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2022, Vol. 26 ›› Issue (19): 2978-2984Effect of RAB39B gene based on CRISPR/Cas9 technology on cartilage differentiation of bone marrow mesenchymal stem cellsMa Dujun1, 2, Peng Liping1, Zhou Ziqiong1, Zhao Jing2, Zhu Houjun2, Jiang Shunwan1, Zhong Jing2, She Ruihao2
Abstract: BACKGROUND: Based on previous research, the research team used proteomics and high-throughput sequencing to screen the correlation between the RAB39B gene and the proliferation and cartilage differentiation of bone marrow mesenchymal stem cells. OBJECTIVE: To investigate the effect of RAB39B gene knockout by CRISPR/Cas9 gene editing technology on cartilage differentiation of bone marrow mesenchymal stem cells. METHODS: CRISPR/Cas9 lentivirus infection method was used to construct stable transfection cell line of RAB39B gene knockdown. Rabbit bone marrow mesenchymal stem cells were divided into normal control group, no-load control group, and RAB39B knockout group. The knockout effect of CRISPR/Cas9 system on RAB39B was evaluated by western blot assay. The proliferation activity of bone marrow mesenchymal stem cells was detected by CCK-8 assay. The apoptosis rate of bone marrow mesenchymal stem cells was detected by flow cytometry. The mRNA expression of COLII and COLX was detected by qRT-PCR. The expression levels of RHOA, LIMK, and Sox9 proteins in chondrogenic differentiation related pathways of bone marrow mesenchymal stem cells were detected by western blot assay. RESULTS AND CONCLUSION: (1) Lentivirus carrying knockout RAB39B gene plasmid was found to be highly expressed in rabbit bone marrow mesenchymal stem cells 48-96 hours after infection. Western blot assay showed that the expression of RAB39B protein in bone marrow mesenchymal stem cells was significantly decreased after RAB39B knockout (P < 0.01). (2) Compared with the normal control group and the empty control group, the cell proliferation activity of the RAB39B gene knockout group was significantly reduced (P < 0.01); the apoptosis rate was increased (P < 0.01); the expression of COLII and COLX mRNA was significantly reduced (P < 0.01); RHOA, LIMK, and Sox9 protein expression was significantly decreased (P < 0.01). (3) These findings indicate that RAB39B gene knockout by CRISPR/Cas9 system can inhibit the proliferation of bone marrow mesenchymal stem cells, promote cell apoptosis, and reduce the chondrocyte differentiation ability, which indicates that RAB39B gene may affect the proliferation and chondrocyte differentiation ability of bone marrow mesenchymal stem cells, and its specific mechanism needs further study. Key words:gene editing, RAB39B gene, bone marrow mesenchymal stem cells, chondrocytes, cartilage differentiation |