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Archive > Volume 28 Issue 5 > 2021,28(5):443-450. DOI:10.3872/j.issn.1007-385X.2021.05.004 Prev Next
Basic Research
Effects of TFDP3 knock-out by CRISPR/Cas9 on biological function of prostate cancer PC3 cells
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Abstract:
[Abstract] Objective: CRISPR/Cas9 technology was used to construct a stable transgenic strain of prostate cancer PC3 cells with TFDP3 gene knock-out (KO) to explore the effect of inhibiting TFDP3 expression on cell cycle, apoptosis and invasion of PC3 cells. Methods: The sgRNAs were screened by bioinformatics, and the sgRNA-cas9 co-transfection lentivirus with TFDP3 gene knockout was constructed by CRISPR/Cas9 technology. The constructed lentivirus was used to infect PC3 cells, and the stable transgenic strain was screened. Flow cytometry was used to detect the cell cycle distribution and apoptosis of cells in KO group (with TFDP3 KO) and control group. Cell migration and invasion capabilities were further detected by Scratch and Transwell assays. Results: Three sgRNAs were obtained through bioinformatics screening. Among them, the sgRNA2 obviously inhibited the prostate cancer gene expression. By using the CRISPR/Cas9 technology, a stable transgenic strain of PC3 prostate cancer cells with low expression of TFDP3 was obtained. The results of Flow cytometry showed that after the expression of TFDP3 gene was inhibited, compared with the control group, the percentage of cells in G0/G1 phase increased while the percentage of cells in G2/M stage decreased in the KO group, and the cell apoptosis rate significantly increased in the KO group (P<0.05); the migration rate of the PC3 cells in the KO group was significantly decreased (24 h migration rate: [44.00±1.60]% vs [65.00±4.40]%, P<0.01); the number of migrated cells in the KO group that passed through the polycarbonate membrane was significantly lower than that of the control group (185.89±11.71 vs 248.33±11.95, P<0.01). Conclusion: In this study, a stable transgenic strain of PC3 prostate cancer cell line with TFDP3 gene KO was constructed through CRISPR/Cas9 technology. It was confirmed that after the expression of TFDP3 gene was inhibited, PC3 cell cycle was blocked and the apoptosis rate was increased. Furthermore, the ability of migration and invasion was significantly weakened, suggesting that TFDP3 is a tumor-promoting gene in prostate cancer.
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Project supported by the National Natural Science Foundation of China (No. 81372747)
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