Construction of Human-derived Chondrocyte PIEZO2 Overexpressing Cell Line and Identification of Osteoarthritis Phenotype
10.13865/j.cnki.cjbmb.2025.05.1073
- VernacularTitle:人源软骨细胞PIEZO2过表达细胞株模拟骨关节炎表型
- Author:
Bo-Yang XU
1
;
Yi-Fei FAN
1
;
Yu-Qing DU
1
;
Meng-Ze SUN
1
;
Jun-Yan WANG
1
;
Jin CHENG
1
;
Ying-Fang AO
1
;
Xiao-Qing HU
1
Author Information
1. 北京大学第三临床医学院,北京大学第三医院运动医学科,北京大学运动医学研究所运动医学关节伤病北京市重点实验室,北京 100191
- Publication Type:Journal Article
- Keywords:
mechanosensitive ion channels;
PIEZO2;
chondrocytes;
osteoarthritis(OA);
CRISPR/Cas9-SAM system
- From:
Chinese Journal of Biochemistry and Molecular Biology
2025;41(6):871-878
- CountryChina
- Language:Chinese
-
Abstract:
To investigate the molecular mechanisms underlying the mechanosensitive ion channel PI-EZO2 in osteoarthritis(OA),we developed a lentiviral vector for endogenous PIEZO2 overexpression and established a stable PIEZO2-high-expressing immortalized human primary chondrocyte line.By map-ping the open reading frame of the PIEZO2 locus and designing sequence-specific sgRNA,we employed the CRISPR/Cas9 synergistic activation mediator(SAM)system to precisely integrate transcriptional ac-tivation elements into the PIEZO2 promoter region.Lentiviral-mediated targeted genomic integration en-sured endogenous PIEZO2 overexpression,confirmed by mCherry fluorescence tracing coupled with flow cytometric sorting,which revealed membrane-specific localization of PIEZO2 protein(localization effi-ciency:78.49％).Quantitative PCR demonstrated a 17-fold upregulation of PIEZO2 mRNA,while Western blotting validated enhanced membrane-localized protein expression.Strikingly,PIEZO2-overex-pressing chondrocytes exhibited hallmark OA metabolic phenotypes compared to wild-type controls:typeⅡ collagen mRNA expression decreased to 50％of baseline levels,whereas matrix metalloproteinase 13(MMP13)mRNA surged by 20-fold.These alterations recapitulated the pathological matrix metabolic phenotype observed in biomechanical OA models induced by cyclic mechanical stress(10％strain,0.5 Hz,8 h/day for 2 consecutive days).Collectively,we successfully generated a human chondrocyte model with stable PIEZO2 overexpression,which faithfully mirrors mechanotransduction-driven OA progression.This engineered cellular system provides a robust platform for dissecting PIEZO2-mediated mechanosig-naling networks and advancing targeted therapeutic discovery.
