ABSTRACT: The aim of the present study was to obtain and characterize an in vitro model for endogenous APOA1 and PON1 long-time up-regulation in hepatocytes that can be further used to decipher the mechanism of their protective action. Cultured human hepatocytes (HuH-7 cell line) were transfected with CRISPR/dCas9 activation plasmids targeting APOA1/PON1 genes. Following selection with specific antibiotics, bulk RNA sequencing was used for the transcriptomic characterization of the transfected hepatocytes. Hepatocytes from human hepatocarcinoma (Huh7 line, Cell Lines Service GmbH, Germany) were cultured in RPMI-1640 supplemented with FBS (10%, v/v), penicillin (100U/mL), and streptomycin (0.1mg/mL). Huh7 cells were seeded into 12 well plates at a density of 70.000 cells/well in complete RPMI media, without antibiotics. At 70-80% confluency, the hepatocytes were transfected using the CRISPR/(d)Cas9 activation plasmids for APOA1, PON1, or Control plasmids (CP) containing resistance genes for Blasticidin, Hygromycin B and Puromycin. 1 µg DNA plasmid/mL and 2.5µL/mL transfection reagent for each transfection condition were used according to the manufacturer’s instructions (SantaCruz Biotech., USA). After 48h, the cells’ media was changed and the cells were left to recover for another 48h. After transfection, the culture media was replaced with RPMI supplemented with 10% FBS, containing selection antibiotics, Hygromycin B (50 µg/mL), Blasticidin S HCl (2 µg/mL), and Puromycin dihydrochloride (1 µg/mL). The culture media with selection antibiotics was changed every 2 days for a period of 12 days, which was sufficient for the selection antibiotics to induce the death of the un-transfected cells. After selection, the medium was replaced with antibiotics-free RPMI and the transfected and selected Huh7 were allowed to grow until confluency. Total RNA was extracted from cells using Trizol (Invitrogen, USA) based on manufacturer’s instructions. To assess RNA purity and integrity, samples were analyzed via 1% agarose gel electrophoresis, and a NanoDrop spectrophotometer was used to confirm sample purity and quantity. Bulk long-RNA sequencing (longRNA-seq) of total RNA isolated from hepatocytes was done by Novogene, UK. The analysis included an additional RNA sample quality control, directional library preparation (rRNA removal), and long-RNA sequencing on NovaSeq X Plus Series (PE150, 12G raw data per sample).
