Objective: To analyze the relationship of the expression of transcription factor MYB targeted regulation by miR-96 to cell invasion and apoptosis in pediatric acute myeloid leukemia (AML).
Methods: A total of 65 children with AML in The 928 Hospital of PLA Joint Logistics Support Forces from January 2017 to November 2019 were selected, including 35 cases diagnosed as primary AML and 30 cases as complete remission AML. Thirty children with immune thrombocytopenia were selected as control group. The clinical characteristics were analyzed and compared between the two groups. The levels of miR-96 and MYB in peripheral blood samples were detected by qRT-PCR and compared between the two groups. The miR-96 mimics and its negative control (NC), inhibitor-miR-96 and its NC transfected HL60 cells induced by liposome (Lipofectamine 2000), respectively, Then the expression levels of MYB were detected with Western blot and compared among four HL60 cell groups. The invasion ability of four HL60 cell groups were detected with Transwell assay. The cell proliferation ability of four HL60 cell groups were detected with MTT at 24 h, 48 h, and 72 h, respectively. The apoptosis rates of four HL60 cell groups were detected with flow cytometry.
Results: Compared with control group, the level of miR-96 in AML children were higher, but MYB lower (P<0.05). Compared with complete remission AML, the level of miR-96 in primary AML was higher, but MYB lower (P<0.05). Western blot analysis showed that, the expression level of MYB in the four HL60 cell groups was different (P<0.05), the lowest was in miR-96 mimics group, followed by miR-96 NC group and inhibitor-miR-96 NC group, and the highest in inhibitor-miR-96 group (P<0.05), while there was no difference between miR-96 NC group and inhibitor-miR-96 NC group (P>0.05). The promotion of over-expression level of miR-96 on the invasion ability of HL 60 cells was confirmed by Transwell assay. MTT assay showed that miR-96 could promote the proliferation of HL60 cells, inhibit the apoptosis of HL60 cells, and the effect was time-dependent manner (r=0.804). The inhibition of miR-96 on HL60 cells apoptosis was also confirmed with flow cytometry.
Conclusion: MiR-96 has significant negative effect on invasion and apoptosis of AML cells by targeting regulation MYB, and it might be a potential novel strategy for pediatric AML treatment.
题目: miR-96调控MYB表达与儿童急性髓系白血病细胞侵袭及凋亡活性相关性分析.
目的: 分析儿童急性髓系白血病（AML）血清miR-96靶向调控沉默转录因子MYB表达水平与侵袭及凋亡活性的相关性.
方法: 选择2017年1月至2019年11月中国人民解放军联勤保障部队第九二八医院确诊AML患儿共65例（其中35例初发，30例完全缓解），同时选择30例免疫性血小板减少患儿作为对照组。分析两组患儿的临床特征。qRT-PCR检测并比较两组患儿外周血标本中miR-96与MYB的表达水平；通过脂质体Lipofectamine 2000介导将miR-96 mimics及其阴性对照（NC）、inhibitor-miR-96及其NC转染至4组HL60细胞，采用Western blot检测各组细胞MYB表达水平；采用Transwell法检测细胞的侵袭能力；分别在24、48和72 h采用四甲基偶氮唑蓝（MTT）细胞增殖试验检测细胞增殖活性；采用流式细胞术检测细胞凋亡率.
结果: 与对照组比较，AML患儿miR-96呈高表达，而MYB呈低表达（P<0.05）。与完全缓解AML患儿相比，初发AML患儿miR-96高表达，MYB呈低表达（P<0.05）。Western blot检测显示，miR-96转染后4组HL60细胞MYB的表达水平存在差异（P<0.05），miR-96 mimics组最低，miR-96 NC组与inhibitor-miR-96 NC组次之，inhibitor-miR-96组最高（P<0.05），而miR-96 NC组与inhibitor-miR-96 NC组无差异（P>0.05）。Transwell法检测显示，miR-96过表达对HL60细胞侵袭具有促进效应。MTT细胞增殖试验显示，miR-96促进HL60细胞增殖，抑制凋亡，具有时间依赖性（r=0.804）。流式细胞术检测结果显示，miR-96抑制HL60细胞凋亡作用.
结论: miR-96通过靶向负调控MYB表达水平影响AML细胞侵袭及凋亡水平，可能成为治疗儿童AML的一个新策略.