Twist-1通过MPL促进髓系白血病细胞增殖和耐药

Twist-1 promotes proliferation and drug resistance of myeloid leukemic cells via MPL

目的:检测Twist-1与骨髓增生性白血病病毒癌基因(myeloproliferative leukemia virus oncogene,MPL)在髓系白血病患者和髓系造血系统恶性肿瘤细胞系中表达的相关性,并探讨Twist-1是否通过MPL对髓系白血病细胞增殖、耐药发挥促进作用。方法:选取中国医学科学院血液病医院2005年1月至2008年12月初次诊断为急性髓系白血病(acute myeloid leukemia,AML)、慢性粒细胞白血病(chronic myeloid leukemia,CML)患者的骨髓标本41例(其中AML 23例、CML 18例),用Real-time PCR检测AML、CML患者以及髓系造血系统恶性肿瘤细胞系中Twist-1和MPL mRNA的表达情况,并分析其相关性。构建MPL过表达载体,制备慢病毒并感染髓系白血病细胞系K562、U937,通过细胞计数实验、集落形成实验以及药物敏感实验评价其对白血病细胞增殖、集落形成能力以及耐药的影响,并进一步确定Twist-1是否通过MPL发挥白血病促进作用。结果:在U937和K562细胞中过表达Twist-1明显增加MPL蛋白水平(P<0.05),敲降Twist-1后MPL mRNA的表达水平明显下降(P<0.01);AML、CML患者骨髓单核细胞(bone marrow mononuclear cells,BMMCs)中Twist-1 mRNA表达与MPL mRNA表达水平呈显著正相关(P<0.05)。过表达MPL使K562和U937细胞对化疗药物柔红霉素及伊马替尼的敏感性显著降低(P<0.01),且提高K562-MPL、U937-MPL的细胞增殖、集落形成数目(均P<0.01);干扰Twist-1并过表达MPL显著降低髓系白血病细胞增殖和集落形成(均P<0.01)。结论:Twist-1通过MPL促进AML、CML白血病细胞的增殖、存活和耐药。

Objective：To investigate the correlation between myeloproliferative leukemia virus oncogene （MPL） and Twist 1 in patients with acute myeloid leukemia （AML） and chronic myeloid leukemia （CML）, and to explore whether MPL contributes to Twist 1 mediated cell proliferation and drug resistance of leukemia cells. Methods： Bone marrow specimens from 41 patients, who were first diagnosed as AML or CML in Hospital of Blood Diseases Affiliated to Chinese Academy of Medical Sciences between January 2005 and December 2008 （23 cases of AML, 18 cases of CML）, were selected in this study. Expressions of Twist 1 mRNA and MPL mRNA in hematopoietic tumor cell lines and bone marrow mononuclear cells （BMMCs） of patients with AML or CML were detected by Real time PCR, and their correlation was analyzed. Lentiviral vector over expressing MPL （pCDH1 MPL） were constructed and transduced into myeloid leukemia cell lines K562 and U937. Effect of MPL on proliferation, colony formation and drug sensitivity of the leukemic cells were evaluated by cell counting, colony formation assay and MTT assay; in addition, whether Twist 1 promote the proliferation of leukemia cells via MPL was further confirmed. Results： Over expression of Twist 1 significantly increased the protein expressions of MPL in U937 and K562 cell lines （P〈0.05） while knock down of Twist 1 significantly decreased the expression of MPL （P〈0.01）. The mRNA expressions of Twist 1 and MPL showed a significant positive correlation in BMMCs of patients with AML and CML （P〈0.05）. Over expression of MPL significantly reduced the drug sensitivity of K562 and U937 cells to daunorubicin and Imatinib （P〈0.01）. Enforced expression of MPL in U937 and K562 cells promoted the cell growth and colony formation （all P〈0.01）; Twist 1 knockdown with MPL over expression significantly impaired cell growth and colony formation of leukemia cells （all P〈0.01）. Conclusion： Twist 1 promoted proliferation, survival and chemotherapy resistance in the leukemia cells of AML and CML via MPL.