Standardization of total RNA extraction and amplification for the analysis of long non-coding RNA (lncRNA) expression

Abstract

Familial hypercholesterolemia (FH) is an autosomal dominant disease, present in all ethnic groups, that affects cholesterol metabolism, reducing the catabolism of LDL particles and resulting in high plasma concentrations of these particles, with genetic bases not yet fully understood. In recent years, more comprehensive technologies have been used for the genotypic research of HF in the world, including studies related to epigenomic mechanisms, such as gene regulation systems by DNA methylation, histone acetylation, and mRNA inhibition/degradation by microRNAs ( miRNAs) and long non-coding RNAs (lncRNAs).lncRNAs are from 200 to thousands of nucleotides, with or without splicing. They can be transcribed from both strands of the human genome, including intergenic and mainly intronic regions, and exhibit different sites of initiation, termination, splicing and expression patterns in different cell types. Additionally, they are molecules capable of regulating gene expression through interaction with chromatin modifier complexes, mRNAs, miRNAs, siRNAs and proteins, characterizing an entire interactive biological process.While miRNAs are small non-coding genes that regulate post-transcriptional gene expression, targeting transcribed mRNAs, lncRNAs regulate gene expression by several mechanisms yet to be clarified. Admittedly, lncRNAs can act through several molecular mechanisms, both in transcriptional regulation and post-transcriptional mechanisms. Regarding transcriptional regulation, it is suggested that the transcription process of lncRNAs itself may be functional by facilitating an open chromatin structure in promoters of protein-coding genes, increasing accessibility to transcription factors and the RNA polymerase enzyme. On the other hand, the lncRNA transcription process can impair the transcription of the protein-coding gene, interfering with the recruitment of RNA polymerase II.Another way of regulating the transcription of protein-coding genes by lncRNAs involves epigenetic control, either through the recruitment of chromatin modifying complexes or in the regulation of DNA methylation levels. Growing evidence has pointed to the involvement of lncRNAs in several human diseases, especially cardiovascular diseases.Recent studies investigate the performance of lncRNAs in dyslipidemia and atherosclerosis through the regulation of lipid metabolism, endothelial dysfunction, inflammation, cell proliferation, migration and apoptosis. In this context, it is worth noting that in addition to genetic characteristics, epigenetic regulation via lncRNAs is also capable of determining the increase in cholesterol and the consequent development of cardiovascular diseases, as a potential tool in the search for a better elucidation of the pathophysiological mechanisms of FH and thus contribute for individualized therapy.Therefore, the present proposal aims to standardize the extraction of total RNA for amplification and analysis of differential expression of lncRNA in plasma from patients with FH. Epigenetic studies are of paramount importance to better understand the pathophysiological mechanisms and enable the development of new methods for the prevention, prognosis and therapy of cardiovascular diseases.