摘要
目的探讨miR-99a对儿童急性髓性白血病(AML)的影响及其作用机制,为寻找新的靶向治疗方法提供理论依据。方法用实时定量聚合酶链反应(qRT-PCR)技术检测57例AMLL患儿及10例对照患儿的骨髓标本中miR-99a的表达;在细胞系水平通过对miR-99a过表达或抑制表达,利用MTT细胞增殖及双染凋亡试验研究miR-99a对AML细胞增殖、凋亡的影响。用在线软件预测miR-99a可能的靶基因,并通过双荧光报告及蛋白印迹技术对其靶基因进行验证。结果miR-99a在AML患儿初诊时高表达,在完全缓解(CR)时低表达;miR-99a促进AML细胞的增殖,抑制其凋亡;采用双荧光报告及蛋白印迹技术在细胞系中验证出CTDSPL可能是miR-99a的靶基因;并通过蛋白印迹技术在AML患儿标本中验证出miR-99a抑制CTDSPL蛋白的表达。结论miR-99a通过靶向负调控CTDSPL基因影响AML细胞增殖及凋亡,靶向抑制miR-99a表达可能成为治疗AML的一个新策略。
Objective This study aimed to explore the functions and molecular mechanisms of the miR-99 a in pediatric acute myeloid leukemia for the theoretical basis of novel targeted therapeutic strategies.Methods MiR-99 a expression levels were measured in 57 cases of children with AML and 10 cases of children with immune thrombocytopenia by qRT- PCR.Effect of miR-99 a on cell proliferation and apoptosis in pediatric myeloid leukemia was evaluated when miR-99 a was over-expressed or downexpressed in AML cell lines.Online miRNA databases were used to predict the miR-99 a target gene.Dual-luciferase assay and western blot assay were performed to verify the target gene.Results MiR-99 a was highly expressed in pediatric AML at diagnosis,while it became significantly lower expression after getting complete remission.MiR-99 a promoted the proliferation and inhibited the apoptosis of AML cells.Online software analysis predicted that the CTDSPL might be the miR-99 a target gene and our data confirmed that with the Dual-luciferase assay and western blot assay.MiR-99 a down regulated the expression of CTDSPL protein in AML patients.Conclusions MiR-99 a had significant effects on proliferation and apoptosis of AML cells by negative regulation targeting CTDSPL,and inhibition of miR-99 a expression might represent a potential novel strategy for pediatric AML treatment.
出处
《中国小儿血液与肿瘤杂志》
CAS
2015年第5期232-236,250,共6页
Journal of China Pediatric Blood and Cancer
基金
广东省自然科学基金(编号:S2013010015359)
广东省医学科学基金(批准号A2012176)
