LRP6-CRISPR prevents activation of hepatic stellate cells and liver fibrogenesis in rats

Linghua Yu; Linlin Wang; Huixing Yi; Xiaojun Wu

LRP6-CRISPR prevents activation of hepatic stellate cells and liver fibrogenesis in rats

Am J Transl Res. 2020 Feb 15;12(2):397-408. eCollection 2020.

Authors

Linghua Yu¹, Linlin Wang², Huixing Yi³, Xiaojun Wu¹

Affiliations

¹ Center for Gastroenterology and Hepatology, Institute of Liver Diseases, The First Affiliated Hospital of Jiaxing College Jiaxing, Zhejiang Province, PR China.
² Department of Basic Medicine Sciences, School of Medicine, Zhejiang University Hangzhou, Zhejiang Province, PR China.
³ Intensive Care Unit, The Second Affiliated Hospital of Zhejiang University Hangzhou, Zhejiang Province, PR China.

PMID: 32194892
PMCID: PMC7061824

Abstract

This study elaborated on the function of Low-density lipoprotein receptor-related protein 6 (LRP6), a critical component of Wnt signaling, in liver fibrosis. This study enrolled sixty-eight patients with liver fibrosis, with ten healthy liver tissue samples, served as the controls. A lentiviral vector expressing LRP6-CRISPR was constructed. Immortalized HSC-T6 cells were transfected with LRP6-CRISPR. A rat model of CCl₄-induced liver fibrosis was established, and rats were injected with lentiviral vectors expressing LRP6-CRISPR. LRP6 expression and fibrosis biomarkers were examined by PCR, Western blot, and immunofluorescence assay, respectively. HSC growth and its ability of migration and invasion were evaluated by MTT and Transwell assay, separately. Wnt signaling activity was examined by Luciferase reporter assay. LRP6 was overexpressed in human fibrotic-liver tissues, and the expression of LRP6 was correlated with liver fibrosis stages. LRP6 knockout with CRISPR suppressed the Wnt signaling activities and consequently repressed HSC activation and relived liver injury in fibrotic-liver model rats. Our data revealed that the knockout of LRP6 weakens the binding of Wnt ligand with its cell surface receptors, the first step of Wnt transduction cascade, and consequently repressed HSC activation.

Keywords: CRISPR-associated protein 9 (Cas9); Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR); Hepatic stellate cell (HSC); Low-density lipoprotein receptor-related protein 6 (LRP6); single-guide RNA (sgRNA).