[ 摘 要 ] 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.

国家自然科学基金资助项目（No. 81372747）