MiR-30d调控ARID5B基因表达在急性髓系白血病发病中的作用机制研究

Malignant proliferation and differentiation retardant of myeloid hematopoietic progenitor cells are the pathogenic basis of acute myeloid leukemia(AML), and recent researches indicated that miRNAs play key roles in cell proliferation, differentiation and apoptosis. Our group found that ARID5B was strongly related to acute leukemia pathogenesis, and it was proved that miR-30d binding site existed in ARID5B 3’UTR region; Furthermore, miR-30d was downregulated in AML patients. Combining our previous work with literature reports which announced the important roles of miR-30d and ARID5B in cell proliferation and differentiation, we speculate that: miR-30d is downregulated in AML, promoting acute myeloid leukemia cell proliferation, inhibiting cell differentiation and apoptosis by targeting ARID5B gene, and consequently involved in AML pathogenesis. This research is aiming to reveal the relationship of miR-30d and ARID5B mRNA expression with AML pathogenesis by RT-PCR; and explore the mechanism of miR-30d functions in AML cell proliferation, differentiation and apoptosis through targeting ARID5B, in a cellular level, using RT-PCR, western blot, plasmid construction, cell transfection, flow cytometry et al. This study is significant to illuminate the roles of miR-30d play in AML pathogenesis, and will provide new potential biomarkers for AML diagnosis and treatment.

髓系造血祖细胞的恶性增殖和分化阻滞是急性髓系白血病（AML）发病的基础，最新研究表明miRNAs在细胞增殖、分化和凋亡中也起着重要作用。课题组前期研究发现ARID5B基因与急性白血病密切相关，并证实ARID5B 3’UTR区存在miR-30d的结合位点；在AML患者中miR-30d表达下调。结合文献报道，我们推测：AML患者中miR-30d表达下调，通过负向调控ARID5B基因，促进AML细胞增殖、抑制分化及凋亡，进而参与AML发病机制。本研究拟运用RT-PCR揭示AML患者miR-30d和ARID5B基因表达与AML发病的关系；并运用RT-PCR、免疫印记杂交、构建质粒、细胞转染、流式细胞术等实验技术，从细胞水平探索miR-30d通过调控ARID5B表达在AML细胞增殖、分化和凋亡中的分子机制。本研究为阐明miRNA在AML发病机制中的作用及寻找相关标志物奠定了基础。

课题组前期研究发现ARID5B基因与急性白血病密切相关，拟研究miR-30d通过调控ARID5B进而参与AML的机制，然而在人群大样本验证中发现miR-30d和ARID5B基因表达不符合预期。后调整研究计划，首次从遗传学角度全面系统的研究ARID5B在AML发生发展中的作用。共纳入569例AML患者和410例健康个体，检测筛选出的ARID5B基因的17个TagSNPs。应用Plink、Haploview、GMDR、SPSS17.0和R软件分析ARID5B基因SNPs与AML易感性、临床表征及临床预后的相关性。研究发现：.1.ARID5B基因SNPs位点主要与男性APL的易感性相关，rs6415872、rs2393726、rs7073837、rs10821936和rs7089424位点的突变基因均会增加男性APL的发病风险。.2.ARID5B基因的单倍型与AML的易感性相关，除[AACCG]单倍型增加了男性M3型AML的患病风险，还发现[CGCCG]单倍型降低了女性M3型AML的患病风险。.3.在男性APL中，rs6415872分别与rs10821936和rs7089424之间存在较强的正相加交互作用，且全部归因于rs6415872 和rs10821936或rs7089424位点交互作用的病例占100%。.4.随机森林分析显示与男性APL易感性相关性最强的位点是rs10821936。.5.ARID5B基因多个SNPs位点与AML多项临床特征具有相关性，提示ARID5B基因可能参与AML的发生发展。.6.ARID5B基因多个SNPs位点与APL和非M3型AML的临床预后具有相关性，其中，rs6415872在APL和非M3型AML都可作为预后一项独立的影响因素。.7.生物信息学预测及细胞实验显示rs6415872位点A等位基因可增强转录因子与ARID5B的结合活性，尤其是PPARG，可能通过增强ARID5B表达进而参与AML发生发展。.本研究提示ARID5B基因在AML的发生发展中起着重要的作用，为AML发病机制的理论研究提供了新的思路。该基因的多个遗传多态性位点可能导致个体患AML风险增加，并具有一定的预后指导意义，可能作为AML的新的分子标志物。