Journal of Shanghai Jiao Tong University (Medical Science) ›› 2023, Vol. 43 ›› Issue (9): 1080-1088.doi: 10.3969/j.issn.1674-8115.2023.09.002

• Basic research • Previous Articles    

Effect of Piezo1 on osteogenic differentiation of mouse bone marrow mesenchymal stem cells C3H10T1/2 based on CRISPR/Cas9

GAO Xin1(), YANG Yiling1(), HUANG Xiangru1, DAI Qinggang2, JIANG Lingyong1()   

  1. 1.Department of Oral and Maxillofacial Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for Stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology, Shanghai Research Institute of Stomatology, Shanghai 200011, China
    2.The 2nd Dental Centre, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; National Center for Stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology, Shanghai 200011, China
  • Received:2023-03-21 Accepted:2023-09-08 Online:2023-09-28 Published:2023-09-28
  • Contact: JIANG Lingyong E-mail:gxgaoxin@sjtu.edu.cn;yangyiling2017@foxmail.com;jianglingyong@sjtu.edu.cn
  • Supported by:
    National Natural Science Foundation of China(82071083);Natural Science Foundation of Shanghai(21ZR1436900);Program of Shanghai Academic/Technology Research Leader(20XD1422300);Cross-disciplinary Research Fund of Shanghai Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine(JYJC202116);Clinical Research Plan of Shanghai Hospital Development Center(SHDC2020CR4084);Project of Biobank of Shanghai Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine(YBKB201919);Original Exploration Project of Shanghai Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine(JYYC003);Biomaterials and Regenerative Medicine Institute Cooperative Research Project of Shanghai Jiao Tong University School of Medicine(2022LHB02);"Two-hundred Talents" Program of Shanghai Jiao Tong University School of Medicine(20221809)

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Abstract:

Objective ·To investigate the effect of Piezo1 on osteogenic differentiation of mouse mesenchymal stem cells C3H10T1/2 cell line based on CRISPR/Cas9 system that can achieve stable gene knockout. Methods ·According to the principle of CRISPR/Cas9 target design principle, two single guide RNAs (sgRNAs) were designed to construct lentivirus expressing Cas9 and lentivirus expressing sgRNA by using Lenti-Cas9-GFP and Lenti-U6-sgRNA-mCherry vectors. After the C3H101/2 cells were transfected with two types of lentiviruses, flow cytometry was used to screen mCherry- and GFP-positive cells. The monoclonal cells were selected, and amplified by PCR and agarose gel electrophoresis, and finally the monoclonal cell line with Piezo1 gene fragment knocked out was obtained. Sequencing, quantitative realtime PCR (qPCR) and immunofluorescence were performed to verify the the knockout efficiency of the constructed Piezol knockout C3H10T1/2 cells (CPK). CCK-8 assay was used to detect the effect of knocking out Piezo1 on cell proliferation; in vitro osteogenic induction differentiation was performed on successfully constructed Piezo1 gene knockout cells, and alkaline phosphatase (ALP) staining and alizarin red staining were used to investigate the effect of Piezo1 on osteogenic ability. Results ·Positive clone was obtained in bacterial fluid of monoclonal cell lines with Piezol knocked out after PCR amplification and agarose gel electrophoresis. Sequencing analysis showed that a stop condon TGA was produced in exon 4 of Piezo1 gene in advance, so that the protein could not be translated correctly. qPCR verified that Piezo1 gene in CPK was inhibited at mRNA level; Immunofluorescence showed that the knockout efficiency of Piezo1 gene in CPK was high, which basically hindered the expression of Piezo1 in cells. CCK-8 assay showed that the cell proliferation ability decreased after knocking out Piezo1 (P<0.05); The results of ALP staining and alizarin red staining showed that the osteogenic ability of cells decreased after knocking out Piezo1(P<0.05). The mRNA expression levels of osteogenetic-related genes such as α 1 type Ⅰ collagen (Col1a1),Runt-related transcription factor 2 (Runx-2),osterix (Osx) and alkaline phosphatase (Alp)in CPK decreased significantly (all P<0.05). Conclusion ·Piezo1 knockout C3H10T1/2 cells based on CRISPR/Cas9 system is constructed successfully and the osteogenic activity of stable Piezo1 knockout cell line is hindered significantly.

Key words: Piezo1, CRISPR/Cas9, mesenchymal stem cell, osteogenesis

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