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q PCR检测组织和细胞中GADD45g和E2F1 m RNA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 m RNA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。
Objective:To investigate the correlation between the expression of E2 F1 and growth arrest and DNA damage inducible protein 45 g(GADD45 g)in patients with acute myeloid leukemia(AML),and to explore whether GADD45 g exerts its induction of DNA damage,cell apoptosis,senescence,cell cycle arrest and drug sensitivity in AML through inhibition of E2 F1.Methods:A total of32 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 m RNA expression of GADD45 g and E2 F1 in above mentioned specimens and cell lines by qPCR,and their correlation was also analyzed.The lentiviral vector over-expressing E2 F1(pLV-E2 F1-RFP)was constructed to prepare recombinant lentivirus,which was then transfected Molm-13 and THP-1 cells that over-expressing GADD45 g.Whether GADD45 g exerts tumor inhibition effect on AML cells through inhibition of E2 F1 was determined by comet assay,Annexin V/7 AAD flow cytometry,β-galactosidase staining and PI staining flow cytometry etc.Results:The mRNA expression of GADD45 g was negatively correlated with E2 F1 in bone marrow of AML patients and AML cell lines(r=–0.663,P<0.01).Over-expression of GADD45 g significantly inhibited the expression of E2 F1 in AML cell lines(all P<0.01).Molm-13 and THP-1 cells that simultaneously over-expressing GADD45 g and E2 F1 were successfully constructed.Compared with the control group,the protein expressions of GADD45 g and E2 F1 in over-expression groups were significantly increased(all P<0.01).Compared with cells over-expressing GADD45 g alone,simultaneous over-expression of both GADD45 g and E2 F1 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 GADD45 g over-expression(all P<0.01).Conclusion:GADD45 g exerts anti-tumor effect in AML via inhibition of E2 F1.
作者
常利珍
赵杨杨
郭丹
王楠
尹静
任倩
尤娜
马小彤
CHANG Lizhen;ZHAO Yangyang;GUO Dan;WANG Nan;YIN Jing;REN Qian;YOU Na;MA Xiaotong(State Key Laboratory ofExperimental Hematology,Institute of Hematology & Hospital of Blood Diseases,Chinese Academy of Medical Sciences & PekingUnion Medical College,Tianjin 300020,China)
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
北大核心
2019年第7期736-742,共7页
Chinese Journal of Cancer Biotherapy
基金
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)~~
