通过CRISPR/Cas9技术抑制TFDP3基因对前列腺癌PC3细胞生物学功能的影响

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....

Effects of TFDP3 knock-out by CRISPR/Cas9 on biological function of prostate cancer PC3 cells

[Abstract] Objective: CRISPR/Cas9 technology was used to construct a stable transgenic strain of prostate cancer PC3 cells withTFDP3 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 knockoutwas constructed by CRISPR/Cas9 technology. The constructed lentivirus was used to infect PC3 cells, and the stable transgenic strainwas screened. Flow cytometry was used to detect the cell cycle distribution and apoptosis of cells in KO group (with TFDP3 KO) andcontrol group. Cell migration and invasion capabilities were further detected by Scratch and Transwell assays. Results: Three sgRNAswere obtained through bioinformatics screening. Among them, the sgRNA2 obviously inhibited the prostate cancer gene expression. Byusing 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, thepercentage of cells in G0/G1 phase increased while the percentage of cells in G2/M stage decreased in the KO group, and the cellapoptosis rate significantly increased in the KO group (P<0.05); the migration rate of the PC3 cells in the KO group was significantlydecreased (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 passedthrough 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 constructedthrough CRISPR/Cas9 technology. It was confirmed that after the expression of TFDP3 gene was inhibited, PC3 cell cycle was blockedand the apoptosis rate was increased. Furthermore, the ability of migration and invasion was significantly weakened, suggesting thatTFDP3 is a tumor-promoting gene in prostate cancer.