RESUMO
Macrophages constitute important immune cell targets of the antifolate methotrexate (MTX) in autoimmune diseases, including rheumatoid arthritis. Regulation of folate/MTX metabolism remains poorly understood upon pro-inflammatory (M1-type/GM-CSF-polarized) and anti-inflammatory (M2-type/M-CSF-polarized) macrophages. MTX activity strictly relies on the folylpolyglutamate synthetase (FPGS) dependent intracellular conversion and hence retention to MTX-polyglutamate (MTX-PG) forms. Here, we determined FPGS pre-mRNA splicing, FPGS enzyme activity and MTX-polyglutamylation in human monocyte-derived M1- and M2-macrophages exposed to 50 nmol/L MTX ex vivo. Moreover, RNA-sequencing analysis was used to investigate global splicing profiles and differential gene expression in monocytic and MTX-exposed macrophages. Monocytes displayed six-eight-fold higher ratios of alternatively-spliced/wild type FPGS transcripts than M1- and M2-macrophages. These ratios were inversely associated with a six-ten-fold increase in FPGS activity in M1- and M2-macrophages versus monocytes. Total MTX-PG accumulation was four-fold higher in M1- versus M2-macrophages. Differential splicing after MTX-exposure was particularly apparent in M2-macrophages for histone methylation/modification genes. MTX predominantly induced differential gene expression in M1-macrophages, involving folate metabolic pathway genes, signaling pathways, chemokines/cytokines and energy metabolism. Collectively, macrophage polarization-related differences in folate/MTX metabolism and downstream pathways at the level of pre-mRNA splicing and gene expression may account for variable accumulation of MTX-PGs, hence possibly impacting MTX treatment efficacy.
Assuntos
Metotrexato , Monócitos , Humanos , Metotrexato/farmacologia , Metotrexato/metabolismo , Monócitos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Fator Estimulador de Colônias de Macrófagos/metabolismo , Processamento Alternativo , Precursores de RNA/metabolismo , Ácido Fólico/farmacologia , Ácido Fólico/metabolismo , Macrófagos/metabolismo , Expressão Gênica , Peptídeo Sintases/genéticaRESUMO
LINC-HELLP, showing chromosomal linkage with the pregnancy-specific HELLP syndrome in Dutch families, reduces differentiation from a proliferative to an invasive phenotype of first-trimester extravillous trophoblasts. Here we show that mutations in LINC-HELLP identified in HELLP families negatively affect this trophoblast differentiation either by inducing proliferation rate or by causing cell cycle exit as shown by a decrease in both proliferation and invasion. As LincRNAs predominantly function through interactions with proteins, we identified the directly interacting proteins using chromatin isolation by RNA purification followed by protein mass spectrometry. We found 22 proteins predominantly clustering in two functional networks, i.e. RNA splicing and the ribosome. YBX1, PCBP1, PCBP2, RPS6 and RPL7 were validated, and binding to these proteins was influenced by the HELLP mutations carried. Finally, we show that the LINC-HELLP transcript levels are significantly upregulated in plasma of women in their first trimester of pregnancy compared with non-pregnant women, whereas this upregulation seems absent in a pilot set of patients later developing pregnancy complications, indicative of its functional significance in vivo.
Assuntos
Síndrome HELLP/genética , Mutação , Primeiro Trimestre da Gravidez/genética , RNA Longo não Codificante/genética , Trofoblastos/citologia , Ciclo Celular , Diferenciação Celular , Proliferação de Células , Feminino , Regulação da Expressão Gênica , Síndrome HELLP/sangue , Humanos , Gravidez , Primeiro Trimestre da Gravidez/sangue , Proteínas/metabolismo , Splicing de RNA , RNA Longo não Codificante/sangue , Ribossomos/metabolismoRESUMO
In humans, the elucidation of the genetics underlying multifactorial diseases such as pre-eclampsia remains complex. Given the current day availability of genome-wide linkage- and expression data pools, we applied pathway-guided genome-wide meta-analysis guided by the premise that the functional network underlying these multifactorial syndromes is under selective genetic pressure. This approach drastically reduced the genomic region of interest, i.e. 2p13 linked with pre-eclampsia in Icelandic families, from 8 679 641 bp (region with linkage) to 45 264 bp (coding exons of prioritized genes) (0.83%). Mutation screening of the candidate genes (n = 13) rapidly reduced the minimal critical region and showed the INO80B gene, encoding a novel winged helix domain (pfam14465) and part of the chromatin-remodeling complex, to be linked to pre-eclampsia. The functional defect in placental cells involved a susceptibility allele-dependent loss-of-gene silencing due to increased INO80B RNA stability as a consequence of differential binding of miR-1324 to the susceptibility allele of rs34174194. This risk allele is located at position 1 in an absolutely conserved 7-mer (UUGUCUG) in the 3-UTR of INO80B immediately downstream of a variant Pumillio Recognition Element (UGUANAAG). These data support that pre-eclampsia genes affect a conserved fundamental mechanism that evolved as a consequence of hemochorial placentation. Functionally, this involves founder-dependent, placentally expressed paralogous genes that regulate an essential trophoblast differentiation pathway but act at different entry points.
Assuntos
Proteínas de Ciclo Celular/genética , Estudos de Associação Genética/métodos , MicroRNAs/genética , Proteínas Nucleares/genética , Pré-Eclâmpsia/genética , Análise Mutacional de DNA/métodos , Feminino , Predisposição Genética para Doença , Humanos , Islândia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Placenta/metabolismo , Polimorfismo de Nucleotídeo Único , GravidezRESUMO
During prolonged hypoxic conditions, endothelial cells change their gene expression to adjust to the low oxygen environment. This process is mainly regulated by the hypoxia-inducible factors, HIF-1α and HIF-2α. Although endothelial cells do not form sprouts during prolonged hypoxic culturing, silencing of HIF-2α partially restores sprout formation. The present study identifies novel HIF-2α-target genes that may regulate endothelial sprouting during prolonged hypoxia. The gene expression profile of primary human microvascular endothelial cells (hMVECs) that were cultured at 20 % oxygen was compared to hMVECs that were cultured at 1 % oxygen for 14 days by using genome-wide RNA-sequencing. The differentially regulated genes in hypoxia were compared to the genes that were differentially regulated upon silencing of HIF-2α in hypoxia. Surprisingly, KEGG pathway analysis showed that metabolic pathways were enriched within genes upregulated in response to hypoxia and enriched within genes downregulated upon HIF-2α silencing. Moreover, 51 HIF-2α-regulated genes were screened for their role in endothelial sprouting in hypoxia, of which four genes ARRDC3, MME, PPARG and RALGPS2 directly influenced endothelial sprouting during prolonged hypoxic culturing. The manipulation of specific downstream targets of HIF-2α provides a new, but to be further evaluated, perspective for restoring reduced neovascularization in several pathological conditions, such as diabetic ulcers or other chronic wounds, for improvement of vascularization of implanted tissue-engineered scaffolds.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Microvasos/citologia , Neovascularização Fisiológica/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Humanos , Oxigênio/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , TransfecçãoRESUMO
OBJECTIVE: To compare available analysis methods for determining fetal fraction on single read next generation sequencing data. This is important as the performance of non-invasive prenatal testing (NIPT) procedures depends on the fraction of fetal DNA. METHODS: We tested six different methods for the detection of fetal fraction in NIPT samples. The same clinically obtained data were used for all methods, allowing us to assess the effect of fetal fraction on the test result, and to investigate the use of fetal fraction for quality control. RESULTS: We show that non-NIPT methods based on body mass index (BMI) and gestational age are unreliable predictors of fetal fraction, male pregnancy specific methods based on read counts on the Y chromosome perform consistently and the fetal sex-independent new methods SeqFF and SANEFALCON are less reliable but can be used to obtain a basic indication of fetal fraction in case of a female fetus. CONCLUSION: We recommend the use of a combination of methods to prevent the issue of reports on samples with insufficient fetal DNA; SANEFALCON to check for presence of fetal DNA, SeqFF for estimating the fetal fraction for a female pregnancy and any Y-based method for estimating the fetal fraction for a male pregnancy. © 2017 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.
Assuntos
Ácidos Nucleicos Livres/isolamento & purificação , Testes para Triagem do Soro Materno/métodos , Feminino , Humanos , Masculino , GravidezRESUMO
Noninvasive prenatal testing using fetal DNA in maternal plasma is an actively researched area. The current generation of tests using massively parallel sequencing is based on counting plasma DNA sequences originating from different genomic regions. In this study, we explored a different approach that is based on the use of DNA fragment size as a diagnostic parameter. This approach is dependent on the fact that circulating fetal DNA molecules are generally shorter than the corresponding maternal DNA molecules. First, we performed plasma DNA size analysis using paired-end massively parallel sequencing and microchip-based capillary electrophoresis. We demonstrated that the fetal DNA fraction in maternal plasma could be deduced from the overall size distribution of maternal plasma DNA. The fetal DNA fraction is a critical parameter affecting the accuracy of noninvasive prenatal testing using maternal plasma DNA. Second, we showed that fetal chromosomal aneuploidy could be detected by observing an aberrant proportion of short fragments from an aneuploid chromosome in the paired-end sequencing data. Using this approach, we detected fetal trisomy 21 and trisomy 18 with 100% sensitivity (T21: 36/36; T18: 27/27) and 100% specificity (non-T21: 88/88; non-T18: 97/97). For trisomy 13, the sensitivity and specificity were 95.2% (20/21) and 99% (102/103), respectively. For monosomy X, the sensitivity and specificity were both 100% (10/10 and 8/8). Thus, this study establishes the principle of size-based molecular diagnostics using plasma DNA. This approach has potential applications beyond noninvasive prenatal testing to areas such as oncology and transplantation monitoring.
Assuntos
DNA/genética , Doenças Fetais/genética , Patologia Molecular/métodos , Diagnóstico Pré-Natal/métodos , Transtornos Cromossômicos/diagnóstico , Transtornos Cromossômicos/genética , Cromossomos Humanos Par 13/genética , Cromossomos Humanos Par 18/genética , Cromossomos Humanos X/genética , DNA/sangue , DNA/química , Síndrome de Down/diagnóstico , Síndrome de Down/genética , Eletroforese Capilar/métodos , Feminino , Doenças Fetais/sangue , Doenças Fetais/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Monossomia/diagnóstico , Monossomia/genética , Gravidez , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Trissomia/diagnóstico , Trissomia/genética , Síndrome da Trissomia do Cromossomo 13 , Síndrome da Trissomía do Cromossomo 18RESUMO
OBJECTIVE: While large fetal copy number aberrations can generally be detected through sequencing of DNA in maternal blood, the reliability of tests depends on the fraction of DNA that originates from the fetus. Existing methods to determine this fetal fraction require additional work or are limited to male fetuses. We aimed to create a sex-independent approach without additional work. METHODS: DNA fragments used for noninvasive prenatal testing are cut only by natural processes; thus, influences on cutting by the packaging of DNA in nucleosomes will be preserved in sequencing. As cuts are expected to be made preferentially in linker regions, the shorter fetal fragments should be enriched for reads starting in nucleosome covered positions. RESULTS: We generated genome-wide nucleosome profiles based on single end sequencing of cell-free DNA. We found a difference between DNA digestion of fetal cell-free DNA and maternal cell-free DNA and used this to calculate the fraction of fetal DNA in maternal plasma for both male and female fetuses. CONCLUSION: Our method facilitates cost-effective noninvasive prenatal testing, as the fetal DNA fraction can be estimated without the need for expensive paired-end sequencing or additional tests. The methodology is implemented as a tool, which we called SANEFALCON (Single reAds Nucleosome-basEd FetAL fraCtiON). It is available for academic and non-profit purposes under Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International Public License. github.com/rstraver/sanefalcon. © 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.
Assuntos
Transtornos Cromossômicos/diagnóstico , DNA/metabolismo , Feto/metabolismo , Nucleossomos/metabolismo , Transtornos Cromossômicos/sangue , Análise Custo-Benefício , DNA/sangue , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Gravidez , Diagnóstico Pré-Natal , Reprodutibilidade dos Testes , Análise de Sequência de DNARESUMO
Genetic disorders can be detected by prenatal diagnosis using Chorionic Villus Sampling, but the 1:100 chance to result in miscarriage restricts the use to fetuses that are suspected to have an aberration. Detection of trisomy 21 cases noninvasively is now possible owing to the upswing of next-generation sequencing (NGS) because a small percentage of fetal DNA is present in maternal plasma. However, detecting other trisomies and smaller aberrations can only be realized using high-coverage NGS, making it too expensive for routine practice. We present a method, WISECONDOR (WIthin-SamplE COpy Number aberration DetectOR), which detects small aberrations using low-coverage NGS. The increased detection resolution was achieved by comparing read counts within the tested sample of each genomic region with regions on other chromosomes that behave similarly in control samples. This within-sample comparison avoids the need to re-sequence control samples. WISECONDOR correctly identified all T13, T18 and T21 cases while coverages were as low as 0.15-1.66. No false positives were identified. Moreover, WISECONDOR also identified smaller aberrations, down to 20 Mb, such as del(13)(q12.3q14.3), +i(12)(p10) and i(18)(q10). This shows that prevalent fetal copy number aberrations can be detected accurately and affordably by shallow sequencing maternal plasma. WISECONDOR is available at bioinformatics.tudelft.nl/wisecondor.
Assuntos
Transtornos Cromossômicos/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala , Diagnóstico Pré-Natal/métodos , Análise de Sequência de DNA , DNA/sangue , Feminino , Feto , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Gravidez , Padrões de Referência , Análise de Sequência de DNA/normas , TrissomiaRESUMO
Preeclampsia is a pregnancy-associated disease with maternal symptoms but placental origin. Epigenetic inheritance is involved in some populations. By sequence analysis of 17 genes in the 10q22 region with maternal effects, we narrowed the minimal critical region linked with preeclampsia in the Netherlands to 444 kb. All but one gene in this region, which lies within a female-specific recombination hotspot, encode DNA- or RNA-binding proteins. One gene, STOX1 (also called C10orf24), contained five different missense mutations, identical between affected sisters, cosegregating with the preeclamptic phenotype and following matrilineal inheritance. Four STOX1 transcripts are expressed in early placenta, including invasive extravillus trophoblast, generating three different isoforms. All contain a winged helix domain related to the forkhead (FOX) family. The largest STOX1 isoform has exclusive nuclear or cytoplasmic expression, indicating activation and inactivation, respectively, of the PI3K-Akt-FOX pathway. Because all 38 FOX proteins and all 8 STOX1 homologs have either tyrosine or phenylalanine at position 153, the predominant Y153H variation is highly mutagenic by conservation criteria but subject to incomplete penetrance. STOX1 is a candidate for preeclampsia controlling polyploidization of extravillus trophoblast.
Assuntos
Cromossomos Humanos Par 10 , Pré-Eclâmpsia/genética , Transativadores/genética , Feminino , Fatores de Transcrição Forkhead , Haplótipos , Humanos , Masculino , Dados de Sequência Molecular , Família Multigênica , Linhagem , Gravidez , Análise de Sequência de DNARESUMO
Hypertrophic cardiomyopathy (HCM) is predominantly caused by mutations in genes encoding sarcomeric proteins. One of the most frequent affected genes is MYBPC3, which encodes the thick filament protein cardiac myosin binding protein C. Despite the prevalence of HCM, disease pathology and clinical outcome of sarcomeric mutations are largely unknown. We hypothesized that microRNAs (miRNAs) could play a role in the disease process. To determine which miRNAs were changed in expression, miRNA arrays were performed on heart tissue from HCM patients with a MYBPC3 mutation (n=6) and compared with hearts of non-failing donors (n=6). 532 out of 664 analyzed miRNAs were expressed in at least one heart sample. 13 miRNAs were differentially expressed in HCM compared with donors (at p<0.01, fold change ≥ 2). The genomic context of these differentially expressed miRNAs revealed that miR-204 (fold change 2.4 in HCM vs. donor) was located in an intron of the TRPM3 gene, encoding an aspecific cation channel involved in calcium entry. RT-PCR analysis revealed a trend towards TRPM3 upregulation in HCM compared with donor myocardium (fold change 2.3, p=0.078). In silico identification of mRNA targets of differentially expressed miRNAs showed a large proportion of genes involved in cardiac hypertrophy and cardiac beta-adrenergic receptor signaling and we showed reduced phosphorylation of cardiac troponin I in the HCM myocardium when compared with donor. HCM patients with MYBPC3 mutations have a specific miRNA expression profile. Downstream mRNA targets reveal possible involvement in cardiac signaling pathways.
Assuntos
Cardiomiopatia Hipertrófica/genética , Proteínas de Transporte/genética , Perfilação da Expressão Gênica , MicroRNAs/genética , Mutação/genética , Miocárdio/metabolismo , Transcriptoma/genética , Adulto , Idoso , Cardiomiopatia Hipertrófica/patologia , Simulação por Computador , Feminino , Genoma Humano/genética , Humanos , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Miocárdio/patologia , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Troponina I/metabolismo , Adulto JovemRESUMO
By using complementary in vitro and ex vivo approaches, we show that the risk allele (Y153H) of the pre-eclampsia susceptibility gene STOX1 negatively regulates trophoblast invasion by upregulation of the cell-cell adhesion protein alpha-T-catenin (CTNNA3). This is effectuated at the crucial epithelial-mesenchymal transition of proliferative into invasive extravillous trophoblast. This STOX1-CTNNA3 interaction is direct and includes Akt-mediated phosphorylated control of nucleo-cytoplasmic shuttling and ubiquitin-mediated degradation as shared with the FOX multigene family. This, to our knowledge, is the first time a genotype associated with pre-eclampsia has been shown to directly limit first trimester extravillous trophoblast invasion, the earliest hallmark of pre-eclampsia.
Assuntos
Proteínas de Transporte/metabolismo , Pré-Eclâmpsia/genética , Trofoblastos/metabolismo , alfa Catenina/metabolismo , Alelos , Proteínas de Transporte/genética , Adesão Celular/genética , Feminino , Fatores de Transcrição Forkhead/metabolismo , Estudos de Associação Genética , Humanos , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/patologia , Gravidez , Proteínas Proto-Oncogênicas c-akt/metabolismo , Trofoblastos/patologia , Regulação para CimaRESUMO
Background: Myxovirus resistance protein A (MxA) is a protein that is upregulated by interferon-beta. Homeostatic MxA mRNA levels are potentially correlated with inflammatory disease activity in multiple sclerosis (MS) and could have an important role in MS pathology. Aim: To investigate the association between myxovirus resistance protein A (MxA) mRNA levels in blood and disease activity and progression in MS over a long-term follow-up period. Methods: Baseline blood MxA mRNA levels were determined in a prospective cohort of 116 untreated patients with a clinically isolated syndrome (CIS) or early relapsing remitting MS (RRMS), and related to long-term relapses, radiological disease activity, clinical scores [Expanded Disability Status Scale (EDSS), timed-25-foot walk (T25FW), 9-hole-peg test (9HPT)], MS type, and disease modifying therapy (DMT) use. Results: Low MxA mRNA levels were associated with the occurrence of ≥9 T2-lesions on MRI imaging and the occurrence of relapses during long-term follow-up (median 11 years, IQR 5.91-13.69 years). MxA mRNA levels were not associated with EDSS, T25FW, 9HPT, and MS subtype. Conclusion: Baseline MxA mRNA levels are associated with long-term development of T2-lesions on MRI-scans in our cohort. This confirms the relevance of the endogenous interferon-beta system in the occurrence of MS disease activity.
RESUMO
BACKGROUND: A recent study reported that an 18-analyte multiplexed plasma panel of signaling proteins differentiated Alzheimer's disease (AD) from controls. This study measured mRNA expression for nine of these promising bio-markers in 23 AD patients and 23 age- and sex-matched controls. METHODS: Total RNA was isolated from PaxGene RNA tubes. Relative mRNA expression levels of CCL5 [RANTES], CSF1, ICAM1, IGFBP6, IL1A, IL3, IL8, PDGFB and TNF were determined by Q-RT-PCR, with GAPDH as housekeeping gene. RESULTS: A panel of five markers (CCL5, CSF1, ICAM1, IL8, TNF) with detectable expression levels in all individuals differed between AD patients and controls (p interaction <0.10). Especially, the relative expression level of CCL5 was lower in AD patients than in controls (p<0.005). Across groups, levels of both CCL5 and TNF were correlated to CSF levels of τ (r=0.39, r=0.32), pτ-181 (r=0.38, r=0.33), and MMSE (r=-0.31, r=-0.33, all p<0.05). CONCLUSIONS: The measured panel, and especially CCL5, could aid in the differentiation of AD from controls.
Assuntos
Doença de Alzheimer/sangue , Quimiocina CCL5/sangue , Regulação para Baixo , Idoso , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Quimiocina CCL5/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismoRESUMO
INTRODUCTION: Tumor-specific genetic aberrations in cell-free DNA (cfDNA) from plasma are promising biomarkers for diagnosis of recurrent head and neck squamous cell carcinoma (HNSCC). However, the sensitivity when using somatic mutations only in cfDNA is suboptimal. Here, we combined detection of copy number aberrations (CNAs), human papillomavirus (HPV) DNA and somatic mutations in a single sequencing workflow. METHODS: Pretreatment plasmas of 40 patients and 20 non-cancer controls were used for analysis. Plasma DNA underwent low-coverage whole genome sequencing (lcWGS) to detect both CNAs and HPV-DNA, and deep sequencing to detect mutations in 12 frequently altered cancer driver genes in HNSCC using the same sequencing library. A specific analysis pipeline line was developed for data mining. The corresponding tumors were analyzed using slightly adapted protocols. RESULTS: Using the developed method, somatic mutations and CNAs were detected in plasma DNA of HNSCC patients in 67% and 52%, respectively. HPV-DNA in plasma was detected in 100% of patients with HPV-positive tumors, and not in plasma of patients with HPV-negative tumors or non-cancer controls. Combined analysis increased the detection rate of tumor DNA in plasma to 78%. The detection rate was significantly associated with the stage of disease of the tumor. Neither HPV status nor location of the primary tumor influenced detection of CNAs or somatic mutations in plasma. CONCLUSIONS: This study demonstrates that the combined analysis of CNAs, HPV and somatic mutations in plasma of HNSCC patients is feasible and contributes to a higher sensitivity of the assay compared to single modality analyses.
Assuntos
Metilação de DNA , DNA/genética , Feto , Genoma Humano , Diagnóstico Pré-Natal , Sulfitos , Feminino , Humanos , GravidezRESUMO
A novel in vitro method is described wherein gene expression profiling is reflective and informative for the way how syncytiotrophoblast cells shed RNA products in vivo in maternal plasma. After controlled denudation, RNA is obtained selectively from the syncytiotrophoblast cells of trisomy 21 placentae. cDNA copies are subsequently analyzed by microarray profiling and cDNA cloning with sequencing. Given the preponderance of 5' mRNA fragments lacking a poly-A tail, the placental RNA products are amplified after polymerase A-mediated tailing by using a method originally designed for small-sized microRNAs. This approach, when combined with cDNA library or microarray expression screening, is a novel in vitro method to screen for syncytiotrophoblast-derived RNA products representative of trisomy 21 placental RNA as present in vivo in maternal plasma.
Assuntos
Síndrome de Down/diagnóstico , Testes Genéticos , Diagnóstico Pré-Natal/métodos , RNA Mensageiro/sangue , Trofoblastos/metabolismo , Clonagem Molecular , Análise Mutacional de DNA , Síndrome de Down/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Troca Materno-Fetal , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez , RNA Mensageiro/isolamento & purificaçãoRESUMO
The number of molecules identified to be involved in communication between placenta and decidua is fast expanding. Previously, we showed that NODAL expressed in maternal endometrial stromal cells is able to affect NODAL and STOX1 expression in placental extravillous trophoblasts. The effect of maternal NODAL on placental NODAL expression is achieved via Activin A, while preliminary data suggests that maternal NODAL affects STOX1 expression in trophoblasts potentially via IGF1. In the current study, T-HESC endometrial stromal cells were treated with siRNAs against NODAL after which IGF1 mRNA expression was determined by quantitative RT-PCR, while IGF1 secretion was measured by ELISA. Recombinant IGF1 and inhibitors of the MAPK and PI3K/AKT pathways were added to SGHPL-5 extravillous trophoblasts after which the effects on STOX1 mRNA and STOX1 protein expression were determined. The effect of IGF1 and the MAPK and PI3K/AKT inhibitors on the invasive capacity of SGHPL-5 cells was investigated by performing invasion assays. We found that T-HESC cells treated with NODAL siRNAs showed significant upregulation of IGF1 mRNA expression and IGF1 protein secretion. Addition of IGF1 to SGHPL-5 cell media significantly upregulated STOX1 mRNA and protein expression. Using inhibitors of the PI3K/AKT and MAPK pathway showed that the effect of IGF1 on STOX1 expression is accomplished via MAPK signaling. Secondly, PI3K inhibition independently leads to reduced STOX1 expression which can be rescued by adding IGF1. IGF1 was unable to influence the invasive capacity of SGHPL-5 cells, while inhibiting the PI3K/AKT pathway did reduce the invasion of these cells. To conclude, here we show that downregulated NODAL expression in endometrial stromal cells, previously associated with pre-eclampsia like symptoms in mice, increases IGF1 secretion. Increased levels of IGF1 lead to increased expression levels of STOX1 in extravillous trophoblasts via the MAPK pathway, hereby identifying a novel signaling cascade involved in maternal-fetal communication.
Assuntos
Proteínas de Transporte/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Proteína Nodal/metabolismo , Trofoblastos/metabolismo , Linhagem Celular , Endométrio/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Nodal/antagonistas & inibidores , Proteína Nodal/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Células Estromais/metabolismoRESUMO
Gemcitabine is a commonly used therapy for many solid tumors. Acquired resistance to this nucleoside analogue, however, diminishes the long-term effectiveness in a majority of patients. To better define the molecular background of gemcitabine resistance, a mouse colon tumor was selected during successive rounds of transplantation with continued treatment of gemcitabine. Expression microarray analysis was applied to determine which genes are consistently and highly overexpressed or underexpressed in the resistant versus the nonresistant tumor. For the statistical interpretation of the microarray data, a parametric model was implemented, which returns model-based differential gene expression (log-) ratios and their uncertainties. This defined a set of 13 genes, putatively responsible for the gemcitabine resistance in solid tumors. One of these, RRM1, was previously identified as an important marker for gemcitabine resistance in human cell lines. Five of the 13 genes, including RRM1, are located within a 3 Mb region at chromosome 7E1 of which four are highly overexpressed, suggesting a chromosomal amplification. Therefore, chromosomal copy number changes were measured, using oligo array comparative genomic hybridization. A narrow and high amplification area was identified on 7E1 that encompassed all five genes. In addition, reduced RNA expression of two other genes at 8E1 encoding COX4I1 and RPL13 could be explained by a decrease in chromosomal copy number on chromosome 8. In conclusion, the array comparative genomic hybridization biologically validates our statistical approach and shows that gemcitabine is capable to select for chromosomally aberrant tumor cells, where changed gene expression levels lead to drug resistance.