[摘要] 目的：检测转录因子E2F1 与生长阻滞和DNA损伤诱导蛋白45g（GADD45g）基因在急性髓系白血病（AML）患者中表达的相关性，探讨GADD45g 是否通过抑制E2F1 诱导AML细胞DNA损伤、凋亡、衰老、周期阻滞和提高药物敏感性。方法：选取2013 年1 月至2016 年12 月中国医学科学院血液病医院初诊为AML患者32 例骨髓标本及AML细胞系U937、HL60、THP-1和Molm-13，用qPCR检测组织和细胞中GADD45g 和E2F1 mRNA的表达水平，并分析其相关性。构建E2F1 过表达载体，并制备重组慢病毒在过表达GADD45g 的Molm-13 和THP-1 细胞中过表达E2F1，通过彗星实验、Annexin V/7AAD流式细胞术、β-半乳糖苷酶染色和PI 染色流式细胞术等确定GADD45g 是否通过抑制E2F1 对AML细胞发挥抑癌作用。结果：AML患者骨髓和细胞系中GADD45g 和E2F1 mRNA的表达呈显著负相关（r=–0.663，P<0.01）。GADD45g 在AML细胞系中过表达显著抑制了E2F1的表达（均P<0.01）。成功构建同时过表达GADD45g 和E2F1 的Molm-13 和THP-1 细胞，与对照组比较，过表达组细胞GADD45g和E2F1 蛋白表达水平均显著升高（均P<0.01）。与过表达GADD45g 的细胞相比，同时过表达GADD45g 和E2F1 细胞的凋亡、衰老率和DNA损伤水平均显著降低（均P<0.01）；在过表达GADD45g 的细胞中过表达E2F1 逆转了GADD45g 诱导的周期阻滞（均P<0.01），进而降低了过表达GADD45g 对化疗药物的敏感性（均P<0.01）。结论：GADD45g 通过抑制E2F1 在AML中发挥抗肿瘤作用。

[Abstract] Objective: To investigate the correlation between the expression of E2F1 and growth arrest and DNA damage inducible protein 45g (GADD45g) in patients with acute myeloid leukemia (AML), and to explore whether GADD45g exerts its induction of DNA damage, cell apoptosis, senescence, cell cycle arrest and drug sensitivity in AML through inhibition of E2F1. Methods: A total of 32 cases of bone marrow specimens from patients initially diagnosed as AML in Hospital of Blood Diseases Affiliated to Chinese Academy of Medical Sciences from January 2013 to December 2016, were selected for this study; In addition, AML cell lines (U937, HL60,THP-1, Molm-13) were also collected for this study. The mRNA expression of GADD45g and E2F1 in above mentioned specimens and cell lines by qPCR, and their correlation was also analyzed. The lentiviral vector over-expressing E2F1 (pLV-E2F1-RFP) was constructed to prepare recombinant lentivirus, which was then transfected Molm-13 and THP-1 cells that over-expressing GADD45g. Whether GADD45g exerts tumor inhibition effect on AML cells through inhibition of E2F1 was determined by comet assay, Annexin V/7AAD flow cytometry, β-galactosidase staining and PI staining flow cytometry etc. Results: The mRNA expression of GADD45g was negatively correlated with E2F1 in bone marrow of AML patients and AML cell lines (r=–0.663, P<0.01). Over-expression of GADD45g significantly inhibited the expression of E2F1 in AML cell lines (all P<0.01). Molm-13 and THP-1 cells that simultaneously over-expressing GADD45g and E2F1 were successfully constructed. Compared with the control group, the protein expressions of GADD45g and E2F1 in over-expression groups were significantly increased (all P<0.01). Compared with cells over-expressing GADD45g alone,simultaneous over-expression of both GADD45g and E2F1 significantly reduced the apoptosis, senescence and DNA damage (all P<0.01), and rescued cell cycle arrest in Molm-13 and THP-1 cells (all P<0.01), thus further reduced the chemo-sensitivity of AML cells caused by GADD45g over-expression (all P<0.01). Conclusion: GADD45g exerts anti-tumor effect in AML via inhibition of E2F1.

国家自然科学基金资助项目（No.81670158, No.81470278, No.81600138, No.81700106）；天津市应用基础与前沿技术研究计划项目（No. 17JCZDJC35100，No. 15JCQNJC10300，No. 17JCQNJC10800）