RESUMO
BACKGROUND: Obesity is prevalent in PR and has been associated with prostate cancer (PCa) mortality and aggressiveness. Polymorphisms (SNPs) rs9930506 and rs9939609 in the FTO gene have been associated with both obesity and PCa. The aim of this work was to ascertain whether the presence of these SNPs is associated with PCa risk and severity in a cohort of Puerto Rican men. METHODS AND FINDINGS: The study population consisted of 513 Puerto Rican men age ranging from 40-79 years old who underwent radical prostatectomy (RP) as the first treatment for PCa and 128 healthy Puerto Rican men age ranging from 40-79 years old. Genomic DNA (gDNA) was extracted and SNPs were determined by Real-Time PCR. PCa severity was defined based on RP stage and Gleason Score. The relationship of FTO SNPs with demographic, clinical characteristics, PCa status and PCa severity were assessed. Logistic regression models with a 95% confidence interval (CI) determined SNPs interaction with PCa risk and severity odds ratio (ORs). RESULTS AND DISCUSSION: BMI, age and PSA were considered as confounders. Hardy-Weinberg equilibrium was present for both SNPs. The heterozygous forms (A/G; T/A) were the most prevalent genotypes and the frequency of alleles and genotypes for both SNPs agreed with those published in 1000 genomes. Results suggest an inverse association between the mutated rs9939609 and the risk of having PCa (OR: 0.53, 95% CI: 0.31-0.92) and a positive association with overweight (OR: 1.05, 95% CI: 0.68-1.62). Importantly, among the cases that were overweight, those with mutated rs9939609 had a greater chance of high severity PCa (OR: 1.39, 95% CI: 0.84-2.32) although these results were not statistical significant upon adjustment. Limitations of the study were the relatively small cohort and lack of access to the weight history of all our subjects. CONCLUSION: Results offer a research line to be followed with an expanded number of subjects that may provide a better statistical significance, to unravel the high mortality rate in this population.
RESUMO
BACKGROUND: Sporothrix schenckii is a pathogenic dimorphic fungus of worldwide distribution. It grows in the saprophytic form with hyaline, regularly septated hyphae and pyriform conidia at 25°C and as the yeast or parasitic form at 35°C. Previously, we characterized a calcium/calmodulin kinase in this fungus. Inhibitors of this kinase were observed to inhibit the yeast cell cycle in S. schenckii. RESULTS: The presence of RNA interference (RNAi) mechanism in this fungus was confirmed by the identification of a Dicer-1 homologue in S. schenckii DNA. RNAi technology was used to corroborate the role of calcium/calmodulin kinase I in S. schenckii dimorphism. Yeast cells were transformed with the pSilent-Dual2G (pSD2G) plasmid w/wo inserts of the coding region of the calcium/calmodulin kinase I (sscmk1) gene. Transformants were selected at 35°C using resistance to geneticin. Following transfer to liquid medium at 35°C, RNAi transformants developed as abnormal mycelium clumps and not as yeast cells as would be expected. The level of sscmk1 gene expression in RNAi transformants at 35°C was less than that of cells transformed with the empty pSD2G at this same temperature. Yeast two-hybrid analysis of proteins that interact with SSCMK1 identified a homologue of heat shock protein 90 (HSP90) as interacting with this kinase. Growth of the fungus similar to that of the RNAi transformants was observed in medium with geldanamycin (GdA, 10 µM), an inhibitor of HSP90. CONCLUSIONS: Using the RNAi technology we silenced the expression of sscmk1 gene in this fungus. RNAi transformants were unable to grow as yeast cells at 35°C showing decreased tolerance to this temperature. The interaction of SSCMK1 with HSP90, observed using the yeast two-hybrid assay suggests that this kinase is involved in thermotolerance through its interaction with HSP90. SSCMK1 interacted with the C terminal domain of HSP90 where effector proteins and co-chaperones interact. These results confirmed SSCMK1 as an important enzyme involved in the dimorphism of S. schenckii, necessary for the development of the yeast phase of this fungus. Also this study constitutes the first report of the transformation of S. schenckii and the use of RNAi to study gene function in this fungus.