Análise da expressão circadiana de genes envolvidos com câncer em leucócitos normais isolados de sangue periférico humano

Abstract

The term circadian rhythm derives from the latin meaning “about a day” and describes endogenous oscillations that occur in organisms through the day with a period of approximately 24 hours. These rhythmic variations are presente in diferente tissues. Modifications in circadian rhythms can cause various diseases such as câncer. Although circadian rhythms and cell cycle control are closely related, there are few known genes that could participate in the molecular pathways that connect these processes. This study aimed to identify genes involved in câncer previously showing circadian expression. In normal leukocytes isolated from human peripheral blood. Samples were collected over 24 hours with intervals of four hours. Alalyses were performed by RT-qPCR using PCR Array plates (RT2 PCR Array ProfilerTM Human Cancer Pathway Finder). The stability analysis performed with six reference genes by geNorm software determined the best pair for nomalization wer Rpl13a and Gapdh. However, four of the six analyzed genes showed temporal variation. Thus, normalizations were performed with Rpl13a and B2m genes. These stability data of reference genes suggest that use only a single program may compromisse the analysis of the expression profile in the research ciecadian rhythms. After normalization with these genes, the PCR Array results showed that 13 (15,5%) of the 84 genes exhibited daily variations in their expression.: Bad, Casp8, Cflar, Fas, Htatip2, Itga1, Mmp9, Plaur, Nme1, Pdgfa, Rb1, Tnf and Tp53. Furthermore, we observed that seven had a peak of expression. During the night phase and six presented during the dirunal phase. The expression profile of these genes show a possible functonal significance. Of the thirteen genes showed variation, three (Casp8, Htatip2 and Mmp9) were selected for validation of expression. Through the day utilizing na expanded sample os individuals. The results indicated that Htatip2 and Mmp9 genes exhibited significant variation in the analyzes. However, it was not possible to confirm the pattern of variation in Casp8. We believe that the data presented are original and can contribute to a better understanding of the pathways regulating hematopoiesis in normal and altered contexto, and its potential contribution to the diagnosis and chronopharmacological therapies.