RESUMO
BACKGROUND: Colorectal cancer (CRC) screening can help to reduce its incidence and mortality. Noninvasive strategies, such as plasma analysis of epigenetic alterations, can constitute important biomarkers of CRC detection. OBJECTIVE: This study aimed to evaluate the plasma methylation status of SEPT9 and BMP3 promoters as biomarkers for detection of CRC and its precursor lesions in a Brazilian population. METHODS: Plasma samples from 262 participants of the CRC screening program of Barretos Cancer Hospital who had a positive fecal occult blood test and underwent colonoscopy and cancer patients were analyzed. Participants were grouped according to the worst lesion detected in the colonoscopy. Cell-free circulating DNA (cfDNA) was bisulfite treated followed by the analysis of SEPT9 and BMP3 methylation status using a droplet digital PCR system (ddPCR). The best methylation cutoff value for group discrimination was calculated by receiver operating characteristic (ROC) curve analysis. RESULTS: Among the 262 participants, 38 were diagnosed with CRC, 46 with advanced adenomas 119 with nonadvanced adenomas, three with sessile serrated lesions, and 13 with hyperplastic polyps. In 43 participants, no lesion was detected in the colonoscopy and were used as controls. The CRC group showed the highest cfDNA concentration (10.4 ng/mL). For the SEPT9 gene, a cutoff of 2.5% (AUC = 0.681) that discriminates between CRC and the control group resulted in CRC sensitivity and specificity of 50% and 90%, respectively. Concerning the BMP3 gene, a cutoff of 2.3% (AUC = 0.576) showed 40% and 90% of sensitivity and specificity for CRC detection, respectively. Combining SEPT9, BMP3 status, and age over 60 years resulted in a better performance for detecting CRC (AUC = 0.845) than the individual gene models, yielding 80% and 81% of sensitivity and specificity, respectively. CONCLUSION: The present study suggests that a combination of SEPT9 and BMP3 plasma methylation, along with age over 60 years, showed the highest performance in detecting CRC in a Brazilian population. These noninvasive biomarkers can potentially serve as useful tools for CRC screening programs.
Assuntos
Adenoma , Ácidos Nucleicos Livres , Neoplasias Colorretais , Humanos , Pessoa de Meia-Idade , Detecção Precoce de Câncer , Brasil/epidemiologia , Metilação de DNA , Septinas/genética , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Sensibilidade e Especificidade , Adenoma/diagnóstico , Adenoma/genética , Biomarcadores Tumorais/genética , Proteína Morfogenética Óssea 3/genéticaRESUMO
Lung adenocarcinoma (LUAD) is a malignant tumor that occurs in the lungs. Numerous reports have substantiated the participation of long non-coding RNAs (lncRNAs) in the tumorigenesis of LUAD. Previously, lncRNA alpha-2-macroglobulin antisense RNA 1 (A2M-AS1) was confirmed to be an important regulator in the biological processes of LUAD and dysregulation of A2M-AS1 was associated with non-small cell lung cancer (NSCLC) progression. However, the precise mechanism of A2M-AS1 in LUAD has not been elucidated. Therefore, our study was designed to investigate the detailed molecular mechanism of A2M-AS1 in LUAD. Herein, the expression of lncRNA A2M-AS1, microRNA (miRNA) miR-587, and bone morphogenetic protein 3 (BMP3) in LUAD cell lines and tissues were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting. The viability, proliferation, migration and invasion of LUAD cells were tested by cell counting kit-8 (CCK-8), colony formation and Transwell assays. In vivo tumor growth was investigated by xenograft animal experiment. Interactions among A2M-AS1, miR-587 and BMP3 were measured by RNA pulldown and luciferase reporter assays. In this study, A2M-AS1 was downregulated in LUAD tissues and cells and related to poor prognosis in LUAD patients. A2M-AS1 overexpression suppressed LUAD cell proliferation, migration and invasion in vitro and inhibited tumor growth in vivo. Mechanistically, A2M-AS1 directly bound with miR-587 to promote BMP3 expression in LUAD cells. Low expression of BMP3 was found in LUAD tissues and cells and was closely correlated with poor prognosis in LUAD patients. BMP3 deficiency reserved the inhibitory influence of A2M-AS1 overexpression on LUAD cell behaviors. Overall, A2M-AS1 inhibits cell growth and aggressiveness via regulating the miR-587/BMP3 axis in LUAD.
Assuntos
Adenocarcinoma de Pulmão , Proteína Morfogenética Óssea 3 , Neoplasias Pulmonares , MicroRNAs , RNA Longo não Codificante , alfa-Macroglobulinas , Animais , Humanos , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , alfa-Macroglobulinas/genética , alfa-Macroglobulinas/metabolismo , Proteína Morfogenética Óssea 3/genética , Proteína Morfogenética Óssea 3/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Invasividade Neoplásica/genética , Invasividade Neoplásica/fisiopatologia , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Metástase Neoplásica/fisiopatologia , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Progressão da DoençaRESUMO
Bone morphogenetic proteins (BMPs) have a major role in tissue development. BMP3 is synthesized in osteocytes and mature osteoblasts and has an antagonistic effect on other BMPs in bone tissue. The main aim of this study was to fully characterize cortical bone and trabecular bone of long bones in both male and female Bmp3-/- mice. To investigate the effect of Bmp3 from birth to maturity, we compared Bmp3-/- mice with wild-type littermates at the following stages of postnatal development: 1 day (P0), 2 weeks (P14), 8 weeks and 16 weeks of age. Bmp3 deletion was confirmed using X-gal staining in P0 animals. Cartilage and bone tissue were examined in P14 animals using Alcian Blue/Alizarin Red staining. Detailed long bone analysis was performed in 8-week-old and 16-week-old animals using micro-CT. The Bmp3 reporter signal was localized in bone tissue, hair follicles, and lungs. Bone mineralization at 2 weeks of age was increased in long bones of Bmp3-/- mice. Bmp3 deletion was shown to affect the skeleton until adulthood, where increased cortical and trabecular bone parameters were found in young and adult mice of both sexes, while delayed mineralization of the epiphyseal growth plate was found in adult Bmp3-/- mice.
Assuntos
Proteína Morfogenética Óssea 3/genética , Osso e Ossos/metabolismo , Osso Cortical/metabolismo , Osteogênese/genética , Fatores Etários , Animais , Biomarcadores , Proteína Morfogenética Óssea 3/metabolismo , Calcificação Fisiológica , Feminino , Expressão Gênica , Lâmina de Crescimento/crescimento & desenvolvimento , Lâmina de Crescimento/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Fatores Sexuais , Microtomografia por Raio-XRESUMO
BACKGROUND: Research on the role of lncRNAs in the process of bone metastasis in breast cancer (BM-BCa) has just begun at an early stage, and an increasing number of lncRNAs have been proved to play a regulatory role in the process of BM-BCa. Our study focused on the balance of osteogenic-osteoclast regulated by lncRNA-SNHG3 in bone metastasis microenvironment. METHODS: SNHG3 level of clinical tissues and breast cancer cell lines was determined by RT-qPCR. ALP staining, ALP activity identification and western blotting of OPG, OSX, RUNX2, BMP2 together with BMP3 was performed to verify the osteogenesis of bone marrow mesenchymal stem cells (BMSCs) both in vitro and in vivo. Exosomes derived from MDA-MB-231 were characterized and sequenced, followed by RT-qPCR. Dual luciferase reporter gene assay was utilized to analyze the binding sites of miR-1273g-3p on SNHG3 and BMP3. RESULTS: Expression of BMP3 was positively regulated by SNHG3 via exosomal miR-1273g-3p. CONCLUSION: The overexpression of SNHG3 in breast cancer cells may be responsible for osteolytic metastasis Thus, knockdown of SNHG3 might be a potential target for improvement of BM-BCa Treatment.
Assuntos
Proteína Morfogenética Óssea 3/genética , Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , RNA Longo não Codificante/metabolismo , Regiões 3' não Traduzidas , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Neoplasias da Mama/patologia , Diferenciação Celular , Exossomos , Feminino , Humanos , Células MCF-7 , Metástase Neoplásica , Osteogênese , Microambiente TumoralRESUMO
INTRODUCTION: Osteoporosis is the most susceptible disease for people over 60. The main cause of osteoporosis is the decreased osteogenic differentiation of mesenchymal stem cells (MSCs). Here we showed that upstream stimulatory factor 2 (USF2)/microRNA-34a (miR-34a)/bone morphogenetic protein 3 (BMP3) axis regulated osteogenic differentiation of BMSCs. MATERIALS AND METHODS: USF2 and miR-34a expression were examined using qPCR. Protein levels of BMP3 and osteogenic markers expression were evaluated using both western blot and qPCR. Activity of ALP was determined by ALP assay kit. Mineralization capacity of hBMSCs was assessed using ARS. Besides, CHIP assay was employed to verify whether USF2 could bind to miR-34a promoter. Finally, RIP assay and dual-luciferase reporter assay were employed to verify whether miR-34a directly bound to BMP3. RESULTS: Our results suggested that miR-34a was upregulated during osteogenic differentiation of BMSCs, and miR-34a overexpression could enhance osteogenic differentiation of BMSCs. USF2 could positively regulate miR-34a expression by interacting with its promoter. USF2 overexpression enhanced osteogenic differentiation of BMSCs, while miR-34a inhibition reversed the effect. Besides, BMP3 was the target of miR-34a. MiR-34a overexpression enhanced osteogenic differentiation of BMSCs, which was abolished by BMP3 overexpression. CONCLUSION: Taken together, USF2 enhanced osteogenic differentiation of BMSCs via downregulating BMP3 by interacting with miR-34a promoter.
Assuntos
Proteína Morfogenética Óssea 3/genética , MicroRNAs , Diferenciação Celular , Células Cultivadas , Humanos , MicroRNAs/genética , Osteogênese/genética , Ativação Transcricional , Fatores Estimuladores UpstreamRESUMO
Botrytis cinerea can attack over 500 genera of vascular plants and is considered the second phytopathogen in the 'top ten' for its economic importance. Traditional fungicides can be ineffective and with increasing fungicide resistance, new sustainable technologies are required. Lately, RNA interference-based fungicides are emerging for their potential uses in crop protection. Therefore, we assessed the potential of this innovative approach targeting the MAP kinase Bmp3 in B. cinerea, a gene involved in saprophytic growth, response to low osmolarity, conidiation, surface sensing, host penetration and lesion formation. After performing a prediction analysis of small interfering RNAs, a 427 nucleotides long dsRNA was selected as construct. We tested the effect of topical applications of dsRNA construct both in vitro by a fungal growth assay in microtiter plates and in vivo on detached lettuce leaves artificially inoculated. In both cases, topical applications of dsRNA led to gene knockdown with a delay in conidial germination, an evident growth retardation and a strong reduction of necrotic lesions on leaves. These results correlated with a strongly reduced expression of Bmp3 gene. In accordance to these findings, the Bmp3 gene could be a promising target for the development of an RNAi-based fungicide against B. cinerea.
Assuntos
Proteína Morfogenética Óssea 3/genética , Botrytis/genética , Proteína Morfogenética Óssea 3/metabolismo , Botrytis/metabolismo , Botrytis/patogenicidade , Fungicidas Industriais/metabolismo , Lactuca/genética , Lactuca/microbiologia , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , RNA de Cadeia Dupla/metabolismo , RNA de Cadeia Dupla/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , VirulênciaRESUMO
Rheumatoid arthritis (RA) is a persistent autoimmune disease. Fibroblast-like synoviocytes (FLS) are a key component of invasive pannus and a pathogenetic mechanism in RA. Expression of bone morphogenetic protein 3 (BMP3) mRNA is reportedly decreased in the arthritic synovium. We previously showed that BMP3 expression is significantly downregulated in the synovial tissues of RA patients and models of adjuvant-induced arthritis (AIA). In the present study, we explored the association between BMP3 and FLS migration and secretion of proinflammatory factors in RA. We found that inhibition of BMP3 expression using BMP3 siRNA increased the proinflammatory chemokines and migration of FLS stimulated with TNF-α. Inhibition of BMP3 expression also increased expression of IL-6, IL-1ß, IL-17A, CCL-2, CCL-3, VCAM-1, MMP-3, and MMP-9, but not TIMP-1, in AIA and RA FLS. Correspondingly, induction of BMP3 overexpression through intra-articular injection of ad-BMP3 diminished arthritis severity in AIA rats. We also found that BMP3 may inhibit activation of TGF-ß1/Smad signaling. These data indicate that BMP3 may suppress the proliferation and migration of FLS via the TGF-ß1/Smad signaling pathway.
Assuntos
Artrite Reumatoide/imunologia , Proteína Morfogenética Óssea 3/metabolismo , Membrana Sinovial/imunologia , Sinoviócitos/imunologia , Animais , Artrite Experimental/diagnóstico , Artrite Experimental/imunologia , Artrite Experimental/patologia , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/patologia , Artrite Reumatoide/cirurgia , Proteína Morfogenética Óssea 3/genética , Movimento Celular/genética , Movimento Celular/imunologia , Células Cultivadas , Quimiocinas/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Cultura Primária de Células , Ratos , Índice de Gravidade de Doença , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Proteínas Smad/metabolismo , Sinovectomia , Membrana Sinovial/patologia , Sinoviócitos/patologia , Fator de Crescimento Transformador beta1/metabolismoRESUMO
INTRODUCTION: We set out to evaluate the performance of a multitarget stool DNA (MT-sDNA) in an average-risk colonoscopy-controlled colorectal cancer (CRC) screening population. MT-sDNA stool test results were evaluated against fecal immunochemical test (FIT) results for the detection of different lesions, including molecularly defined high-risk adenomas and several other tumor characteristics. METHODS: Whole stool samples (n = 1,047) were prospectively collected and subjected to an MT-sDNA test, which tests for KRAS mutations, NDRG4 and BMP3 promoter methylation, and hemoglobin. Results for detecting CRC (n = 7), advanced precancerous lesions (advanced adenoma [AA] and advanced serrated polyps; n = 119), and non-AAs (n = 191) were compared with those of FIT alone (thresholds of 50, 75, and 100 hemoglobin/mL). AAs with high risk of progression were defined by the presence of specific DNA copy number events as measured by low-pass whole genome sequencing. RESULTS: The MT-sDNA test was more sensitive than FIT alone in detecting advanced precancerous lesions (46% (55/119) vs 27% (32/119), respectively, P < 0.001). Specificities among individuals with nonadvanced or negative findings (controls) were 89% (791/888) and 93% (828/888) for MT-sDNA and FIT testing, respectively. A positive MT-sDNA test was associated with multiple lesions (P = 0.005), larger lesions (P = 0.03), and lesions with tubulovillous architecture (P = 0.04). The sensitivity of the MT-sDNA test or FIT in detecting individuals with high-risk AAs (n = 19) from individuals with low-risk AAs (n = 52) was not significantly different. DISCUSSION: In an average-risk screening population, the MT-sDNA test has an increased sensitivity for detecting advanced precancerous lesions compared with FIT alone. AAs with a high risk of progression were not detected with significantly higher sensitivity by MT-sDNA or FIT.
Assuntos
Adenoma/diagnóstico , Pólipos do Colo/diagnóstico , Neoplasias Colorretais/diagnóstico , DNA/análise , Fezes/química , Hemoglobinas/análise , Adenoma/genética , Adenoma/metabolismo , Adenoma/patologia , Idoso , Proteína Morfogenética Óssea 3/genética , Pólipos do Colo/genética , Pólipos do Colo/metabolismo , Pólipos do Colo/patologia , Colonoscopia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Detecção Precoce de Câncer , Feminino , Hemoglobinas/metabolismo , Humanos , Imunoquímica , Masculino , Pessoa de Meia-Idade , Proteínas Musculares/genética , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
BACKGROUND: BMP3 gene is often found hypermethylated and hence inactivated in several types of cancers including colorectal cancer (CRC), indicating that it has a suppressor role in carcinogenesis. Though BMP3 is a reliable biomarker for screening CRC, the molecular mechanism of BMP3 in carcinogenesis remains largely unknown. METHODS: The expression level of BMP3 was examined by immunohistochemistry staining and western blot. Methylation-specific PCR (MSP) and real-time quantitative MSP were used to test the hypermethylation status of BMP3 gene. Analyses of BMP3 function in colon cancer cell proliferation, migration, invasion, and apoptosis were performed using HCT116 and KM12 cells. BMP3 was further knocked down or overexpressed in CRC cells, and the effects on cell growth of xenograft tumors in nude mice were assessed. Co-immunoprecipitation and immunofluorescence staining were used to analyze the association between BMP3 and BMPR2 or BMP3 and ActRIIB. Microarray analysis was performed to identify most differentially expressed genes and pathways regulated by BMP3. The BMP3-regulated SMAD2-dependent signaling pathway and TAK1/JNK signal axes were further investigated by quantitative PCR and western blot. RESULTS: BMP3 gene was hypermethylated and its expression was downregulated in both CRC tissues and cell lines. Expressing exogenous BMP3 in HCT116 inhibited cell growth, migration, and invasion and increased rate of apoptosis both in vitro and in vivo. However, shRNA-mediated attenuation of endogenous BMP3 in KM12 reversed such inhibitory and apoptotic effects. Furthermore, BMP3 could bind to ActRIIB, an activin type II receptor at the cellular membrane, thereby activating SMAD2-dependent pathway and TAK1/JNK signal axes to regulate downstream targets including caspase-7, p21, and SMAD4 that play crucial roles in cell cycle control and apoptosis. CONCLUSIONS: Our study reveals a previously unknown mechanism of BMP3 tumor suppression in CRC and provides a rationale for future investigation of BMP3 as a potential target for the development of novel therapeutic agents to fight CRC.
Assuntos
Proteína Morfogenética Óssea 3/metabolismo , Neoplasias Colorretais/patologia , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Proteína Morfogenética Óssea 3/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Metilação de DNA , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , MAP Quinase Quinase 4/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Transplante de Neoplasias , Proteína Smad2/genética , Proteína Smad2/metabolismoRESUMO
OBJECTIVES: Pancreatic cystic lesions (PCLs) may be precancerous. Those likely to harbor high-grade dysplasia (HGD) or pancreatic cancer (PC) are targets for surgical resection. Current algorithms to predict advanced neoplasia (HGD/PC) in PCLs lack diagnostic accuracy. In pancreatic tissue and cyst fluid (CF) from PCLs, we sought to identify and validate novel methylated DNA markers (MDMs) that discriminate HGD/PC from low-grade dysplasia (LGD) or no dysplasia (ND). METHODS: From an unbiased whole-methylome discovery approach using predefined selection criteria followed by multistep validation on case (HGD or PC) and control (ND or LGD) tissues, we identified discriminant MDMs. Top candidate MDMs were then assayed by quantitative methylation-specific polymerase chain reaction on archival CF from surgically resected PCLs. RESULTS: Of 25 discriminant MDMs identified in tissue, 13 were selected for validation in 134 CF samples (21 cases [8 HGD, 13 PC], 113 controls [45 ND, 68 LGD]). A tree-based algorithm using 2 CF-MDMs (TBX15, BMP3) achieved sensitivity and specificity above 90%. Discrimination was significantly better by this CF-MDM panel than by mutant KRAS or carcinoembryonic antigen, with areas under the receiver operating characteristic curve of 0.93 (95% confidence interval: 0.86-0.99), 0.71 (0.57-0.85), and 0.72 (0.60-0.84), respectively. Cutoffs for the MDM panel applied to an independent CF validation set (31 cases, 56 controls) yielded similarly high discrimination, areas under the receiver operating characteristic curve = 0.86 (95% confidence interval: 0.77-0.94, P = 0.2). DISCUSSION: Novel MDMs discovered and validated in tissue accurately identify PCLs harboring HGD/PC. A panel of 2 MDMs assayed in CF yielded results with potential to enhance current risk prediction algorithms. Prospective studies are indicated to optimize and further evaluate CF-MDMs for clinical use.
Assuntos
Carcinoma Ductal Pancreático/genética , Cistadenoma Seroso/genética , Metilação de DNA/genética , Cisto Pancreático/genética , Neoplasias Intraductais Pancreáticas/genética , Neoplasias Pancreáticas/genética , Lesões Pré-Cancerosas/genética , Idoso , Proteína Morfogenética Óssea 3/genética , Antígeno Carcinoembrionário/metabolismo , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/patologia , Líquido Cístico/metabolismo , Cistadenoma Seroso/diagnóstico , Cistadenoma Seroso/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Cisto Pancreático/diagnóstico , Cisto Pancreático/patologia , Neoplasias Intraductais Pancreáticas/diagnóstico , Neoplasias Intraductais Pancreáticas/patologia , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/patologia , Reação em Cadeia da Polimerase , Lesões Pré-Cancerosas/diagnóstico , Lesões Pré-Cancerosas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Proteínas com Domínio T/genéticaRESUMO
Detecting cfDNA in plasma or serum could serve as a 'liquid biopsy', for circulating tumor DNA with aberrant methylation patterns offer a possible method for early detection of several cancers which could avoid the need for tumor tissue biopsies. Bone Morphogenetic Protein 3 (BMP3) was identified as a candidate tumor suppressor gene putatively down-regulated in colorectal cancer (CRC). In this study, we aimed to assess the potential role of BMP3 promoter methylation changes in plasma DNA for detection of colorectal cancerous and precancerous lesions. Plasma DNA samples were extracted from 50 patients with histologically diagnosed polyps or tumor and 50 patients reported negative for polyps or tumors. The procedure consists of bisulfite conversion of the extracted DNA, purification of bis-DNA, and BMP3 methylation status analysis by using the bisulfite specific high resolution melting analysis. This study demonstrated that there was a significantly higher frequency of BMP3 methylated DNA in plasma in patients with polyps versus healthy controls with a sensitivity and specificity of 40 and 94%, respectively. In conclusion, our results demonstrated that BMP3 DNA methylation in plasma had not have sufficient sensitivity and it should be used in combination with other biomarkers for the detection of CRC.
Assuntos
Proteína Morfogenética Óssea 3/genética , DNA Tumoral Circulante/genética , Neoplasias Colorretais/genética , Metilação de DNA , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , DNA Tumoral Circulante/sangue , Neoplasias Colorretais/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras GenéticasRESUMO
A study was carried out to characterize and explore the expression profile of BMP 3 gene in control broiler and control layer chicken. The total open reading frame of BMP 3 (1389 bp) was cloned and sequenced. The control broiler and control layer chicken showed variation at nucleotide and amino acid level with reference gene (Gallus gallus, NCBI Acc. No. NM_001034819). When compared to reference gene, the control broiler showed four nucleotide differences (c.192A>G, c.519C>T, 903G>A and 960C>G), while, control layer showed variation at c.33G>C, 192A>G, 858G>A, 904G>A, 960C>G and 1257C>T making six differences in total. However, between control broiler and control layer lines, nucleotide differences was observed at c.33G>C, 519T>C, 858G>A, 903A>G, 904G>A and 1257C>T. The change at amino acid level between reference and control broiler was p.D320N and with control layer chicken, it was p.D302N and p.D320N. On the other hand, a single amino acid difference (p.D302N) was observed between the control broiler and control layer chicken lines. The phylogenetic study displayed a close relationship between broiler and layer lines and reference gene and also with other avian species resulting in a cluster formation. These cluster in turn displayed a distant link with the mammalian species. The expression profile of BMP 3 gene exhibited a variation at different stages of embryonic development and also at post embryonic period among the lines with control layer showing higher expression than that of broiler chicken. The protein was also detected in bone marrow tissue of broiler and layer lines by western blotting. It is concluded that the BMP 3 gene sequence differed at nucleotide and amino acid level among the lines and the gene expressed differentially at different periods of embryonic development and also at post hatch period.
Assuntos
Proteína Morfogenética Óssea 3/genética , Galinhas/genética , Animais , Composição de Bases , Sequência de Bases , Filogenia , TranscriptomaRESUMO
Using our novel surgical model of simultaneous intestinal adaptation "A" and neointestinal regeneration "N" conditions in individual rats to determine feasibility for research and clinical application, we further utilized next generation RNA sequencing (RNA-Seq) here in normal control tissue and both conditions ("A" and "N") across time to decipher transcriptome changes in neoregeneration and adaptation of intestinal tissue at weeks 1, 4, and 12. We also performed bioinformatics analyses to identify key growth factors for improving intestinal adaptation and neointestinal regeneration. Our analyses indicate several interesting phenomena. First, Gene Ontology and pathway analyses indicate that cell cycle and DNA replication processes are enhanced in week 1 "A"; however, in week 1 "N", many immune-related processes are involved. Second, we found some growth factors upregulated or downregulated especially in week 1 "N" versus "A". Third, based on each condition and time point versus normal control tissue, we found in week 1 "N" BMP2, BMP3, and NTF3 are significantly and specifically downregulated, indicating that the regenerative process may be inhibited in the absence of these growth factors. This study reveals complex growth factor regulation in small neointestinal regeneration and intestinal adaptation and provides potential applications in tissue engineering by introducing key growth factors identified here into the injury site.
Assuntos
Ciclo Celular/fisiologia , Perfilação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular , Intestinos/fisiologia , Regeneração/fisiologia , Animais , Proteína Morfogenética Óssea 2/biossíntese , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 3/biossíntese , Proteína Morfogenética Óssea 3/genética , Ontologia Genética , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: The compromise of blood brain barrier (BBB) integrity is often associated with human hemorrhage stroke and neurodegeneration diseases, including retina diseases, such as age-related macular degeneration and diabetic retinopathy. Brain pericytes play pivotal roles in regulation and maintenance of BBB integrity. However, the mechanisms underlying brain pericyte development to establish BBB integrity remain unclear. METHODS: Zebrafish transgenic lines Tg(flk1:GFP; gata1:dsRed), Tg(flk1:GFP), Tg(fli1:GFP) and Tg(BRE:GFP) were used in this work. The functional studies of bmp3 were performed by mopholino oligonucleotide (MO) injection, dye-based permeability assay, RT-PCR, in vivo imaging, immunofluorescence staining and statics analysis. RESULTS: Here we report that bmp3 regulates BBB integrity in zebrafish brain by promoting pericyte development. Knockdown of bmp3 with injection of bmp3-MO causes intracerebral hemorrhage in zebrafish embryos. Meanwhile, disruption of bmp3 function by bmp3-MO injection impairs cerebral pericyte coverage in zebrafish embryos. Mechanistically, knockdown of bmp3 disrupts the pattern and activities of BMP signaling in zebrafish brain, thus probably disrupting the balance of TGFß/BMP signaling in zebrafish embryos. CONCLUSION: In summary, our data shows that bmp3 regulates BBB integrity potentially by promoting pericyte development.
Assuntos
Barreira Hematoencefálica/embriologia , Proteína Morfogenética Óssea 3/metabolismo , Embrião não Mamífero/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Proteína Morfogenética Óssea 3/genética , Humanos , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: Colorectal cancer (CRC) is one of the most common cancers in the western world. Screening is an efficient method of reducing cancer-related mortality. Molecular biomarkers for cancer in general and CRC in particular have been proposed, and hypermethylated DNA from stool or blood samples are already implemented as biomarkers for CRC screening. We aimed to evaluate the performance of proven hypermethylated DNA promoter regions as plasma based biomarkers for CRC detection. METHODS: We conducted a cross-sectional case-control study of 193 CRC patients and 102 colonoscopy-verified healthy controls. Using methylation specific polymerase chain reaction, we evaluated 30 DNA promoter regions previously found to be CRC specific. We used multivariable logistic regression with stepwise backwards selection, and subsequent leave-pair-out cross validation, to calculate the optimism corrected area under the receiver operating characteristics curve (AUC) for all stage as well as early stage CRC. RESULTS: None of the individual DNA promoter regions provided an overall sensitivity above 30% at a reasonable specificity. However, seven hypermethylated promoter regions (ALX4, BMP3, NPTX2, RARB, SDC2, SEPT9, and VIM) along with the covariates sex and age yielded an optimism corrected AUC of 0.86 for all stage CRC and 0.85 for early stage CRC. Overall sensitivity for CRC detection was 90.7% at 72.5% specificity using a cut point value of 0.5. CONCLUSIONS: Individual hypermethylated DNA promoter regions have limited value as CRC screening markers. However, a panel of seven hypermethylated promoter regions show great promise as a model for CRC detection.
Assuntos
Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Metilação de DNA/genética , Idoso , Biomarcadores Tumorais/sangue , Proteína Morfogenética Óssea 3/genética , Proteína C-Reativa/genética , Estudos de Casos e Controles , Neoplasias Colorretais/sangue , Estudos Transversais , Proteínas de Ligação a DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas/genética , Curva ROC , Receptores do Ácido Retinoico/genética , Sindecana-2/genética , Fatores de Transcrição/genéticaRESUMO
AIMS: Mechanical factors play significant roles in neointimal hyperplasia after vein grafting, but the mechanisms are not fully understood. Here, we investigated the roles of microRNA-33 (miR-33) in neointimal hyperplasia induced by arterial mechanical stretch after vein grafting. METHODS AND RESULTS: Grafted veins were generated by the 'cuff' technique. Neointimal hyperplasia and cell proliferation was significantly increased, and miR-33 expression was decreased after 1-, 2-, and 4-week grafts. In contrast, the expression of bone morphogenetic protein 3 (BMP3), which is a putative target of miR-33, and the phosphorylation of smad2 and smad5, which are potential downstream targets of BMP3, were increased in the grafted veins. miR-33 mimics/inhibitor and dual luciferase reporter assay confirmed the interaction of miR-33 and BMP3. miR-33 mimics attenuated, while miR-33 inhibitor accelerated, proliferation of venous smooth muscle cells (SMCs). Moreover, recombinant BMP3 increased SMC proliferation and P-smad2 and P-smad5 levels, whereas BMP3-directed siRNAs had the opposite effect. Then, venous SMCs were exposed to a 10%-1.25 Hz cyclic stretch (arterial stretch) by using the FX4000 cyclic stretch loading system in vitro to mimic arterial mechanical conditions. The arterial stretch increased venous SMC proliferation and repressed miR-33 expression, but enhanced BMP3 expression and smad2 and smad5 phosphorylation. Furthermore, perivascular multi-point injection in vivo demonstrated that agomiR-33 not only attenuates BMP3 expression and smad2 and smad5 phosphorylation, but also slows neointimal formation and cell proliferation in grafted veins. These effects of agomiR-33 on grafted veins could be reversed by local injection of BMP3 lentivirus. CONCLUSION: The miR-33-BMP3-smad signalling pathway protects against venous SMC proliferation in response to the arterial stretch. miR-33 is a target that attenuates neointimal hyperplasia in grafted vessels and may have potential clinical applications.
Assuntos
Proliferação de Células , Veias Jugulares/metabolismo , Veias Jugulares/transplante , Mecanotransdução Celular , MicroRNAs/metabolismo , Neointima , Regiões 3' não Traduzidas , Animais , Antagomirs/genética , Antagomirs/metabolismo , Sítios de Ligação , Proteína Morfogenética Óssea 3/genética , Proteína Morfogenética Óssea 3/metabolismo , Células Cultivadas , Hiperplasia , Veias Jugulares/patologia , Masculino , MicroRNAs/genética , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Músculo Liso Vascular/transplante , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Miócitos de Músculo Liso/transplante , Fosforilação , Interferência de RNA , Ratos Sprague-Dawley , Proteína Smad2/metabolismo , Proteína Smad5/metabolismo , Estresse Mecânico , Fatores de Tempo , TransfecçãoRESUMO
BACKGROUND: Fecal immunochemical test (FIT) screening detects most asymptomatic colorectal cancers. Combining FIT screening with stool-based genetic biomarkers increases sensitivity for cancer, but whether DNA biomarkers (biomarkers) differ for cancers detected versus missed by FIT screening has not been evaluated in a community-based population. AIMS: To evaluate tissue biomarkers among Kaiser Permanente Northern California patients diagnosed with colorectal cancer within 2 years after FIT screening. METHODS: FIT-negative and FIT-positive colorectal cancer patients 50-77 years of age were matched on age, sex, and cancer stage. Adequate DNA was isolated from paraffin-embedded specimens in 210 FIT-negative and 211 FIT-positive patients. Quantitative allele-specific real-time target and signal amplification assays were performed for 7 K-ras mutations and 10 aberrantly methylated DNA biomarkers (NDRG4, BMP3, SFMBT2_895, SFMBT2_896, SFMBT2_897, CHST2_7890, PDGFD, VAV3, DTX1, CHST2_7889). RESULTS: One or more biomarkers were found in 414 of 421 CRCs (98.3%). Biomarker expression was not associated with FIT status, with the exception of higher SFMBT2_897 expression in FIT-negative (194 of 210; 92.4%) than in FIT-positive cancers (180 of 211; 85.3%; p = 0.02). There were no consistent differences in biomarker expression by FIT status within age, sex, stage, and cancer location subgroups. CONCLUSIONS: The biomarkers of a currently in-use multi-target stool DNA test (K-ras, NDRG4, and BMP3) and eight newly characterized methylated biomarkers were commonly expressed in tumor tissue specimens, independent of FIT result. Additional study using stool-based testing with these new biomarkers will allow assessment of sensitivity, specificity, and clinical utility.
Assuntos
Proteína Morfogenética Óssea 3/genética , Neoplasias Colorretais , Fezes , Genes ras/genética , Proteínas Musculares/genética , Proteínas do Tecido Nervoso/genética , Idoso , Doenças Assintomáticas , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Proteína Morfogenética Óssea 3/análise , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Metilação de DNA , Feminino , Perfilação da Expressão Gênica , Marcadores Genéticos , Humanos , Imunoquímica/métodos , Imunoquímica/estatística & dados numéricos , Masculino , Programas de Rastreamento/métodos , Programas de Rastreamento/estatística & dados numéricos , Pessoa de Meia-Idade , Proteínas Musculares/análise , Mutação , Proteínas do Tecido Nervoso/análise , PrevalênciaRESUMO
BACKGROUND: Stool DNA testing in patients with inflammatory bowel disease (IBD) may detect colorectal cancer and advanced precancers with high sensitivity; less is known about the presence of DNA markers in small IBD lesions, their association with metachronous neoplasia, or contribution to stool test positivity. METHODS: At a single center in 2 blinded phases, we assayed methylated bone morphogenic protein 3, methylated N-Myc downstream-regulated gene 4, and mutant KRAS in DNA extracted from paraffin-embedded benign lesions, and matched control tissues of patients with IBD, who were followed for subsequent colorectal dysplasia. Stool samples from independent cases and controls with lesions <1 cm or advanced neoplasms were assayed for the same markers. RESULTS: Among IBD lesions (29 low-grade dysplasia, 19 serrated epithelial change, and 10 sessile serrated adenoma/polyps), the prevalence of methylation was significantly higher than in mucosae from 44 matched IBD controls (P < 0.0001 for methylated bone morphogenic protein 3 or methylated N-Myc downstream-regulated gene 4). KRAS mutations were more abundant in serrated epithelial change than all other groups (P < 0.001). Subsequent dysplasia was not associated with DNA marker levels. In stools, the sensitivity of methylated bone morphogenic protein 3 as a single marker was 60% for all lesions <1 cm, 63% for low-grade dysplasia ≥1 cm and 81% for high-grade dysplasia/colorectal cancer, all at 91% specificity (P < 0.0001). CONCLUSIONS: Selected DNA markers known to be present in advanced IBD neoplasia can also be detected in both tissues and stools from IBD patients with small adenomas and serrated lesions. Mutant KRAS exfoliated from serrated epithelial change lesions might raise false-positive rates. These findings have relevance to potential future applications of stool DNA testing for IBD surveillance.
Assuntos
Adenoma/genética , Colite Ulcerativa/complicações , Neoplasias do Colo/diagnóstico , Neoplasias do Colo/genética , Doença de Crohn/complicações , Metilação de DNA , DNA/análise , Vigilância da População , Adenoma/complicações , Adenoma/patologia , Adulto , Biomarcadores Tumorais/genética , Proteína Morfogenética Óssea 3/genética , Estudos de Casos e Controles , Neoplasias do Colo/complicações , Neoplasias do Colo/patologia , Método Duplo-Cego , Fezes/química , Feminino , Dosagem de Genes , Humanos , Mucosa Intestinal/química , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Sensibilidade e EspecificidadeRESUMO
BACKGROUND/AIMS: Aberrant DNA methylation has a specific role in field cancerization. Certain molecular markers, including secreted frizzled-related protein 2 (SFRP2), tissue factor pathway inhibitor 2 (TFPI2 ), N-Myc downstream-regulated gene 4 (NDRG4) and bone morphogenic protein 3 (BMP3), have previously been shown to be hypermethylated in colorectal cancer (CRC). We aim to examine field cancerization in CRC based on the presence of aberrant DNA methylation in normal-appearing tissue from CRC patients. METHODS: We investigated promoter methylation in 34 CRC patients and five individuals with normal colonoscopy results. CRC patients were divided into three tissue groups: tumor tissue, adjacent and nonadjacent normal-appearing tissue. The methylation status (positive: methylation level >20%) of SFRP2 , TFPI2 , NDRG4 , and BMP3 promoters was investigated using methylation-specific PCR. RESULTS: The methylation frequencies of the SFRP2 , TFPI2 , NDRG4 and BMP3 promoters in tumor/adjacent/nonadjacent normal-appearing tissue were 79.4%/63.0%/70.4%, 82.4%/53.6%/60.7%, 76.5%/61.5%/69.2%, 41.2%/35.7%/50.0%, respectively. The methylation levels of the SFRP, TFPI2, NDRG4 and BMP3 promoters in tumor tissues were significantly higher than those in normal-appearing tissue (SFRP2, p=0.013; TFPI2, p<0.001; NDRG4, p=0.003; BMP3, p=0.001). No significant correlation was observed between the methylation levels of the promoters and the clinicopathological variables. CONCLUSIONS: The field effect is present in CRC and affects both the adjacent and nonadjacent normal-appearing mucosa.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias do Colo/genética , Metilação de DNA/genética , Regiões Promotoras Genéticas/genética , Proteína Morfogenética Óssea 3/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Estudos de Casos e Controles , Colo/patologia , Neoplasias do Colo/patologia , Colonoscopia , Feminino , Glicoproteínas/genética , Humanos , Mucosa Intestinal/patologia , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Proteínas Musculares/genética , Proteínas do Tecido Nervoso/genética , Reação em Cadeia da PolimeraseRESUMO
OBJECTIVES: Inflammation in diseases such as rheumatoid arthritis (RA) stimulates osteoclast-mediated articular bone erosion and inhibits osteoblast-mediated bone formation, leading to a net loss of bone. Pro-inflammatory cytokines and antagonists of the Wnt signalling pathway have been implicated in the inhibition of osteoblast differentiation and activity in RA, contributing to the erosive process and impairing erosion healing. Importantly, osteoblast differentiation and function are also regulated by the osteogenic bone morphogenetic protein (BMP) signalling pathway, which is antagonized by BMP3. We therefore examined the potential role of BMP3 in inflammatory arthritis. METHOD: Two murine models of RA, K/BxN serum transfer arthritis (STA) and antigen-induced arthritis (AIA), were used to establish the temporal expression of BMP3 and the cellular sources of BMP3 mRNA and protein in inflammatory arthritis. To determine the effects of inflammation on the expression of BMP3 in osteoblasts, murine calvarial osteoblasts were treated with pro-inflammatory cytokines and BMP3 expression was assessed. RESULTS: In both murine models of RA, BMP3 mRNA and protein are highly expressed by osteoblasts lining inflammation-bone interfaces late in the course of arthritis. Synovial tissues are not a significant source of BMP3. BMP3 expression is induced in osteocalcin-expressing osteoblasts in vitro following stimulation by tumour necrosis factor (TNF). CONCLUSIONS: These data implicate BMP3 as a novel factor that may act locally to contribute to the erosive process and inhibit the repair of articular bone in RA through inhibition of osteoblast differentiation and function.