Tumoral , Neoplasias do Colo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Imidazóis/farmacologia , Melanoma/genética , Camundongos Nus , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oximas/farmacologia , Piridonas/farmacologia , Pirimidinonas/farmacologia , Neoplasias da Glândula Tireoide/genética , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Am J Physiol Cell Physiol ; 319(2): C268-C276, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32459505

RESUMO

DNA methylation, a critical epigenetic mechanism, plays an important role in governing gene expressions during biological processes such as aging, which is well known to be accelerated in hyperglycemia (diabetes). In the present study, we investigated the effects of glucose on whole genome DNA methylation in small [human retinal microvascular endothelial cells (HRECs)] and large [human umbilical vein endothelial cells (HUVECs)] vessel endothelial cell (EC) lines exposed to basal or high glucose-containing media for variable lengths of time. Using the Infinium EPIC array, we obtained 773,133 CpG sites (probes) for analysis. Unsupervised clustering of the top 5% probes identified four distinct clusters within EC groups, with significant methylation differences attributed to EC types and the duration of cell culture rather than glucose stimuli alone. When comparing the ECs incubated for 2 days versus 7 days, hierarchical clustering analyses [methylation change >10% and false discovery rate (FDR) <0.05] identified 17,354 and 128 differentially methylated CpGs for HUVECs and HRECs, respectively. Predominant DNA hypermethylation was associated with the length of culture and was enriched for gene enhancer elements and regions surrounding CpG shores and shelves. We identified 88 differentially methylated regions (DMRs) for HUVECs and 8 DMRs for HRECs (all FDR <0.05). Pathway enrichment analyses of DMRs highlighted involvement of regulators of embryonic development (i.e., HOX genes) and cellular differentiation [transforming growth factor-ß (TGF-ß) family members]. Collectively, our findings suggest that DNA methylation is a complex process that involves tightly coordinated, cell-specific mechanisms. Such changes in methylation overlap genes critical for cellular differentiation and embryonic development.


Assuntos
Envelhecimento/genética , Ilhas de CpG/genética , Metilação de DNA/genética , Células Endoteliais/metabolismo , Envelhecimento/patologia , Ilhas de CpG/efeitos dos fármacos , DNA/genética , Metilação de DNA/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Epigênese Genética , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Homeobox/genética , Genoma Humano/genética , Glucose/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Regiões Promotoras Genéticas/efeitos dos fármacos
3.
Artigo em Inglês | MEDLINE | ID: mdl-32120263

RESUMO

5-lipoxygenase (5-LO), coded by the ALOX5 gene, is expressed in leukocytes and catalyzes the formation of leukotrienes, pro-inflammatory lipid mediators. Leukotrienes are central to immune responses, but are also involved in inflammatory disorders and 5-LO expression is associated with leukemia stem cell survival. It is therefore important to understand mechanisms that control 5-LO expression. This study investigated the control of 5-LO expression and leukotriene biosynthesis following the maturation of human monocytic cells. MonoMac-1 (MM1) and THP-1 cells were incubated for up to 72 h with or without LPS and TGF-ß. LPS, but not TGF-ß, increased CD14 expression in both MM1 and THP-1 cells. Incubation with LPS (100 ng/ml) and TGF-ß (1 ng/ml) synergistically increased the capacity of MM1 cells to produce 5-LO products from undetectable levels to 40±5 pmol/106 cells. 5-LO product biosynthesis in THP-1 cells increased 25-fold. A synergistic effect of LPS and TGF-ß was measured with increases in 5-LO mRNA of 54- and 13-fold in MM1 and THP-1 cells, respectively. 5-LO protein expression increased significantly in both MM1 and THP-1 cells. ALOX5 promoter activity was significantly elevated >2-fold in both cell lines following LPS treatment, but TGF-ß was without effect. The main 5-LO products were cysteinyl-leukotrienes, however LPS and TGF-ß did not impact on the capacity of the cells to metabolize leukotriene A4. Overall, this study demonstrates that receptor-mediated stimulation of MM1 and THP-1 cells by LPS is associated with increased 5-LO expression. This represents a new mechanism by which leukotriene biosynthesis can be modulated by pathological agents.


Assuntos
Araquidonato 5-Lipoxigenase/genética , Araquidonato 5-Lipoxigenase/metabolismo , Lipopolissacarídeos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Sinergismo Farmacológico , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Regiões Promotoras Genéticas/efeitos dos fármacos , Células THP-1 , Fator de Crescimento Transformador beta1/farmacologia
4.
PLoS One ; 15(3): e0229754, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32134978

RESUMO

PURPOSE: To determine whether SRD5A2 promoter methylation is associated with cancer progression during androgen deprivation therapy (ADT) in CRPC. PATIENTS AND METHODS: In a Local CRPC cohort, 42 prostatic specimens were collected from patients who were diagnosed as CRPC and underwent transurethral resection of the prostate (TURP) at Massachusetts General Hospital (MGH). In a metastatic CRPC (Met CRPC) cohort, 12 metastatic biopsies were collected from CRPC patients who would be treated with abiraterone plus dutasteride (Clinical Trial NCT01393730). As controls, 36 benign prostatic specimens were collected from patients undergoing prostate reduction surgery for symptoms of bladder outlet obstruction secondary to benign prostatic hyperplasia (BPH). The methylation status of cytosine-phosphate-guanine (CpG) site(s) at SRD5A2 promoter regions was tested. RESULTS: Compared with benign prostatic tissue, CRPC samples demonstrated higher SRD5A2 methylation in the whole promoter region (Local CRPC cohort: P < 0.001; Met CRPC cohort: P <0.05). In Local CRPC cohort, a higher ratio of methylation was correlated with better OS (R2 = 0.33, P = 0.013). Hypermethylation of specific regions (nucleotides -434 to -4 [CpG# -39 to CpG# -2]) was associated with a better OS (11.3±5.8 vs 6.4±4.4 years, P = 0.001) and PFS (8.4±5.4 vs 4.5±3.9 years, P = 0.003) with cutoff value of 37.9%. Multivariate analysis showed that SRD5A2 methylation was associated with OS independently (whole promoter region: P = 0.035; specific region: P = 0.02). CONCLUSION: Our study demonstrate that SRD5A2 methylation in promoter regions, specifically at CpG# -39 to -2, is significantly associated with better survival for CRPC patients treated with ADT. Recognition of epigenetic modifications of SRD5A2 may affect the choices and sequence of available therapies for management of CRPC.


Assuntos
3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , Inibidores de 5-alfa Redutase/uso terapêutico , Androstenos/uso terapêutico , Dutasterida/uso terapêutico , Proteínas de Membrana/genética , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Metilação de DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Humanos , Masculino , Prognóstico , Regiões Promotoras Genéticas/efeitos dos fármacos , Neoplasias de Próstata Resistentes à Castração/diagnóstico , Resultado do Tratamento
5.
Oncol Rep ; 43(3): 827-838, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32020216

RESUMO

Restoration of normal DNA promoter methylation and expression states of cancer­related genes may be an option for the prevention as well as the treatment of several types of cancer. Constitutional promoter methylation of BRCA1 DNA repair associated (BRCA1) gene is linked with a high risk of developing breast and ovarian cancer. Furthermore, hypomethylation of the proto­oncogene Î³ synuclein (SNCG) is associated with the metastasis of breast and ovarian cancer and reduced disease­free survival (DFS). In the present study, we evaluated the potential of curcumin to re­express hypermethylated BRCA1 and to suppress hypomethylated SNCG in triple­negative breast cancer (TNBC) cell line HCC­38, the estrogen receptor­negative/progesterone receptor­negative (ER­/PR­) cell line UACC­3199, and the ER+/PR+ cell line T47D. The cells were treated with 5 and 10 µM curcumin for 6 days and with 5­aza­2'­deoxycytidine (5'­aza­CdR) for 48 h. Methylation­specific PCR and bisulfite pyrosequencing assays were used to assess DNA promoter methylation while gene expression levels were analyzed using quantitative real­time PCR and immunoblotting. We found that curcumin treatment restored BRCA1 gene expression by reducing the DNA promoter methylation level in HCC­38 and UACC­3199 cells and that it suppressed the expression of SNCG by inducing DNA promoter methylation in T47D cells. Notably, 5'­aza­CdR restored BRCA1 gene expression only in UACC­3199, and not in HCC­38 cells. Curcumin­induced hypomethylation of the BRCA1 promoter appears to be realized through the upregulation of the ten­eleven translocation 1 (TET1) gene, whereas curcumin­induced hypermethylation of SNCG may be realized through the upregulation of the DNA methyltransferase 3 (DNMT3) and the downregulation of TET1. Notably, miR­29b was found to be reversely expressed compared to TET1 in curcumin­ and 5'­aza­CdR­treated cells, suggesting its involvement in the regulation of TET1. Overall, our results indicate that curcumin has an intrinsic dual function on DNA promoter methylation. We believe that curcumin may be considered a promising therapeutic option for treating TNBC patients in addition to preventing breast and ovarian cancer, particularly in cancer­free females harboring methylated BRCA1.


Assuntos
Proteína BRCA1/genética , Curcumina/farmacologia , DNA (Citosina-5-)-Metiltransferases/genética , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , gama-Sinucleína/genética , Azacitidina/farmacologia , Linhagem Celular Tumoral , Metilação de DNA/efeitos dos fármacos , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , MicroRNAs/genética , Oxigenases de Função Mista/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia
6.
Environ Toxicol Pharmacol ; 75: 103331, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32004919

RESUMO

Skeletal fluorosis is a chronic metabolic bone disease caused by excessive exposed to fluoride. Recent studies have shown that fluoride causes abnormal bone metabolism through disrupting the expression of Bone Morphogenetic Proteins (BMPs). However, the relationship between fluoride and BMPs is not fully understood, and the mechanism of fluoride on BMPs expression is still unclear. This study investigated the dose-time effects of fluoride on BMP-2 and BMP-7 levels and DNA methylation status of the promoter regions of these two genes in peripheral blood of rats. Eighty Wistar male rats were randomly divided into four groups and treated for 1 month and 3 months with distilled water (control), 25 mg/L, 50 mg/L or 100 mg/L of sodium fluoride (NaF). Rats exposed to fluoride had higher protein expression of BMP-2 and BMP-7 in plasma at 1 month and 3 months. An increase in BMP-2 expression was also observed with an increase of fluoride exposure time. Significant hypomethylation was observed in 2 CpG sites (CpGs) of BMP-2 and 1 CpG site of BMP-7 promoter regions in the fluoride treatment groups. It concludes that fluoride has a dose-response effect on BMP-2 in fluorosis rats, and fluoride-induced hypomethylation of specific CpGs may play an essential role in the regulation of BMP-2 and BMP-7 expression in rats.


Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 7/metabolismo , Fluoretos/toxicidade , Animais , Metilação de DNA , Masculino , Regiões Promotoras Genéticas/efeitos dos fármacos , Ratos , Ratos Wistar
7.
Cell Physiol Biochem ; 54(2): 195-210, 2020 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-32083406

RESUMO

BACKGROUND/AIMS: Idiopathic pulmonary fibrosis (IPF) is a specific form of progressive and chronic interstitial lung disease of unknown cause. IPF is characterized by excessive deposition of extracellular matrix (ECM) and destructive pathological remodeling due to epithelial-to-mesenchymal transition (EMT). Eventually, lung interstitium thickens and stiffens and breathing becomes difficult. It has been well established that the transforming growth factor-ß1 (TGF-ß1)/Smad signaling pathway plays a critical role in the pathogenesis of pulmonary fibrosis. TGF-ß1-mediated activation of mitogen activated protein kinase (MAPK) family affects Smad signaling. p90RSK is a serine/threonine kinase and is activated by the extracellular signal-regulated kinase (ERK) signaling pathway. However, the roles played by p90RSK in TGF-ß1 signaling and the pathogenesis of pulmonary fibrosis remain unknown. METHODS: We investigated whether p90RSK regulates the pathogenesis of pulmonary fibrosis using in vitro and in vivo systems and Western blotting, real-time quantitative PCR, transcriptional activity assays and immunofluorescence studies. RESULTS: Pharmacological inhibition of p90RSK by FMK or inhibition of p90RSK with adenoviral vector encoding a dominant negative form of p90RSK suppressed TGF-ß1-induced ECM accumulation and EMT in lung epithelial cells and fibroblasts. Interestingly, FMK significantly inhibited TGF-ß1-induced Smad3 nuclear translocation and smad binding element-dependent transcriptional activity, but not Smad3 phosphorylation. Furthermore, in a mouse model of bleomycin-induced lung fibrosis, FMK ameliorated pulmonary fibrosis. CONCLUSION: These findings indicate that p90RSK plays critical roles in pulmonary fibrosis, which suggests it be viewed as a novel therapeutic target for the treatment of lung fibrosis.


Assuntos
Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Proteína Smad3/metabolismo , Animais , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Matriz Extracelular/metabolismo , Humanos , Isoquinolinas/farmacologia , Cetonas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Piridinas/farmacologia , Pirróis/farmacologia , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Proteína Smad3/antagonistas & inibidores , Proteína Smad3/genética , Ativação Transcricional/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
8.
PLoS One ; 15(1): e0227646, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31940421

RESUMO

The serum concentration of thyrotropin (thyroid stimulating hormone, TSH) is drastically reduced by small increase in the levels of thyroid hormones (T3 and its prohormone, T4); however, the mechanism underlying this relationship is unknown. TSH consists of the chorionic gonadotropin α (CGA) and the ß chain (TSHß). The expression of both peptides is induced by the transcription factor GATA2, a determinant of the thyrotroph and gonadotroph differentiation in the pituitary. We previously reported that the liganded T3 receptor (TR) inhibits transactivation activity of GATA2 via a tethering mechanism and proposed that this mechanism, but not binding of TR with a negative T3-responsive element, is the basis for the T3-dependent inhibition of the TSHß and CGA genes. Multiple GATA-responsive elements (GATA-REs) also exist within the GATA2 gene itself and mediate the positive feedback autoregulation of this gene. To elucidate the effect of T3 on this non-linear regulation, we fused the GATA-REs at -3.9 kb or +9.5 kb of the GATA2 gene with the chloramphenicol acetyltransferase reporter gene harbored in its 1S-promoter. These constructs were co-transfected with the expression plasmids for GATA2 and the pituitary specific TR, TRß2, into kidney-derived CV1 cells. We found that liganded TRß2 represses the GATA2-induced transactivation of these reporter genes. Multi-dimensional input function theory revealed that liganded TRß2 functions as a classical transcriptional repressor. Then, we investigated the effect of T3 on the endogenous expression of GATA2 protein and mRNA in the gonadotroph-derived LßT2 cells. In this cell line, T3 reduced GATA2 protein independently of the ubiquitin proteasome system. GATA2 mRNA was drastically suppressed by T3, the concentration of which corresponds to moderate hypothyroidism and euthyroidism. These results suggest that liganded TRß2 inhibits the positive feedback autoregulation of the GATA2 gene; moreover this mechanism plays an important role in the potent reduction of TSH production by T3.


Assuntos
Fator de Transcrição GATA2/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Tireotropina/metabolismo , Animais , Linhagem Celular , Fator de Transcrição GATA2/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Genes Reporter/efeitos dos fármacos , Genes Reporter/genética , Subunidade alfa de Hormônios Glicoproteicos , Homeostase/efeitos dos fármacos , Ligantes , Camundongos , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ratos , Receptores dos Hormônios Tireóideos/genética , Hormônios Tireóideos/metabolismo , Tireotrofos/metabolismo , Tireotropina/análise , Tireotropina/sangue , Tireotropina Subunidade beta/genética , Tireotropina Subunidade beta/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , Tri-Iodotironina/metabolismo
9.
Nat Commun ; 11(1): 320, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31949157

RESUMO

Endocrine therapy resistance frequently develops in estrogen receptor positive (ER+) breast cancer, but the underlying molecular mechanisms are largely unknown. Here, we show that 3-dimensional (3D) chromatin interactions both within and between topologically associating domains (TADs) frequently change in ER+ endocrine-resistant breast cancer cells and that the differential interactions are enriched for resistance-associated genetic variants at CTCF-bound anchors. Ectopic chromatin interactions are preferentially enriched at active enhancers and promoters and ER binding sites, and are associated with altered expression of ER-regulated genes, consistent with dynamic remodelling of ER pathways accompanying the development of endocrine resistance. We observe that loss of 3D chromatin interactions often occurs coincidently with hypermethylation and loss of ER binding. Alterations in active A and inactive B chromosomal compartments are also associated with decreased ER binding and atypical interactions and gene expression. Together, our results suggest that 3D epigenome remodelling is a key mechanism underlying endocrine resistance in ER+ breast cancer.


Assuntos
Sítios de Ligação , Neoplasias da Mama/genética , Cromatina/metabolismo , Epigênese Genética , Receptores Estrogênicos/química , Receptores Estrogênicos/metabolismo , Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/metabolismo , Fator de Ligação a CCCTC/química , Fator de Ligação a CCCTC/metabolismo , Cromatina/química , Cromatina/genética , Metilação de DNA , Epigênese Genética/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas , Sequenciamento Completo do Genoma
10.
J Sci Food Agric ; 100(4): 1486-1494, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31756772

RESUMO

BACKGROUND: We reported previously that maternal betaine promotes hepatic insulin-like growth factor (IGF2) expression in F1 offspring rats through hypermethylation of the IGF2/H19 imprinting control region (ICR). It remains unknown whether this acquired trait can be transmitted to the F2 generation. This study aimed to determine whether dietary betaine supplementation to grand dams affects the hepatic IGF2 expression in F2 rat offspring and how it is related to alterations in DNA methylation. F2 rat offspring derived from grand dams fed basal or betaine-supplemented diet (10 g kg-1 ) were examined at weaning. Serum IGF2 concentration was measured with enzyme-linked immunosorbent assay (ELISA). Hepatic expression of IGF2, together with other proliferation and apoptosis markers, was determined by using quantitative polymerase chain reaction (qPCR), western blot, and immunohistochemistry. The methylation status of the IGF2/H19 ICR and the promoters of IGF2 gene were detected by methylated DNA immunoprecipitation quantitative polymerase chain reaction (MeDIP-qPCR). RESULTS: The maternal betaine-induced up-regulation of hepatic IGF2 expression in F1 rat offspring was transmitted to the F2 generation. The F2 rats from the betaine group demonstrated enhanced hepatic IGF2 expression at both mRNA and protein levels, in association with higher serum IGF2 concentration. No alterations were observed in the ICR methylation of the IGF2/H19 locus, and hypomethylation was detected in promoters of IGF2 gene in betaine group. CONCLUSION: These results indicate that maternal betaine enhances hepatic IGF2 expression in F2 rat offspring through modification of DNA methylation on IGF2 promoters. © 2019 Society of Chemical Industry.


Assuntos
Betaína/administração & dosagem , Metilação de DNA/efeitos dos fármacos , Fator de Crescimento Insulin-Like II/genética , Fígado/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Animais , Suplementos Nutricionais/análise , Feminino , Fator de Crescimento Insulin-Like II/metabolismo , Fígado/metabolismo , Masculino , Fenômenos Fisiológicos da Nutrição Materna , Linhagem , Gravidez , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Ratos , Ratos Sprague-Dawley
11.
Gene ; 733: 144274, 2020 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-31809844

RESUMO

Bone regeneration has been a challenge for both researchers and clinicians. In the field of tissue engineering, much effort has been made to identify cell sources including stem cells. The present study aimed to induce trans-differentiation from adipocytes to osteoblasts using epigenetic modifiers; 5-aza-dC and/or trichostatin-A (TSA). 3 T3-L1 preadipocytes were treated with TSA (100 nM) and then with Wnt3a (50 ng/ml). Microscopic observation showed trans-differentiated cell morphology. Methylation-specific PCR and immunoblotting were performed to analyze the DNA methylation and histone acetylation patterns. The gene expression was determined by real-time PCR. Based on these in vitro experiments, in vivo mouse experiments supplemented the possibility of trans-differentiation by epigenetic modification. TSA induced the acetylation of lysine9 on histone H3, and a sequential Wnt3a treatment stimulated the expression of bone marker genes in adipocytes, suppressing adipogenesis and stimulating osteogenesis. Furthermore, TSA induced DNA hypomethylation, and a combined treatment with TSA and 5-aza-dC showed a synergistic effect in epigenetic modifications. The number of adipocytes and DNA methylation patterns of old (15 months) and young (6 weeks) mice were significantly different, and TSA and sequential Wnt3a treatments increased bone formation in the old mice. Collectively, our results confirmed cell trans-differentiation via epigenetic modifications and osteogenic signaling from adipocytes to osteoblasts for the bone regeneration in vitro and in vivo, and indicated that histone acetylation could induce DNA hypomethylation, enhancing the chance of trans-differentiation.


Assuntos
Adipócitos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Osteoblastos/metabolismo , Células 3T3-L1 , Acetilação , Adipócitos/efeitos dos fármacos , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Ilhas de CpG , Desmetilação do DNA/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Decitabina/metabolismo , Decitabina/farmacologia , Epigênese Genética/efeitos dos fármacos , Epigênese Genética/genética , Epigenômica/métodos , Inibidores de Histona Desacetilases/farmacologia , Histonas/genética , Histonas/metabolismo , Ácidos Hidroxâmicos/metabolismo , Ácidos Hidroxâmicos/farmacologia , Camundongos , Osteoblastos/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/genética
12.
Toxicol Lett ; 319: 175-186, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31733319

RESUMO

Information on the effects of gibberellic acid (gibberellin A3, GA3) on ovarian follicle development is limited. In our present study, 21-day-old female Wistar rats were exposed to GA3 by gavage (25, 50, and 100 mg/kg body weight, once per day) for eight weeks to evaluate the influence of GA3 on ovarian follicle development. After treatment, significant (P < 0.05) increases (to 40.17 % and 44.5 %, respectively) in atretic follicle proportions and significant decreases (to 19.49 % and 17.86 %, respectively) in corpus luteum proportions were observed in the 50 and 100 mg/kg treatment groups compared to the control group. Significant (P < 0.05) increases (to 31.3 % and 42.0 %, respectively) in follicle apoptosis were observed in the 50 and 100 mg/kg treatment groups by transmission electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays. Significantly increased expression of caspase-3, caspase-8, caspase-9 and Fas was observed by real-time PCR and Western blotting. Bisulfite sequencing PCR (BSP) revealed obviously decreased total methylation percentages of the caspase-3 promoter region in the two treatment groups. Real-time quantitative PCR also showed significantly decreased mRNA expression of DNA methyltransferase (Dnmt) 3a and Dnmt3b. Further in vitro studies showed that a DNA methylation inhibitor could enhance the GA3-induced increase in the mRNA expression of caspase-3. Overall, our present study indicates that GA3 administration from weaning until sexual maturity can affect ovarian follicle development by inducing apoptosis and suggests that signaling through the Fas-mediated apoptotic pathway may be an important underlying mechanism of this apoptosis. In addition, GA3-induced aberrant DNA methylation patterns might be partly responsible for upregulation of caspase-3 gene expression.


Assuntos
Apoptose/efeitos dos fármacos , Caspase 3/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Proteína de Domínio de Morte Associada a Fas/biossíntese , Giberelinas/toxicidade , Células da Granulosa/efeitos dos fármacos , Ovário/citologia , Transdução de Sinais/efeitos dos fármacos , Animais , DNA (Citosina-5-)-Metiltransferases/biossíntese , DNA (Citosina-5-)-Metiltransferases/genética , Proteína de Domínio de Morte Associada a Fas/efeitos dos fármacos , Feminino , Folículo Ovariano/efeitos dos fármacos , Ovário/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Ratos , Ratos Wistar , Superovulação/efeitos dos fármacos
13.
Cell Mol Life Sci ; 77(2): 351-363, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31222373

RESUMO

Cancer stem cells (CSC) are highly associated with poor prognosis in cancer patients. Our previous studies report that isorhapontigenin (ISO) down-regulates SOX2-mediated cyclin D1 induction and stem-like cell properties in glioma stem-like cells. The present study revealed that ISO could inhibit stem cell-like phenotypes and invasivity of human bladder cancer (BC) by specific attenuation of expression of CD44 but not SOX-2, at both the protein transcription and degradation levels. On one hand, ISO inhibited cd44 mRNA expression through decreases in Sp1 direct binding to its promoter region-binding site, resulting in attenuation of its transcription. On the other hand, ISO also down-regulated USP28 expression, which in turn reduced CD44 protein stability. Further studies showed that ISO treatment induced miR-4295, which specific bound to 3'-UTR activity of usp28 mRNA and inhibited its translation and expression, while miR-4295 induction was mediated by increased Dicer protein to enhance miR-4295 maturation upon ISO treatment. Our results provide the first evidence that ISO has a profound inhibitory effect on human BC stem cell-like phenotypes and invasivity through the mechanisms distinct from those previously noted in glioma stem-like cells.


Assuntos
Receptores de Hialuronatos/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Estilbenos/farmacologia , Regiões 3' não Traduzidas/efeitos dos fármacos , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular Tumoral , Ciclina D1/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Células-Tronco , Transcrição Genética/efeitos dos fármacos , Ubiquitina Tiolesterase/metabolismo , Neoplasias da Bexiga Urinária
14.
Chemosphere ; 242: 125186, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31675590

RESUMO

Cadmium (Cd) is considered as a carcinogenic chemical with potential to endanger normal cellular functioning. The present study was aimed to investigate the impact of Cd on the expression of two oncogenic epigenetic regulators, viz., protein arginine methyltransferase 5 (PRMT5) and the polycomb repressive complex 2 (PRC2) member enhancer of Zeste homolog 2 (EZH2). Our results indicate that Cd at 1 µM concentration increases the viability of HepG2 and MCF7 cells and significantly upregulates the expression of PRMT5 and EZH2, leading to an increased global level of symmetric dimethylarginine (SDMA), H4R3me2s, and H3K27me3. The luciferase reporter assay showed that the promoter activity of PRMT5 and EZH2 is significantly enhanced in both cell lines. Furthermore, Cd exposure induces global DNA hypomethylation due to a decrease in DNA methyltransferases (DNMTs) expression. Methylation-specific and bisulfite sequencing PCR reveal that the proximal promoters of PRMT5 and EZH2, which harbour CpG islands, are almost demethylated when exposed to Cd. The Cd exposure also increases the protein level of transcription factors NFYA and E2F1; consistently, the two transcription factors are found to be enriched at the PRMT5 and EZH2 promoter in chromatin immunoprecipitation experiments. The alterations induced by Cd in the two cancer cell lines were also observed in a non-cancerous cell line (HEK-293). In conclusion, we propose that Cd increases the expression of two oncogenic methyltransferases, possibly with a DNA methylation-dependent mechanism. Further studies focused on the epigenetic alterations induced by Cd would provide mechanistic insights on the carcinogenicity of this metal toxicant at the molecular level.


Assuntos
Cádmio/farmacologia , Metilação de DNA/efeitos dos fármacos , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Fatores de Transcrição , Fator de Ligação a CCAAT/efeitos dos fármacos , Fator de Ligação a CCAAT/metabolismo , Cádmio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ilhas de CpG , Fator de Transcrição E2F1/efeitos dos fármacos , Fator de Transcrição E2F1/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/efeitos dos fármacos , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Células HEK293 , Humanos , Células MCF-7 , Metiltransferases/efeitos dos fármacos , Metiltransferases/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteína-Arginina N-Metiltransferases/efeitos dos fármacos , Proteína-Arginina N-Metiltransferases/genética , Fatores de Transcrição/metabolismo
15.
RNA ; 26(1): 10-18, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31601735

RESUMO

Assessing variations in mRNA stability typically involves inhibiting transcription either globally or in a gene-specific manner. Alternatively, mRNA pulse-labeling strategies offer a means to calculate mRNA stability without inhibiting transcription. However, key stress-responsive cell signaling pathways, which affect mRNA stability, may themselves be perturbed by the approaches used to measure mRNA stability, leading to artifactual results. Here, we have focused on common strategies to measure mRNA half-lives in yeast and determined that commonly used transcription inhibitors thiolutin and 1,10 phenanthroline inhibit TORC1 signaling, PKC signaling, and partially activate HOG signaling. Additionally, 4-thiouracil (4tU), a uracil analog used in mRNA pulse-labeling approaches, modestly induces P-bodies, mRNA-protein granules implicated in storage and decay of nontranslating mRNA. Thiolutin also induces P-bodies, whereas phenanthroline has no effect. Doxycycline, which controls "Tet On/Tet Off" regulatable promoters, shows no impact on the above signaling pathways or P-bodies. In summary, our data argues that broad-acting transcriptional inhibitors are problematic for determining mRNA half-life, particularly if studying the impacts of the TORC1, HOG, or PKC pathway on mRNA stability. Regulatable promoter systems are a preferred approach for individual mRNA half-life studies, with 4tU labeling representing a good approach to global mRNA half-life analysis, despite modestly inducing P-bodies.


Assuntos
Estabilidade de RNA/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Citoplasma/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Meia-Vida , Fenantrolinas/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Pirrolidinonas/farmacologia , RNA Fúngico/química , RNA Fúngico/efeitos dos fármacos , RNA Mensageiro/química , RNA Mensageiro/efeitos dos fármacos , Saccharomyces cerevisiae/fisiologia , Estresse Fisiológico
16.
Mol Cell Endocrinol ; 499: 110588, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31541682

RESUMO

The presence of insulin receptor (IR) on insulin-secreting beta cells suggests an autocrine regulatory role for insulin in its own signalling. Congenital beta cell-specific IR knockout (ßIRKO) mouse studies have demonstrated the development of age-dependent glucose intolerance. We investigated the role of beta cell IR signalling specifically during postnatal life following undisturbed prenatal pancreatic development and maturation. We utilized a tamoxifen-inducible mouse insulin 1 promoter (MIP) driven Cre recombinase IR knockout mouse model (MIP-ßIRKO) to achieve partial knockout of IR in islets and determine the functional role of beta cell IR in adult mice fed a control normal diet (ND) or 60% high-fat diet (HFD). At 24 weeks of age, MIP-ßIRKO ND mice maintained glucose tolerance, insulin release, and unchanged beta cell mass when compared to control ND mice. In contrast, 24-week-old MIP-ßIRKO mice demonstrated significant glucose intolerance and lower insulin release after 18 weeks of HFD feeding. A reduction in beta cell soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein expression, phosphorylated AktS473 and P70S6K1T389, and glucose transporter 2 (GLUT2) expression were also identified in MIP-ßIRKO HFD islets. Overall, the postnatal knockout of beta cell IR in HFD-fed mice resulted in decreased expression of beta cell glucose-sensing and exocytotic proteins and a reduction in intracellular signalling. These findings highlight that IR expression in the adult islet is required to maintain beta cell function under hyperglycemic stress.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/citologia , Receptor de Insulina/genética , Animais , Animais Recém-Nascidos , Células Cultivadas , Intolerância à Glucose/tratamento farmacológico , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas/efeitos dos fármacos , Transdução de Sinais , Tamoxifeno/farmacologia
17.
Mol Carcinog ; 59(1): 24-31, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31579968

RESUMO

Hexavalent chromium is recognized as a human carcinogen. Our previous studies revealed that lung cancer (LC) in chromate-exposed workers (chromate LC) had molecular features of frequent microsatellite instability (MSI), repression of MLH1 level, and aberrant DNA methylation of several tumor-suppressor genes, including MLH1. In the present study, we quantitatively investigated MLH1-promoter methylation status using bisulfite pyrosequencing of paired tumorous/nontumorous tissues from chromate and nonchromate LCs to determine the effect of chromate exposure on MLH1-promoter methylation. The methylation level of MLH1 promoter was significantly higher in chromate LC tumors (P < .001) than nonchromate LC tumors and, among chromate LC, significantly higher in tumorous tissue than nontumorous tissue (P = .004). Moreover, the methylation level of MLH1 promoter in normal lung tissue tended to be higher in chromate LC than nonchromate LC (P = .062). In addition, LC with reduced levels of MLH1 showed significantly higher methylation levels of MLH1 promoter than LC exhibiting normal MLH1 levels (P = .019). Moreover, immunohistochemical analyses determined that levels of SUV39H1, an H3K9me2-related methyltransferase, were higher in chromate LC than nonchromate LC (P = .076). Furthermore, we evaluated three DNA double-strand break-repair genes (MRE11, RAD50, and DNA-PKcs) as possible targets of MSI by fragment-length polymorphism analysis, revealing the mutation frequency of RAD50 as significantly higher in chromate LC than nonchromate LC (P = .047). These results suggest that chromate exposure might induce MLH1 hypermethylation in LC as a mechanism of chromate-induced carcinogenesis.


Assuntos
Cromatos/efeitos adversos , Metilação de DNA/efeitos dos fármacos , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/genética , Proteína 1 Homóloga a MutL/genética , Idoso , Carcinogênese/induzido quimicamente , Carcinogênese/genética , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Humanos , Instabilidade de Microssatélites/efeitos dos fármacos , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/efeitos dos fármacos
18.
Genes (Basel) ; 10(12)2019 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-31783675

RESUMO

Prenyl diphosphate synthase subunit 2 (PDSS2) is the first key enzyme in the CoQ10 biosynthesis pathway, and contributes to various metabolic and nephritic diseases. It has been reported that PDSS2 is downregulated in several types of tumors and acts as a potential tumor suppressor gene to inhibit the proliferation and migration of cancer cells. However, the regulatory mechanism of PDSS2 expression remains elusive. In the present study, we first identified and characterized the PDSS2 promoter region. We established four different luciferase reporter constructs which mainly cover the 2 kb region upstream of the PDSS2 gene transcription initiation site. Series luciferase reporter assay demonstrated that all four constructs have prominent promoter activity, and the core promoter of PDSS2 is mainly located within the 202 bp region near its transcription initiation site. Transcription factor binding site analysis revealed that the PDSS2 promoter contains binding sites for canonical transcription factors such as Sp1 and GATA-1. Overexpression of Sp1 significantly inhibited PDSS2 promoter activity, as well as its endogenous expression, at both mRNA and protein levels in lung cancer cells. Site-directed mutagenesis assay further confirmed that the Sp1 binding sites are essential for proximal prompter activity of PDSS2. Consistently, a selective Sp1 inhibitor, mithramycin A, treatment repressed the PDSS2 promoter activity, as well as its endogenous expression. Chromatin immunoprecipitation (ChIP) assay revealed that Sp1 binds to the PDSS2 promoter in vivo. Of note, the expression of Sp1 and PDSS2 are negatively correlated, and higher Sp1 expression with low PDSS2 expression is significantly associated with poor prognosis in lung cancer. Taken together, our results strongly suggest the essential role of Sp1 in maintaining the basic constitutive expression of PDSS2, and the pathogenic implication of Sp1-mediated PDSS2 transcriptional repression in lung cancer cells.


Assuntos
Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Neoplasias Pulmonares/mortalidade , Fator de Transcrição Sp1/genética , Células A549 , Alquil e Aril Transferases/química , Alquil e Aril Transferases/efeitos dos fármacos , Sítios de Ligação , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Plicamicina/análogos & derivados , Plicamicina/farmacologia , Prognóstico , Regiões Promotoras Genéticas/efeitos dos fármacos , Fator de Transcrição Sp1/metabolismo , Análise de Sobrevida , Sítio de Iniciação de Transcrição
19.
Acta Biochim Pol ; 66(4): 619-625, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31826047

RESUMO

The CD146 (also known as MCAM, MUC-18, Mel-CAM) was initially reported in 1987, as a protein crucial for the invasiveness of malignant melanoma. Recently, it has been confirmed that CD146 has been involved in progression and poor overall survival of many cancers including breast cancer. Importantly, in independent studies, CD146 was reported to be a trigger of epithelial to mesenchymal transition in breast cancer cells. The goal of our current study was to verify the potential involvement of epigenetic mechanism behind the regulation of CD146 expression in breast cancer cells, as it has been previously reported in prostate cancer. First, we analysed the response of breast cancer cell lines, differing in the initial CD146 mRNA and protein content, to epigenetic modifier, 5-aza-2-deoxycytidine, and subsequently the methylation status of CD146 gene promoter was investigated, using direct bisulfite sequencing. We observed that treatment with demethylating agent led to induction of CD146 expression in all analysed breast cancer cell lines, both at mRNA and protein level, what was accompanied by increased expression of selected mesenchymal markers. Importantly, CD146 gene promoter analysis showed aberrant CpG island methylation in 2 out of 3 studied breast cancer cells lines, indicating epigenetic regulation of CD146 gene expression. In conclusion, our study revealed, for the first time, that aberrant methylation maybe involved in expression control of CD146, a very potent EMT inducer in breast cancer cells. Altogether, the data obtained may provide the basis for novel therapies as well as diagnostic approaches enabling sensitive and very accurate detection of breast cancer cells.


Assuntos
Neoplasias da Mama/genética , Metilação de DNA/efeitos dos fármacos , Decitabina/farmacologia , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Antígeno CD146/genética , Ilhas de CpG/efeitos dos fármacos , Progressão da Doença , Epigênese Genética/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Regiões Promotoras Genéticas/efeitos dos fármacos , Análise de Sequência de DNA
20.
BMC Biotechnol ; 19(1): 79, 2019 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-31747926

RESUMO

BACKGROUND: Drought is one of the most harmful abiotic stresses for plants, leading to reduced productivity of several economically important crops and, consequently, considerable losses in the agricultural sector. When plants are exposed to stressful conditions, such as drought and high salinity, they modulate the expression of genes that lead to developmental, biochemical, and physiological changes, which help to overcome the deleterious effects of adverse circumstances. Thus, the search for new specific gene promoter sequences has proved to be a powerful biotechnological strategy to control the expression of key genes involved in water deprivation or multiple stress responses. RESULTS: This study aimed to identify and characterize the GmRD26 promoter (pGmRD26), which is involved in the regulation of plant responses to drought stress. The expression profile of the GmRD26 gene was investigated by qRT-PCR under normal and stress conditions in Williams 82, BR16 and Embrapa48 soybean-cultivars. Our data confirm that GmRD26 is induced under water deficit with different induction folds between analyzed cultivars, which display different genetic background and physiological behaviour under drought. The characterization of the GmRD26 promoter was performed under simulated stress conditions with abscisic acid (ABA), polyethylene glycol (PEG) and drought (air dry) on A. thaliana plants containing the complete construct of pGmRD26::GUS (2.054 bp) and two promoter modules, pGmRD26A::GUS (909 pb) and pGmRD26B::GUS (435 bp), controlling the expression of the ß-glucuronidase (uidA) gene. Analysis of GUS activity has demonstrated that pGmRD26 and pGmRD26A induce strong reporter gene expression, as the pAtRD29 positive control promoter under ABA and PEG treatment. CONCLUSIONS: The full-length promoter pGmRD26 and the pGmRD26A module provides an improved uidA transcription capacity when compared with the other promoter module, especially in response to polyethylene glycol and drought treatments. These data indicate that pGmRD26A may become a promising biotechnological asset with potential use in the development of modified drought-tolerant plants or other plants designed for stress responses.


Assuntos
Ácido Abscísico/farmacologia , Soja/genética , Biotecnologia/métodos , Secas , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Soja/efeitos dos fármacos , Estresse Fisiológico/genética , Estresse Fisiológico/fisiologia
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