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本研究旨在探讨DOT1L基因对人白血病THP-1细胞增殖的影响。针对DOT1L mRNA的第732-752靶位点设计并合成shRNA,构建pLKO-Tet-On条件性干扰载体,制备慢病毒并感染THP-1细胞,用强力霉素(Dox)诱导干扰载体表达;应用RT-PCR的方法验证干扰效率;采用改良MTT法检测干扰DOT1L基因后对THP-1细胞增殖能力的影响;用细胞集落形成实验观察干扰后细胞集落形成的能力;用流式细胞术分析细胞周期的变化。结果表明:THP-1细胞干扰后DOT1L基因的表达显著降低;DOT1L基因的表达下调抑制了细胞的增殖能力、集落形成能力并将细胞周期阻滞在G0/G1期。结论:利用shRNA靶向干扰DOT1L基因的表达,可以有效抑制人急性单核细胞白血病细胞THP-1的增殖。
This study aimed to investigate the effect of DOT1L gene on the proliferation of human leukemia THP-1 cells. To design and synthesize shRNA targeting 732-752 of DOT1L mRNA, we constructed pLKO-Tet-On conditional interference vector, prepared lentivirus and infected THP-1 cells, and induced the expression of interfering vector with Doxycycline (Dox) RT-PCR method to verify the interference efficiency; using modified MTT method to detect interference DOT1L gene on the proliferation of THP-1 cells; using colony formation assay to observe the interference of cell colony formation; using flow cytometry analysis of cell cycle The change. The results showed that the expression of DOT1L gene was significantly decreased after THP-1 cells interference. The down-regulation of DOT1L gene inhibited the proliferation and colony-forming ability of cells and blocked the cell cycle in G0 / G1 phase. Conclusion: shRNA targeting targeting DOT1L gene expression can effectively inhibit the proliferation of human acute monocytic leukemia cells THP-1.