Circulating levels of miR-150 are associated with poorer outcomes of A/H1N1 infection.

BACKGROUND: Overproduction of pro-inflammatory cytokines and chemokines is frequently associated with severe clinical manifestations in patients infected with influenza A/H1N1 virus. Micro-RNAs (miRNAs) are highly conserved small non-coding RNA molecules that post-transcriptionally regulate gene expression and are potential biomarkers and therapeutic targets in different inflammatory conditions. METHODS: We studied the circulating and miRNA profiles in critically ill A/H1N1 patients, A/H1N1 patients with milder disease, asymptomatic housemates and healthy controls. Cytokine, chemokine and growth factors that were potential targets of differentially expressed miRNAs were assessed. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and interactome analysis of these miRNAs were also performed. RESULTS: Critically ill patients exhibited a significant over-expression of circulating miR-150 (p<0.005) when compared to patients with milder disease. miR-29c, miR-145 and miR-22 were differentially expressed in patients with severe A/H1N1 disease whereas miR-210, miR-126 and miR-222 were downregulated in individuals exposed to the A/H1N1 virus. Significant correlations (p<0.05) between circulating levels of miR-150 with IL-1ra, IL-2, IL-6, CXCL8, IFN-γ, CXCL10 and G-CSF were detected, particularly in critically ill patients. CONCLUSION: The up-regulation of miR-150 is associated with poorer outcomes of A/H1N1 infection. The differential expression of miRNAs related with immune processes in severe A/H1N1 disease supports the potential role of these miRNAs as biomarkers of disease progression.

Hypercontrols in genotype-phenotype analysis reveal ancestral haplotypes associated with essential hypertension.

The angiotensinogen gene locus has been associated with essential hypertension in most populations analyzed to date. Increased plasma angiotensinogen levels have been proposed as an underlying cause of essential hypertension in whites; however, differences in the genetic regulation of plasma angiotensinogen levels have also been reported for other populations. The aim of this study was to analyze the relationship between angiotensinogen gene polymorphisms and haplotypes with plasma angiotensinogen levels and the risk of essential hypertension in the Mexican population. We genotyped 9 angiotensinogen gene polymorphisms in 706 individuals. Four polymorphisms, A-6, C4072, C6309, and G12775, were associated with increased risk, and the strongest association was found for the C6309 allele (χ(2)=23.9; P=0.0000009), which resulted in an odds ratio of 3.0 (95% CI: 1.8-4.9; P=0.000006) in the recessive model. Two polymorphisms, A-20C (P=0.003) and C3389T (P=0.0001), were associated with increased plasma angiotensinogen levels but did not show association with essential hypertension. The haplotypes H1 (χ(2)=8.1; P=0.004) and H5 (χ(2)=5.1; P=0.02) were associated with essential hypertension. Using phylogenetic analysis, we found that haplotypes 1 and 5 are the human ancestral haplotypes. Our results suggest that the positive association between angiotensinogen gene polymorphisms and haplotypes with essential hypertension is not simply explained by an increase in plasma angiotensinogen concentration. Complex interactions between risk alleles suggest that these haplotypes act as "superalleles."

Paradoxial changes in the expression of estrogen receptor alpha in breast cancer multicellular spheroids.

Multicellular spheroids are excellent models for the analysis of cancer behavior. Just like small avascular tumors, they present a marked zonal heterogeneity which influences gene expression and thus, growth and response to chemotherapy. In the present paper, we sought to analyze the effects of three-dimensional culture in the expression and distribution of estrogen receptor alpha. Using MCF-7 breast cancer cells, we found that multicellular spheroids in estrogen-containing medium presented a paradoxical regulation of estrogen receptor alpha, with a decrease in protein expression and a marked increase in mRNA steady-state levels. Immunohistochemistry showed that only sparse cells in the periphery of the spheroid expressed estrogen receptor, in sharp contrast with progesterone receptor, which was more extensively expressed and HIF-alpha, which was expressed in the central core of the spheroid. This could mean that both hypoxia and ERA activation by estrogen participate in the expression heterogeneity of this hormone receptor in breast cancer These results are important to considerate in the analysis and interpretation of immunohistochemistry of ERA and downstream targets in samples of solid tumors.

Genomic medicine in Mexico: initial steps and the road ahead.

Mexico faces important demographic and epidemiological transitions with significant implications to patterns of disease, disability, and death. On the one hand, there are problems of underdevelopment and, on the other, the emerging challenges of the chronic and degenerative diseases of the industrialized world. For these diseases, prevention becomes a key strategy for alleviating a major burden to the economy and health of the Mexican population. Genomic medicine has become a priority to the Mexican government as a means of finding new strategies to tackle common diseases. In 2000, strategic planning for genomic medicine began, from a feasibility study and a multi-institutional consortium effort, to the creation of a National Institute of Genomic Medicine by the Mexican congress in 2004. Current research programs in genomic medicine in Mexico include the construction of a haplotype map of the Mexican population, several genome-wide association studies for common diseases, such as diabetes, obesity, cardiovascular disease, and cancer, as well as translational medicine projects that include biomarkers discovery for several kinds of cancer, pharmacogenomics, and nutrigenomics. Although this strategy has been successful, there are challenges that still need to be addressed, including increased investment in science and technology to stimulate a more vigorous and competitive research environment, development of more effective basic and clinical research synergies, recruitment and training of more human resources in genomic medicine, developing mechanisms to stimulate translational research, and developing a more modern regulatory framework to ensure that genomic medicine will successfully contribute to improve healthcare in the Mexican population.

[Global expression analysis in uterine cervical cancer: metabolic pathways and altered genes]. / Análisis de expresión global del cáncer cérvico uterino: rutas metabólicas y genes alterados.

High risk human papillomavirus (HPV) infection is considered to be the most important etiological factor of Cervical Uterine Cancer. In order to determine the global expression pattern and to identify possible molecular markers of cervical cancer, cDNA arrays with probe sets complementary to 8,000 human genes were used to examine the expression profiles among 5 cell lines derived from human cervical cancer, three HPV16(+) tumor samples and three normal cervical tissues HPV(-). The levels of expression of different cellular processes were identified. Hierarchical clustering was performed and the gene expression using RT-PCR was confirmed. Two genes were found to be consistently overexpressed in invasive cervical cancer biopsies; one of them, IL-6 was previously reported to be overexpressed in cervical cancer and one novel gene, MMP10, previously not known to be related to cervical cancer. Hierarchical clustering of the expression data revealed that samples with common HPV type infection grouped together, maybe this could mean that differences between HPV types could be indirectly determined by expression profiles.

Characterization of the global profile of genes expressed in cervical epithelium by Serial Analysis of Gene Expression (SAGE).

BACKGROUND: Serial Analysis of Gene Expression (SAGE) is a new technique that allows a detailed and profound quantitative and qualitative knowledge of gene expression profile, without previous knowledge of sequence of analyzed genes. We carried out a modification of SAGE methodology (microSAGE), useful for the analysis of limited quantities of tissue samples, on normal human cervical tissue obtained from a donor without histopathological lesions. Cervical epithelium is constituted mainly by cervical keratinocytes which are the targets of human papilloma virus (HPV), where persistent HPV infection of cervical epithelium is associated with an increase risk for developing cervical carcinomas (CC). RESULTS: We report here a transcriptome analysis of cervical tissue by SAGE, derived from 30,418 sequenced tags that provide a wealth of information about the gene products involved in normal cervical epithelium physiology, as well as genes not previously found in uterine cervix tissue involved in the process of epidermal differentiation. CONCLUSION: This first comprehensive and profound analysis of uterine cervix transcriptome, should be useful for the identification of genes involved in normal cervix uterine function, and candidate genes associated with cervical carcinoma.

Gene identification by cDNA arrays in HPV-positive cervical cancer.

BACKGROUND: One of the most frequent malignancies in women worldwide is carcinoma of the uterine cervix. High-risk human papillomavirus (HPV) infection is considered the most important etiological factor of uterine cervical cancer. Our aim was to identify novel cellular genes that could potentially act as predictive molecular markers for human cervical cancer by means of cDNA arrays. METHODS: We used cDNA arrays to examine the expression profiles of six cell lines derived from human cervical cancer, three HPV+ tumor samples and three normal (HPV-) epithelium tissues. Data normalization was performed and the top overexpressed genes were obtained. Hierarchical cluster was performed and, to validate some of the differentially expressed genes between normal and carcinogenic samples, semi-quantitative RT-PCR, in situ hybridization and immunohistochemistry were performed in tissue samples. RESULTS: Four genes were demonstrated to be consistently overexpressed in invasive cervical cancer biopsies; three novel genes not previously related to cervical cancer: MMP10, Lamc2 and Claudin 1. Moreover, overexpression of IL6 and VEGF was corroborated. CONCLUSIONS: The identification of characteristic molecular changes in cervical cells by carcinogenesis and HPV infection can lead to a better understanding of cervical cancer. cDNA arrays are beginning to provide new possible molecular markers for prognosis and diagnosis. This technology could eventually help to elucidate the biological differences of the particular mechanisms associated with each different HPV-type infection and those with a poor prognosis.

Microarray comparative genomic hybridization detection of chromosomal imbalances in uterine cervix carcinoma.

BACKGROUND: Chromosomal Comparative Genomic Hybridization (CGH) has been applied to all stages of cervical carcinoma progression, defining a specific pattern of chromosomal imbalances in this tumor. However, given its limited spatial resolution, chromosomal CGH has offered only general information regarding the possible genetic targets of DNA copy number changes. METHODS: In order to further define specific DNA copy number changes in cervical cancer, we analyzed 20 cervical samples (3 pre-malignant lesions, 10 invasive tumors, and 7 cell lines), using the GenoSensor microarray CGH system to define particular genetic targets that suffer copy number changes. RESULTS: The most common DNA gains detected by array CGH in the invasive samples were located at the RBP1-RBP2 (3q21-q22) genes, the sub-telomeric clone C84C11/T3 (5ptel), D5S23 (5p15.2) and the DAB2 gene (5p13) in 58.8% of the samples. The most common losses were found at the FHIT gene (3p14.2) in 47% of the samples, followed by deletions at D8S504 (8p23.3), CTDP1-SHGC- 145820 (18qtel), KIT (4q11-q12), D1S427-FAF1 (1p32.3), D9S325 (9qtel), EIF4E (eukaryotic translation initiation factor 4E, 4q24), RB1 (13q14), and DXS7132 (Xq12) present in 5/17 (29.4%) of the samples. CONCLUSION: Our results confirm the presence of a specific pattern of chromosomal imbalances in cervical carcinoma and define specific targets that are suffering DNA copy number changes in this neoplasm.

Análisis de expresión global del cáncer cérvico uterino: rutas metabólicas y genes alterados / Global expression analysis in uterine cervical cancer: Metabolic pathways and altered genes

High risk human papillomavirus (HPV) infection is considered to be the most important etiological factor of Cervical Uterine Cancer. In order to determine the global expression pattern and to identify possible molecular markers of cervical cancer, cDNA arrays with probe sets complementary to 8,000 human genes were used to examine the expression profiles among 5 cell lines derived from human cervical cancer, three HPV16(+) tumor samples and three normal cervical tissues HPV(-). The levels of expression of different cellular processes were identified. Hierarchical clustering was performed and the gene expression using RT-PCR was confirmed. Two genes were found to be consistently overexpressed in invasive cervical cancer biopsies; one of them, IL-6 was previously reported to be overexpressed in cervical cancer and one novel gene, MMP10, previously not known to be related to cervical cancer. Hierarchical clustering of the expression data revealed that samples with common HPV type infection grouped together, maybe this could mean that differences between HPV types could be indirectly determined by expression profiles.

La infección por virus de papiloma de alto riesgo (VPH) es considerada como el factor etiológico más importante del cáncer cérvico uterino (CaCU). Con el fin de determinar el patrón de expresión global e identificar algunos posibles genes marcadores del CaCU, se utilizaron microhileras de DNA que contenían 8,000 secuencias que codificaban para transcritos diferentes, para estudiar los perfiles de expresión de cinco líneas celulares derivadas de CaCU, tres muestras tumorales conteniendo VPH 16 y tres muestras normales negativas para la presencia de VPH. Se identificaron los niveles de expresión de genes relacionados con diferentes rutas metabólicas. Se llevó a cabo el análisis de agrupamiento jerárquico y posteriormente se confirmó la sobrexpresión de dos genes mediante RT-PCR. Estos dos genes se encontraron sobrexpresados en biopsias tumorales cervicales. Uno de ellos, el gen de IL6, que ha sido previamente reportado en relación con CaCU, así como el gen de la matriz-metaloproteasa 10 (MMP10) por primera vez relacionado con esta neoplasia. El análisis de agrupamiento jerárquico, además, reveló que las muestras que contienen el mismo tipo viral están asociadas, sugiriendo posibles diferencias en expresión entre tipos virales.

Chromosomal imbalances in four new uterine cervix carcinoma derived cell lines.

BACKGROUND: Uterine cervix carcinoma is the second most common female malignancy worldwide and a major health problem in Mexico, representing the primary cause of death among the Mexican female population. High risk human papillomavirus (HPV) infection is considered to be the most important risk factor for the development of this tumor and cervical carcinoma derived cell lines are very useful models for the study of viral carcinogenesis. Comparative Genomic Hybridization (CGH) experiments have detected a specific pattern of chromosomal imbalances during cervical cancer progression, indicating chromosomal regions that might contain genes that are important for cervical transformation. METHODS: We performed HPV detection and CGH analysis in order to initiate the genomic characterization of four recently established cervical carcinoma derived cell lines from Mexican patients. RESULTS: All the cell lines were HPV18 positive. The most prevalent imbalances in the cell lines were gains in chromosomes 1q23-q32, 3q11.2-q13.1, 3q22-q26.1, 5p15.1-p11.2, this alteration present as a high copy number amplification in three of the cell lines, 7p15-p13, 7q21, 7q31, 11q21, and 12q12, and losses in 2q35-qter, 4p16, 6q26-qter, 9q34 and 19q13.2-qter. CONCLUSIONS: Analysis of our present findings and previously reported data suggest that gains at 1q31-q32 and 7p13-p14, as well as losses at 6q26-q27 are alterations that might be unique for HPV18 positive cases. These chromosomal regions, as well as regions with high copy number amplifications, coincide with known fragile sites and known HPV integration sites. The general pattern of chromosomal imbalances detected in the cells resembled that found in invasive cervical tumors, suggesting that the cells represent good models for the study of cervical carcinoma.

Changes in retinoblastoma gene expression during cervical cancer progression.

The role of tumour suppressor genes in the development of human cancers has been studied extensively. In viral carcinogenesis, the inactivation of suppressor proteins such as retinoblastoma (pRb) and p53, and cellular oncogenes overexpression, such as c-myc, has been the subject of a number of investigations. In uterine-cervix carcinomas, where high-risk human papillomavirus (HPV) plays an important role, pRb and p53 are inactivated by E7 and E6 viral oncoproteins, respectively. However, little is known about the in situ expression of some of these proteins in pre-malignant and malignant cervical tissues. On the other hand, it has also been demonstrated that c-myc is involved in cervical carcinogenesis, and that pRb participates in the control of c-myc gene expression. By using immunostaining techniques, we investigated pRb immunodetection pattern in normal tissues, squamous intraepithelial lesions (SILs) and invasive carcinomas from the uterine cervix. Our data show low pRb detection in both normal cervical tissue and invasive lesions, but a higher expression in SILs. C-Myc protein was observed in most of the cellular nuclei of the invasive lesions, while in SILs was low. These findings indicate a heterogeneous pRb immunostaining during the different stages of cervical carcinogenesis, and suggest that this staining pattern could be a common feature implicated in the pathogenesis of uterine-cervix carcinoma.

Estrategias de análisis global. Hacia el manejo genético de las neoplasias / Global analysis strategies. In direction to genetic management of neoplastic diseases

La investigación biomédica en las enfermedades oncológicas, particularmente enfocada al estudio y entendimiento de los mecanismos moleculares involucrados en la transformación celular, está permitiendo el desarrollo de nuevas estrategias de diagnóstico y tratamiento más eficaces. La obtención y aplicación práctica de los resultados derivados del estudio de la genética molecular del cáncer, ha avanzado paralelamente con el desarrollo de herramientas tecnológicas que permiten obtener una visión global de diversos procesos celulares, tanto en condiciones normales como en los procesos patológicos. Esta combinación de investigación y aplicación tecnológica, ha creado metodologías que en estos momentos nos permiten analizar en su conjunto los tres principales niveles de la Genética Molecular: el genoma (DNA, archivo de la información genética), el transcriptoma (RNA, expresión de la información genética) y finalmente el proteoma (proteínas, aspecto funcional de la información genética). La información obtenida gracias a estos avances ha empezado a modificar nuestra visión fundamental sobre las enfermedades oncológicas; y sumándose a las herramientas de análisis tradicional, prometen modificar de manera importante la forma en que clasificamos, detectamos, diagnosticamos y tratamos a las diversas neoplasias. En esta revisión se presentan algunas de estas metodologías de análisis global, que involucran los tres niveles de organización genética: el genoma, con el proyecto genoma humano, la hibridación genómica comparativa y el pintado cromosómico, el transcriptoma, con el análisis en serie de expresión génica y microarreglos de DNA y el proteoma, con el análisis bidimensional de proteínas y los microarreglos de anticuerpos. En cada caso, además de una breve descripción de cada uno de los métodos, se expone el impacto de ellos en el estudio y manejo de las enfermedades neoplásicas.