Effects of TFDP3 knock-out by CRISPR/Cas9 on biological function of prostate cancer PC3 cells
目的： 通过CRISPR/Cas9技术构建前列腺癌PC3细胞TFDP3基因敲除的稳转株，探讨抑制TFDP3表达对PC3细胞周期、凋亡、迁移和侵袭能力的影响。 方法： 通过生物信息学筛选sgRNA，通过CRISPR/Cas9技术、构建抑制TFDP3基因表达的sgRNA-Cas9共转染慢病毒，感染PC3细胞后筛选获取稳转细胞株。通过流式细胞术对TFDP3基因敲除（knock-out，KO）实验组与空白对照组进行细胞周期和凋亡检测，并进一步通过划痕实验和Transwell实验进行细胞迁移和侵袭能力检测。 结果： 通过生物信息学筛选获得3条sgRNA，其中sgRNA2有明显的抑制前列腺癌细胞基因表达的功能；通过CRISPR/Cas9技术成功构建了基于CRISPR/Cas9介导的TFDP3低表达的PC3细胞稳转株。抑制TFDP3基因表达后，相比于对照组，KO组中G0/G1期细胞百分比增加、G2/M期细胞百分比下降（P<0.05或P<0.01），细胞凋亡率显著升高（P<0.05），细胞迁移率明显下降[24 h迁移率：(44.00±1.60)% vs (65.00±4.40)%，P<0.01]，穿过聚碳酸酯膜的侵袭细胞数明显下降[（185.89±11.71）vs （248.33±11.95）个，P<0.01]。 结论： 通过CRISPR/Cas9技术抑制TFDP3基因表达后，PC3细胞发生周期阻滞、凋亡率也有所增加、迁移和侵袭能力显著减弱，提示TFDP3是一个前列腺癌促癌基因。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.
图 6 TFDP3基因敲除后PC3细胞周期和凋亡的改变
Figure 6. Changes of PC3 cell cycle and apoptosis after TFDP3 gene KO
A:The results of flow cytometry showed that the apoptotic rate of PC3 cells in the KO group was higher than that in the NC group; B: Compared with the NC group, the percentage of the cells in the G1 phase was significantly higher in the KO group,while the percentage of cells in the S phase and G2/M phase decreased, indicating that cells were blocked at G1 phase after TFDP3 KO (NC: Normal control group; KO: TFDP3 gene KO group)
图 7 TFDP3对前列腺癌细胞PC3迁移能力的影响
Figure 7. The effect of TFDP3 on the metastatic ability of prostate cancer PC3 cells
** P <0.01A: Migration of the PC3 cells in the NC and KO groups at 0, 8 and 24 h detected by scratching test (magnification: 100 times); B: Invasion of PC3 cells in the NC and KO groups detected by Transwell assay (magnification: 200 times); NC: normal control group; KO: TFDP3 gene KO group
表 1 TFDP3的sgRNA靶位点的核苷酸序列
Table 1. Nucleotide sequences of sgRNA targeting TFDP3
Target sgRNA sequence 5’- guide sequence Oligo sequence sgRNA1 GCCGGGCAGCACAACAGGAA CACC
sgRNA2 GCCGTCTTTCCATGAAGGTC CACC
sgRNA3 GGAGGTGTGTTCACGACGGC CACC
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