ETV4 plays a role on the primary events during the adenoma-adenocarcinoma progression in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the most common cancers worldwide; it is the fourth leading cause of death in the world and the third in Brazil. Mutations in the APC, DCC, KRAS and TP53 genes have been associated with the progression of sporadic CRC, occurring at defined pathological stages of the tumor progression and consequently modulating several genes in the corresponding signaling pathways. Therefore, the identification of gene signatures that occur at each stage during the CRC progression is critical and can present an impact on the diagnosis and prognosis of the patient. In this study, our main goal was to determine these signatures, by evaluating the gene expression of paired colorectal adenoma and adenocarcinoma samples to identify novel genetic markers in association to the adenoma-adenocarcinoma stage transition. METHODS: Ten paired adenoma and adenocarcinoma colorectal samples were subjected to microarray gene expression analysis. In addition, mutations in APC, KRAS and TP53 genes were investigated by DNA sequencing in paired samples of adenoma, adenocarcinoma, normal tissue, and peripheral blood from ten patients. RESULTS: Gene expression analysis revealed a signature of 689 differentially expressed genes (DEG) (fold-change> 2, p< 0.05), between the adenoma and adenocarcinoma paired samples analyzed. Gene pathway analysis using the 689 DEG identified important cancer pathways such as remodeling of the extracellular matrix and epithelial-mesenchymal transition. Among these DEG, the ETV4 stood out as one of the most expressed in the adenocarcinoma samples, further confirmed in the adenocarcinoma set of samples from the TCGA database. Subsequent in vitro siRNA assays against ETV4 resulted in the decrease of cell proliferation, colony formation and cell migration in the HT29 and SW480 colorectal cell lines. DNA sequencing analysis revealed KRAS and TP53 gene pathogenic mutations, exclusively in the adenocarcinomas samples. CONCLUSION: Our study identified a set of genes with high potential to be used as biomarkers in CRC, with a special emphasis on the ETV4 gene, which demonstrated involvement in proliferation and migration.

Germline variants in DNA repair genes associated with hereditary breast and ovarian cancer syndrome: analysis of a 21 gene panel in the Brazilian population.

BACKGROUND: The Hereditary Breast and Ovarian Cancer Syndrome (HBOC) occurs in families with a history of breast/ovarian cancer, presenting an autosomal dominant inheritance pattern. BRCA1 and BRCA2 are high penetrance genes associated with an increased risk of up to 20-fold for breast and ovarian cancer. However, only 20-30% of HBOC cases present pathogenic variants in those genes, and other DNA repair genes have emerged as increasing the risk for HBOC. In Brazil, variants in ATM, ATR, CHEK2, MLH1, MSH2, MSH6, POLQ, PTEN, and TP53 genes have been reported in up to 7.35% of the studied cases. Here we screened and characterized variants in 21 DNA repair genes in HBOC patients. METHODS: We systematically analyzed 708 amplicons encompassing the coding and flanking regions of 21 genes related to DNA repair pathways (ABRAXAS1, ATM, ATR, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, MLH1, MRE11, MSH2, MSH6, NBN, PALB2, PMS2, PTEN, RAD50, RAD51, TP53 and UIMC1). A total of 95 individuals with HBOC syndrome clinical suspicion in Southeast Brazil were sequenced, and 25 samples were evaluated for insertions/deletions in BRCA1/BRCA2 genes. Identified variants were assessed in terms of population allele frequency and their functional effects were predicted through in silico algorithms. RESULTS: We identified 80 variants in 19 genes. About 23.4% of the patients presented pathogenic variants in BRCA1, BRCA2 and TP53, a frequency higher than that identified among previous studies in Brazil. We identified a novel variant in ATR, which was predicted as pathogenic by in silico tools. The association analysis revealed 13 missense variants in ABRAXAS1, BARD1, BRCA2, CHEK2, CDH1, MLH1, PALB2, and PMS2 genes, as significantly associated with increased risk to HBOC, and the patients carrying those variants did not present large insertions or deletions in BRCA1/BRCA2 genes. CONCLUSIONS: This study embodies the third report of a multi-gene analysis in the Brazilian population, and addresses the first report of many germline variants associated with HBOC in Brazil. Although further functional analyses are necessary to better characterize the contribution of those variants to the phenotype, these findings would improve the risk estimation and clinical follow-up of patients with HBOC clinical suspicion.

miR-450a Acts as a Tumor Suppressor in Ovarian Cancer by Regulating Energy Metabolism.

Dysregulation of miRNA expression is associated with multiple diseases, including cancers, in which small RNAs can have either oncogenic or tumor suppressive functions. Here we investigated the potential tumor suppressive function of miR-450a, one of the most significantly downregulated miRNAs in ovarian cancer. RNA-seq analysis of the ovarian cancer cell line A2780 revealed that overexpression of miR-450a suppressed multiple genes involved in the epithelial-to-mesenchymal transition (EMT). Overexpression of miR-450a reduced tumor migration and invasion and increased anoikis in A2780 and SKOV-3 cell lines and reduced tumor growth in an ovarian tumor xenographic model. Combined AGO-PAR-CLIP and RNA-seq analysis identified a panel of potential miR-450a targets, of which many, including TIMMDC1, MT-ND2, ACO2, and ATP5B, regulate energetic metabolism. Following glutamine withdrawal, miR-450a overexpression decreased mitochondrial membrane potential but increased glucose uptake and viability, characteristics of less invasive ovarian cancer cell lines. In summary, we propose that miR-450a acts as a tumor suppressor in ovarian cancer cells by modulating targets associated with glutaminolysis, which leads to decreased production of lipids, amino acids, and nucleic acids, as well as inhibition of signaling pathways associated with EMT. SIGNIFICANCE: miR-450a limits the metastatic potential of ovarian cancer cells by targeting a set of mitochondrial mRNAs to reduce glycolysis and glutaminolysis.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/13/3294/F1.large.jpg.

The Ala55Val and -866G>A polymorphisms of the UCP2 gene could be biomarkers for weight loss in patients who had Roux-en-Y gastric bypass.

OBJECTIVE: The aim of this study was to investigate whether the Ala55Val and -866G>A polymorphisms of the UCP2 gene are related to weight loss and changes in body composition after bariatric surgery performed by Roux-en-Y gastric bypass (RYGB). METHODS: This longitudinal study enrolled obese patients submitted to RYGB. Data regarding weight (kg), body mass index (kg/m2), fat-free mass (FFM; kg), fat mass (kg), weight loss (kg and %), and percent excess weight loss were collected from both preoperative and 1-y postoperative medical records. Polymorphisms were genotyped by allelic discrimination using real-time polymerase chain reaction and TaqMan-predesigned single nucleotide polymorphism Genotyping Assay kits (Applied Biosystems, Foster City, CA, USA). The t test was used to compare variables between genotypes of each polymorphism to analyze the dominant and recessive models. Linear regression models were used to adjust the effects of initial weight, age, and sex on the variation of weight and body composition (P < 0.05). RESULTS: We analyzed 150 severely obese individuals (age 47.2 ± 10.5 y; 80% women). Genotype analysis showed a greater prevalence of heterozygous GA (41.3%) for -866G>A polymorphism and CT (39.3%) for Ala55Val polymorphism. Individuals who carried the T (CT+TT) and A (GA+AA) mutated alleles for Ala55Val and -866G>A, respectively, showed a higher weight and FFM loss. CONCLUSION: The mutated alleles T for Ala55Val and A for -866G>A polymorphism could be biomarkers of weight loss 1 y after RYGB.

HOX genes: potential candidates for the progression of laryngeal squamous cell carcinoma.

Laryngeal squamous cell carcinoma (LSCC) is a very aggressive cancer, considered to be a subtype of the head and neck squamous cell carcinoma (HNSCC). Despite significant advances in the understanding and treatment of cancer, prognosis of patients with LSCC has not improved recently. In the present study, we sought to understand better the genetic mechanisms underlying LSCC development. Thirty-two tumor samples were collected from patients undergoing surgical resection of LSCC. The samples were submitted to whole-genome cDNA microarray analysis aiming to identify genetic targets in LSCC. We also employed bioinformatic approaches to expand our findings using the TCGA database and further performed functional assays, using human HNSCC cell lines, to evaluate viability, cell proliferation, and cell migration after silencing of selected genes. Eight members of the homeobox gene family (HOX) were identified to be overexpressed in LSCC samples when compared to normal larynx tissue. Quantitative RT-PCR analysis validated the overexpression of HOX gene family members in LSCC. Receiver operating characteristic (ROC) statistical method curve showed that the expression level of seven members of HOX gene family can distinguish tumor from nontumor tissue. Correlation analysis of clinical and gene expression data revealed that HOXC8 and HOXD11 genes were associated with the differentiation degree of tumors and regional lymph node metastases, respectively. Additionally, siRNA assays confirmed that HOXC8, HOXD10, and HOXD11 genes might be critical for cell colony proliferation and cell migration. According to our findings, several members of the HOX genes were overexpressed in LSCC samples and seem to be required in biological processes involved in tumor development. This suggests that HOX genes might play a critical role in the physiopathology of LSCC tumors.

The interaction of cultural consonance and a polymorphism in the 2A serotonin receptor in relation to depression in Brazil: failure to replicate previous findings.

OBJECTIVES: To replicate a previously identified gene-environment interaction between a genetic polymorphism in the serotonin 2A receptor and cultural consonance in family life in relation to depressive symptoms (Dressler et al., 2009). METHODS: A sample of 402 individuals in a sample drawn from four different economic strata in Ribeirão Preto, Brazil was interviewed and genotyped. RESULTS: Cultural consonance in family life has an inverse association with depressive symptoms (beta = -0.439, P < 0.001) and with high depressive symptoms (OR = 2.36, P < 0.001), but the interaction with genotype was not statistically significant. CONCLUSION: The previously identified gene-environment interaction was not replicated. Limitations of the study are discussed. Am. J. Hum. Biol. 28:936-940, 2016. © 2016Wiley Periodicals, Inc.

Placenta-Enriched LincRNAs MIR503HG and LINC00629 Decrease Migration and Invasion Potential of JEG-3 Cell Line.

LINC00629 and MIR503HG are long intergenic non-coding RNAs (lincRNAs) mapped on chromosome X (Xq26), a region enriched for genes associated with human reproduction. Genes highly expressed in normal reproductive tissues and cancers (CT genes) are well known as potential tumor biomarkers. This study aimed to characterize the structure, expression, function and regulation mechanism of MIR503HG and LINC00629 lincRNAs. According to our data, MIR503HG expression was almost exclusive to placenta and LINC00629 was highly expressed in placenta and other reproductive tissues. Further analysis, using a cancer cell lines panel, showed that MIR503HG and LINC00629 were expressed in 50% and 100% of the cancer cell lines, respectively. MIR503HG was expressed predominantly in the nucleus of JEG-3 choriocarcinoma cells. We observed a positively correlated expression between MIR503HG and LINC00629, and between the lincRNAs and neighboring miRNAs. Also, both LINC00629 and MIR503GH could be negatively regulated by DNA methylation in an indirect way. Additionally, we identified new transcripts for MIR503HG and LINC00629 that are relatively conserved when compared to other primates. Furthermore, we found that overexpression of MIR503HG2 and the three-exon LINC00629 new isoforms decreased invasion and migration potential of JEG-3 tumor cell line. In conclusion, our results suggest that lincRNAs MIR503HG and LINC00629 impaired migration and invasion capacities in a choriocarcinoma in vitro model, indicating a potential role in human reproduction and tumorigenesis. Moreover, the MIR503HG expression pattern found here could indicate a putative new tumor biomarker.

Gene expression analysis of laryngeal squamous cell carcinoma.

Laryngeal squamous cell carcinoma (LSCC) is one of the most common malignancies of the head and neck tumors Zhang et al., 2013 [1]). Previous studies have associated its occurrence with social activities, such as tobacco and alcohol consumption (Hashibe et al., 2007a [2]; Hashibe et al., 2007b [3]; Shangina et al., 2006 [4]). Here, we performed a genome-wide gene expression profiling in thirty-one patients positively diagnosed for LSCC, in order to investigate new targets involved in tumorigenesis.

Genetic polymorphisms associated with the inflammatory response in bacterial meningitis.

BACKGROUND: Bacterial meningitis (BM) is an infectious disease that results in high mortality and morbidity. Despite efficacious antibiotic therapy, neurological sequelae are often observed in patients after disease. Currently, the main challenge in BM treatment is to develop adjuvant therapies that reduce the occurrence of sequelae. In recent papers published by our group, we described the associations between the single nucleotide polymorphisms (SNPs) AADAT +401C > T, APEX1 Asn148Glu, OGG1 Ser326Cys and PARP1 Val762Ala and BM. In this study, we analyzed the associations between the SNPs TNF -308G > A, TNF -857C > T, IL-8 -251A > T and BM and investigated gene-gene interactions, including the SNPs that we published previously. METHODS: The study was conducted with 54 BM patients and 110 healthy volunteers (as the control group). The genotypes were investigated via primer-introduced restriction analysis-polymerase chain reaction (PIRA-PCR) or polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) analysis. Allelic and genotypic frequencies were also associated with cytokine and chemokine levels, as measured with the x-MAP method, and cell counts. We analyzed gene-gene interactions among SNPs using the generalized multifactor dimensionality reduction (GMDR) method. RESULTS: We did not find significant association between the SNPs TNF -857C > T and IL-8 -251A > T and the disease. However, a higher frequency of the variant allele TNF -308A was observed in the control group, associated with changes in cytokine levels compared to individuals with wild type genotypes, suggesting a possible protective role. In addition, combined inter-gene interaction analysis indicated a significant association between certain genotypes and BM, mainly involving the alleles APEX1 148Glu, IL8 -251 T and AADAT +401 T. These genotypic combinations were shown to affect cyto/chemokine levels and cell counts in CSF samples from BM patients. CONCLUSIONS: In conclusion, this study revealed a significant association between genetic variability and altered inflammatory responses, involving important pathways that are activated during BM. This knowledge may be useful for a better understanding of BM pathogenesis and the development of new therapeutic approaches.

The Influence of genetic factors on peripartum depression: A systematic review.

BACKGROUND: This systematic review aimed to explore the potential influence of genetic factors on the symptoms of peripartum depression and to critically analyze the methodologies employed by the examined studies. METHODS: A systematic review of the literature indexed prior to July 2014 identified 200 articles. After applying the inclusion and exclusion criteria, 39 papers were included. RESULTS: The papers predominantly featured a molecular genetic approach (n=35), and the majority examined polymorphisms (n=27). Most studies used samples of Caucasians living in high income countries. The results suggest that the influence of genetic factors become more consistent when methodological variations among the studies are considered. Environmental stressors are also important variables that influence the relationship between genetic factors and peripartum depressive states. In addition, differences in the influence of genetic factors were observed depending upon the precise time point during pregnancy or the postpartum period that was examined in the studies. The late stages of pregnancy and the early postpartum period were times of greater genetic vulnerability. LIMITATIONS: This study was limited by the small number of papers reviewed and by the lack of information regarding whether the effects of genetics on peripartum depression are specific to certain ethnicities and/or stressors. CONCLUSIONS: Genetic studies of perinatal depression reinforce a pathophysiological role of the hormonal changes inherent in the childbirth period. However, the distinction between depressive episodes that begin during pregnancy from those that begin during the postpartum period can still be useful to improve our understanding of the physiopathology of depressive disorders.

GPC3 reduces cell proliferation in renal carcinoma cell lines.

BACKGROUND: Glypican 3 (GPC3) is a member of the family of glypican heparan sulfate proteoglycans (HSPGs). The GPC3 gene may play a role in controlling cell migration, negatively regulating cell growth and inducing apoptosis. GPC3 is downregulated in several cancers, which can result in uncontrolled cell growth and can also contribute to the malignant phenotype of some tumors. The purpose of this study was to analyze the mechanism of action of the GPC3 gene in clear cell renal cell carcinoma. METHODS: Five clear cell renal cell carcinoma cell lines and carcinoma samples were used to analyze GPC3 mRNA expression (qRT-PCR). Then, representative cell lines, one primary renal carcinoma (786-O) and one metastatic renal carcinoma (ACHN), were chosen to carry out functional studies. We constructed a GPC3 expression vector and transfected the renal carcinoma cell lines, 786-O and ACHN. GPC3 overexpression was analyzed using qRT-PCR and immunocytochemistry. We evaluated cell proliferation using MTT and colony formation assays. Flow cytometry was used to evaluate apoptosis and perform cell cycle analyses. RESULTS: We observed that GPC3 is downregulated in clear cell renal cell carcinoma samples and cell lines compared with normal renal samples. GPC3 mRNA expression and protein levels in 786-O and ACHN cell lines increased after transfection with the GPC3 expression construct, and the cell proliferation rate decreased in both cell lines following overexpression of GPC3. Further, apoptosis was not induced in the renal cell carcinoma cell lines overexpressing GPC3, and there was an increase in the cell population during the G1 phase in the cell cycle. CONCLUSION: We suggest that the GPC3 gene reduces the rate of cell proliferation through cell cycle arrest during the G1 phase in renal cell carcinoma.