In vitro silencing of YRNAs, a potential new tool for studying a epigenetic mechanisms recently described in allergy

Resumo

Background : As allergic diseases are becoming more frequent reaching a concern of public health, new methods for its research are required. To increase the spectrum of laboratory approaches to the disease, cell line studies need to be improved with new sophis-ticated techniques that mimic pathological states to deepen in their molecular mechanisms. Small non- coding RNAs (snRNAs) develop important functions related to epigenetic regulation. YRNAs are a group of snRNAs involved in the initiation of DNA replication and RNA stability that regulate gene expression. They have been pre-viously related to autoimmunity and cancer but, up to now, never before in Allergy. Our studies have provided differential expression profiles of YRNAs in allergic patients. We have developed a strategy for transiently silencing these YRNAs in cell culture. Method : Transient silencing of the YRNAs YRNA- 621, YRNA- 253 and YRNA- 294 were performed in the cell line Jurkat, used as a model of T lymphocyte. We used Dicer- Substrate Short Interfering RNAs (DSiRNAs) that are 27mer duplex RNAs that demonstrate in-creased potency in RNA interference compared to traditional, 21mer siRNAs, provided by Integrated DNA Technologies, designed against our YRNAs of interest. Cells were plated at a density of 2.5 × 10 5 cells per mL and trans-fected with DSiRNAs designed for each YRNA or the Scrambled Control at a final concentration of 50 nmol/L. Briefly, DSiRNAs were diluted in of Opti- MEM® and mixed with Lipofectamine® RNAiMAX diluted in Opti- MEM®. The complexes of DsiRNA- Lipofectamine® RNAiMAX were added directly to the cells and mixed gently. Complexes did not have to be removed following transfection. Cells were incubated at 37°C in a CO 2 incubator for 3 days post transfection before assaying for silencing gene expression. Results : With this method, we have obtained successful and signifi-cant silencing of the three different YRNAs that are the object of our study. When compared to the scrambled transfected control, the levels of the specific transcript were reduced an 85% in the case of YRNA- 621, a 32% in the case of YRNA- 253 and a 41% in the case of YRNA- 294. Conclusion : We have developed an easy and affordable method to silence small non coding RNAs in the cell line Jurkat, providing new perspectives in this cell line to study molecular mechanisms related to allergic diseases in vitro.