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Gene silencing of ANGPTL3 suppresses α1-antitrypsin and lectin mannose-binding 1 expression in the hepatoma Huh7 cell line

ABSTRACT: Background: Angiopoietin-like 3 (ANGPTL3) is a hepatokine acting as negative regulator of hepatic and endothelial lipases. Vupanorsen, an antisense oligonucleotide targeting ANGPTL3, reduced triglycerides up to 60% in a phase 2b trial, but caused dose-dependent increases in hepatic fat fraction. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plasma levels also correlates with liver fat accumulation. Methods: To further elucidate the biological activity of ANGPTL3 on lipid metabolism in the liver we performed a RNAseq analysis from human hepatoma Huh7 cells transfected with siRNA-ANGPTL3, siRNA-PCSK9 and double siRNA-ANGPTL3/PCSK9. Results: RNA-seq analysis detected 192 genes out of 13,945 transcripts differential ex-pressed in response to ANGPTL3-siRNA compared to scramble-siRNA controls. In cells transfected with PCSK9-siRNA, 88 genes were differentially expressed, while dual transfection with both ANGPTL3- and PCSK9-siRNA resulted in 219 differentially ex-pressed genes. When ANGPTL3 gene expression was silenced we observed a compensa-tory induction of proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA and pro-tein expression. Moreover, PCSK9 silencing increased ANGPTL3 mRNA expression. Bi-oinformatic analysis revealed that gene silencing of ANGPTL3 or both ANGPTL3/PCSK9 suppresses SERPINA1, gene encoding α1-antitrypsin, and lectin mannose-binding 1 ex-pression (LMAN1). These data were confirmed by western blot and qRT-PCR analysis. Conclusions: Our data identified two key genes SERPINA1 and LMAN1, involved in endoplasmic reticulum stress, lipid accumulation and liver damage, that are downregu-lated in response to gene silencing of ANGPTL3 in Huh7 hepatoma cell line.

ORGANISM(S): Homo sapiens

PROVIDER: GSE312289 | GEO | 2026/02/02

REPOSITORIES: GEO

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Project description:Background: Hepatic knockdown of the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene or the angiopoietin-like 3 (ANGPTL3) gene has been demonstrated to reduce blood low-density lipoprotein cholesterol (LDL-C) levels, and hepatic knockdown of the angiotensinogen (AGT) gene has been demonstrated to reduce blood pressure. Genome editing can productively target each of these three genes in hepatocytes in the liver, offering the possibility of durable “one-and-done” therapies for hypercholesterolemia and hypertension. However, concerns around making permanent gene sequence changes via DNA strand breaks might hinder acceptance of these therapies. Epigenome editing offers an alternative approach to gene inactivation, via silencing of gene expression by methylation of the promoter region, but the long-term durability of epigenome editing remains to be established. Objectives and Results: We assessed the ability of epigenome editing to durably reduce the expression of the human PCSK9, ANGPTL3, and AGT genes in HuH-7 hepatoma cells. Cells treated with the CRISPRoff epigenome editor and PCSK9 guide RNAs were maintained for up to 124 cell doublings and demonstrated durable knockdown of gene expression and increased CpG dinucleotide methylation in the promoter, exon 1, and intron 1 regions. In contrast, cells treated with CRISPRoff and ANGPTL3 guide RNAs experienced only transient knockdown of gene expression. Cells treated with CRISPRoff and AGT guide RNAs also experienced transient knockdown of gene expression; although initially there was increased CpG methylation throughout the early part of the gene, this methylation was geographically heterogeneous—transient in the promoter, and stable in intron 1.Conclusions: This work demonstrates precise and durable gene regulation via methylation, supporting a new therapeutic approach for protection against cardiovascular disease via knockdown of genes such as PCSK9. However, the durability of knockdown with methylation changes is not generalizable across target genes, likely limiting the therapeutic potential of epigenome editing compared to other modalities.

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Gene silencing of ANGPTL3 suppresses α1-antitrypsin and lectin mannose-binding 1 expression in the hepatoma Huh7 cell line

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