miR-196b在MDS疾病进展及白血病转化中的作用研究

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell malignancies and can transform to acute myeloid leukemia, which progression is mainly related to the disorder of cell proliferation and apoptosis, and the abnormal expressions of microRNAs play an important role in MDS, but its related pathogenesis is not yet well-known. Our previous microarray and clinical data showed that the miRNA-196b expression was elevated in MDS patients, and its expression also showed a increasing trend when the risk of MDS increases. Moreover, some published references showed that miRNA-196b played a key role in the cell growth differentiation and tumorigenesis. Therefore, we hypothesize that miRNA-196b may regulate its target gene and lead the disorder of MDS cell proliferation and apoptosis, and participate in the development of MDS and transformation to leukemia, and has the potential to be an independent prediction indicator of prognosis for MDS. We will use bioinformatics technology to predict the target genes of miRNA-196b in MDS, and then confirm target genes by double luciferase reporter gene detection method. Gene transfection technology will also be used to explore the mechanisms that miRNA-196b may influence MDS cell proliferation and apoptosis through regulating its target genes. In addition, we will detect the miRNA-196b expressions among larger MDS clinical samples, and especially the same patients samples with different periods, analyze the important clinic significance of miRNA-196b high expression and the dynamic changes of its expression in disease progression in MDS patients, and determine whether miRNA-196b can act as independent prediction index of prognosis and high danger of evolution to acute myeloid leukemia.

MDS是造血干细胞克隆性疾病，可转化为白血病，其发生发展涉及多种致病因素导致的细胞增殖和凋亡紊乱，其中miRNAs异常表达起重要作用，但机制不明。我们芯片筛选及临床验证显示miR-196b在MDS患者中高表达，随着危险度增加其表达呈递增趋势。文献报道miR-196b与细胞生长分化及肿瘤发生发展有关。本项目假设：miR-196b通过调控其靶基因导致MDS细胞增殖凋亡紊乱，参与MDS疾病进展及白血病转化，可成为预测危险度尤其是白血病转化高度危险性的指标。本项目拟通过生物信息学技术预测miR-196b的靶基因，双荧光素酶报告基因方法验证靶基因，利用基因转染技术探讨miR-196b调控其靶基进而影响MDS细胞增殖凋亡机理；扩大临床样本，增加同一患者疾病进展中不同时期样本，分析miR-196b在MDS患者高表达及疾病进展中动态变化的临床意义，确定其可成为预测危险度尤其是白血病转化高度危险性的指标。

骨髓增生异常综合征（MDS）是一组起源于造血血干细胞的恶性克隆性疾病，具有向急性髓系白血病（AML）转化的高度危险性。大量研究表明微小RNA(miRNA)在造血干细胞增殖、分化过程中发挥重要作用，因此推测miRNA可能通过调控造血干细胞的发育参与MDS的发生发展。我们利用芯片技术筛选MDS和健康供者骨髓之间的差异miRNA，随后扩大样本量利用实时荧光定量技术（qRT-PCR）对差异miRNA进行验证，利用用细胞转染技术分析miRNA对MDS细胞增殖和凋亡的影响，通过靶基因在线分析软件预测miRNA靶基因及查阅文献寻找其上游可能的调控机制，并对靶基因在MDS中的表达量进行检测。芯片检测出376个差异miRNA，其中miR-196b-5p、miR-550、miR-99a、miR-320家族在MDS患者中均显著上调，而miR-639则显著下调。随后扩大样本后的qRT-PCR实验结果显示，与对照组相比，miR-196b在MDS患者组中表达量同样显著升高。随后在MDS患者治疗前后对比中，我们发现在治疗后的患者骨髓中miR-196b-5p表达显著下降。同时我们还进一步验证了其他特异性miRNA包括miR-550a, miR-639，miR-99a，miR-320家族，除miR-639表达显著降低以外,其余miRNA均显著高表达。我们检测miR-196b-5p上游可能的调控基因TFF1在MDS中的表达量，发现TFF1在MDS中表达显著降低。我们共预测到125个miR-196b-5p靶基因，经查阅文献选取6个靶基因进行验证，结果显示DICER1，ANXA1，GATA6在MDS患者骨髓中表达显著降低；在细胞转染实验中，我们发现miR-196b-5p表达上调会抑制MDS-L细胞凋亡同时促进MDS-L增殖；经双荧光素酶实验验证, DICER1是miR-196b-5p的直接靶基因。综上所述，MDS患者骨髓中miR-196b-5p表达量和 MDS危险度显著相关，可以作为MDS向AML转化的生物学标志物，并且负性调控其可能的下游靶基因DICER1来参与MDS的发生发展。并且其他特异性miRNA（miR-550a，miR-639，miR-99a，miR-320家族）同样显示出参与MDS发生发展的趋势，可以作为进一步研究的重点。本研究对完善MDS发病机制以及探查其相关治疗分子靶点具有重要意义。