Knockdown do fator de transcrição NRF2 em células embrionárias renais humanas (HEK293T) pelo método de transfecção com plasmídeo

Abstract

Cell culture is an important tool for the study of molecular mechanisms involved in biological processes, allowing the visualization of the response to different stimuli presenting results quickly and effectively compared to in vivo studies. For this reason, techniques have been developed to elucidate such mechanisms, such as the interference RNA methodology. These RNA fragments act by means of genomic alterations in specific targets through the formation of enzymatic complexes such as RNA-induced silencing complex (RISC), which allow alterations in cellular gene expression and show the contribution of that cellular factor within the molecular response cascades. The most cost-effective technique for gene silencing, is Knockdown(KD), i.e., the decrease of gene expression of a target gene, is through plasmid transfection via liposome to which successfully expresses the small interfering RNA fragments (siRNA) in the target cell. The present study proposed a transfection methodology in culture of HEK293T embryonic renal tubular cells, using the Escort™ IV Transfection Reagent, which is the liposome itself, with cationic characteristics, interacting with electronegative molecules such as nucleic acids, This binding helps to incorporate the plasmid pLV-CMV-SV40-Puro shNrf2, aiming to create Nrf2 knockdown clones - HEK293T Nrf2-KD competent. After stabilization, the knockdown cells underwent a cellular selection process with puromycin antibiotic, an amino nucleoside whose mechanism of action is to interfere in the transfer of peptides in the ribosome. The transfected cells, which had a resistance gene included in the plasmid, resisted gradual concentrations of selection, reaching the construction of a homogeneous population of HEK293T Nrf2-knockdown clones.Soon after, the stabilized cells in final selection concentration were collected for analysis in RT-qPCR for evidencing the success of gene expression suppression, evidencing the silencing by siRNA transfection. The results show a 50% decrease in Nrf2 expression compared to the control group, attesting to the success of gene silencing. Thus, it can be said that the methodology proposed by this work fulfills its role in achieving Nrf2 knockdown in HEK293T cells and shows itself as an effective methodology with low cost for transfection processes, being possible to adapt it for knockdown of other genes through the design and cellular insertion of other plasmids with different genes of interest. Moreover, it opens the margin for future studies of compounds that use this pathway of molecular signaling, such as the study ofphytochemical products that have in the literature indications of action by this molecular pathway of Nrf2, such studies will highlight the therapeutic potential of new substances and increase the therapeutic scope for injuries from oxidative stress endogenous and exogenous