Longstanding Transcriptional Activation of APOA1 and PON1 in Human Hepatocytes by CRISPR/dCas9 Technology—Transcriptomic Profile and Crosstalk with Endothelial Cells

Authors:
Preprint
Publisher website
Google Scholar
Abstract
Apolipoprotein A1 (APOA1) and paraoxonase 1 (PON1) are key proteins of high-density lipoprotein (HDL). 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, RNA sequencing was used for the transcriptomic characterization of the transfected hepatocytes. The functionality of the secreted APOAI/PON1 was evaluated as the capacity of the conditioned medium (CM) from transfected HuH-7 to modulate the oxidative and inflammatory stress in TNFα-activated primary human umbilical endothelial cells (HUVEC). The results showed that: (1) a robust, long-time up-regulation (46 days) of endogenous APOA1/PON1 was obtained after CRISPR/dCas9 transfection and antibiotics selection; (2) APOA1/PON1 up-regulation led to a modified transcriptomic profile and increased the expression of several antioxidant genes in transfected hepatocytes as demonstrated by RNAseq analysis; (3) secreted APOA1/PON1 were functional as demonstrated by the CM ability to reduce the levels of reactive oxygen species and inflammatory markers (VCAM-1, MCP-1) in TNF-α-activated HUVEC. In conclusion, we achieved an experimental model of successful long-term up-regulation of endogenous APOA1 and PON1 in human hepatocytes. The targeted proteins are secreted in a functional form and can be used for deciphering their complex mechanism of protective action in various pathological conditions.

This publication has 30 references indexed in Scilit: