沉默DOT1L基因对急性单核细胞白血病细胞THP-1增殖的影响

摘要

This study was aimed to investigate the influence of short hairpin RNA (shRNA) on proliferation of human leukemia cell line THP-1.The shRNA targeting the site 732-752 of DOT1L mRNA was designed and chemically synthesized,then a single-vector lentiviral,tet-inducible shRNA-DOT1L system(Plko-Tet-On) was generated.Thereafter,the THP-1 cells with lentivirus were infected to create stable cell line with regulatable shRNA expression.The expression of DOT1L in the THP-1 cell line was assayed by RT-PCR.Effect of shRNA-DOT1L on the proliferation of THP-1 cells was detected with MTT method,and the change of colony forming potential of THP-1 cells was analyzed by colony forming unit test.Cell cycle distribution was tested by flow cytometry.The results indicated that the expression of DOT1L was statistically lower than that in the control groups.The proliferation and colony forming capacity of THP-1 cells were significantly inhibited.The percentage of cells at G0/G1 phase increased in THP-1/shRNA cells treated with Dox while the percentage of cells at S phase significantly decreased as compared with that in the control group.It is concluded that the shRNA targeting DOT1L can effectively inhibit the proliferation of acute monocytic leukemia cell line THP-1.%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.