共缺失区域特异性miRNA在骨髓增生异常综合征中的作用及调控机制

Myelodysplastic syndromes (MDS) are a heterogeneous group of bone marrow failure disorders by an ineffective hematopoiesis associated with cytopenia(s), with possible further progression to acute myeloid leukemia. Though progress has occurred in the prognostic assessment and evaluation of treatment response while the pathogenesis of MDS is exceedingly complex and mechanisms of MDS remain unclear. So it is crucial to address the mechanisms of MDS disease in order to deliver new approach for the treatment. Our previous study shows that, there is an existing common deleted region (CDR) of 5q deletion in MDS patients, which provided us novel vision of 5q- MDS study. Two candidate genes (SPARC and RPS14) located in CDR are proved to be significantly down regulated in patients with 5q- MDS, while our scientific data also show that these two genes are partially involved in MDS disease progress indicating that there are some other factors such as post-transcriptional regulations also contributing to the disease progress...MiRNAs have been widely involved in post-transcriptional gene regulation that have been demonstrated to play key roles in oncogenesis by silencing various target genes. Our study shows that expression of microRNA-143 in patients with 5q- MDS and 5q- MDS cell line (MUTZ-8) was significantly down-regulated comparing with non 5q- MDS patient samples and non 5q- MDS cell lines which indicating miR-143 might be the missing factor part from SPARC and RPS14 genes on the pathogenesis of MDS. Here we hypothesize: Through the down regulation of miR-143, the target genes of miR-143 has been up regulated combined with SPARC and RPS14 genes down regulation, which results in the body loss the suppress strength of MDS cells...Based on our previous study, which including the establishment of MDS cell lines and MDS mice model, we aim to elucidate the regulation mechanism of miR-143 in MDS cells through regulation of miR-143 expression in different cell lines and mice model as well as monitoring the response of various treatments. We will also identify the miR-143 target genes using gene expression array and co-transfected detection techniques in order to fully understand molecular regulation mechanisms under the phenotype of miR-143 on the pathogenesis of myelodysplastic syndromes. This project is closely allied and complementary to our ongoing project funded by the NSFC on the genetic basis of the 5q- MDS. We do believe that this study will help to enrich the molecular pathogenesis of myelodysplastic syndromes and provide novel information for future gene therapy of myelodysplastic syndromes.

骨髓增生异常综合征(MDS)是起源于造血干/祖细胞的恶性血液病，阐明该病发病机制、寻找治疗新方法具有重要意义。本课题组研究发现，MDS患者5号染色体长臂缺失(5q-)存在共缺失区域(CDR)，该区域抑癌基因SPARC与RPS14表达显著下降，参与部分MDS疾病发生，提示存在转录后调控因素共同参与MDS发生。我们在前期实验中发现，CDR区域特异性miRNA miR-143在人MDS细胞株及5q- MDS患者中表达显著降低。由此假设，miR-143下调通过调控靶基因异常表达而削弱其对MDS细胞的生长抑制作用，参与MDS发生。本项目拟在前期实验的基础上，以人MDS细胞株及我们前期建立的人MDS荷瘤小鼠为模型，从体内外水平，探讨miR-143对MDS细胞生长的影响及其机制，筛选并验证其发挥作用的靶基因，试图明确miR-143在MDS发病中的重要作用，为寻找MDS治疗新靶点奠定理论依据。

本研究以转化为急性白血病的MDS细胞株为细胞模型，首次研究miR-143对MDS转白的影响。课题组首先构建了miR-143的过表达载体--慢病毒载体pmirGLO-miR-143，该载体能够高效转染MDS细胞skm-1，经FCM检测转染效率在60%以上，pmirGLO-miR-143转染skm-1细胞后，可使miRNA-143表达量较转染前明显升高。研究发现，miR-143表达增高对skm-1细胞增殖有抑制作用，并可使skm-1细胞凋亡率增高。RT-qPCR检测转染后miR-143在靶细胞中稳定表达，上调后Caspase3,Caspase8,Fas 蛋白表达增加，而ERK5蛋白表达下降。提示miR-143的表达增加，使癌基因ERK5沉默，激活了凋亡通路基因的表达。提示miR-143起到抑癌作用。同时，构建pEZX-anti-miR-143载体，转染后可高效率降低SKM-1细胞的miR-143基因表达；miR-143基因沉默后通过调控靶基因异常表达，从而削弱其对MDS细胞的生长抑制作用。但其机制不清，本研究还需进一步阐明，对其作用通路及机制进行研究。.使用靶基因预测软件DIANAmT. miRanda miRWalk Targetscan靶基因预miRNA-143的靶基因，取这几个预测软件所预测靶基因的交集发现MLLT3基因3’-UTR一段序miRNA-143相匹配。提示MLLT3可能为miR-143的一个靶基因，miR-143通过调节MLLT3的表达也激活了凋亡通路基因的表达，因此有必要对miR-143及MLLT3之间的关系进行研究。.本项目发现共缺失区域miR-143基因为抑癌基因，通过沉默癌基因ERK5起到抑制肿瘤细胞增殖的作用。并将证实MLLT3为miR-143的一个靶基因。.结论：共缺失区域miR-143基因在skm-1细胞中高表达可以调控其靶基因的表达从而激活激活凋亡通路，使skm-1细胞凋亡增加，而miR-143低表达的skm-1细胞模型中，细胞凋亡减少。.成果：该研究结果正在整理成文，即将投递SCI杂志，文章可望在2014年发表。.意义：该项目研究结果丰富了对MDS尤其对转白的MDS发病机制的认识，为开发针对共缺失区域基因的靶向治疗提供了理论依据。