1号染色体短臂不稳定-miRNA-320b低表达-上调VEGF参与多发性骨髓瘤发病的机制研究

This project is a continuation of previous work.Genomic instability causes genetic abnormalities,the mutations of some oncogenes and tumor suppressor genes and tumorigenesis. 1p deletion is one of the most common genetic abnormalities with deletion rate being about 25-30% in MM. The influence of 1p deletion on prognosis in MM patients catchs international attention in recent years. We performed I-FISH on purified 138+ plasma cells from 318 newly diagnosed MM patients.The probes covered the following loci:1p12,1p13.3, 1p21,1p32.3 using BAC clones RP11-418J17,RP11-180N18,RP11-776K10 and RP11-278J17 that contain FAM46C,AHCYL1,Cdc14A and CDKN2C genes.The result showed that 1p deletion was mainly large size of deletion and a adverse prognostic factor in patients with multiple myeloma.There is now strong evidence that microRNA is closely related to the pathogenesis of tumor incluing multiple myeloma(MM).That 50% of human microRNA genes are frequently located at chromosome fragile sites and genomic regions related to cancers indicates there is some key cancer associated-genes in these regions.Among 16 microRNAs located in chromosome 1p according to the NCBI Map View database,we detected 9 tumor related- microRNAs in CD138+ cells of 25 del（1p）MM patients and 77 MM patients without del(1p).The result showed microRNA-320b,located in chromosome fragile region 1p13.1 was down-regulated in CD138+ cells in del(1p) MM patients compared with controls.Some evidences show that 1p13 is a important fragile region of the short arm of chromosome 1.Cytokines and signaling pathways play a pivotal role in promoting growth and survival of MM cells.We detected expression of IL-6、VEGF and SDF-1αafter overexpression the microRNA-320b by pre-microRNA-320b transfected into H929 and RPMI-8226 using lipofectamine 2000.The result shows the expression of VEGF is down-regulated in H929 and RPMI-8226 ,but no change in IL-6和SDF-1α.In this project ,we will further investigate the role of low expression of microRNA-320b participating in the pathogenesis of myeloma by upregulating VEGF and confirm VEGF is the microRNA-320b-targeted gene.Taken together,this project will provide the theoretical and experimental evidences for exploring microRNA-320b and microRNA-320b-targeted genes as a new potential therapeutic target.

基因组不稳定可导致遗传学异常事件的发生，致使某些癌基因和抑癌基因突变和肿瘤发生。1p缺失率约为25-30%，是MM发生频率较高的遗传学异常，其对预后的影响近年受到国际上的重视。本课题以此为主线，前期针对1p 4个区域制作了FISH探针在318例初治MM患者中检测，结果发现1p缺失多为累及多个区域的大片段缺失，1p缺失的患者PFS和OS明显缩短。microRNA与肿瘤发病密切相关，50%的microRNA位于癌症相关的基因组区或染色体脆性位点。申请人通过NCBI Map View数据库选择1p区域的9个肿瘤相关的microRNA在102例MM患者中筛查，结果定位于1p13.1的microRNA-320b在1p缺失MM患者中表达明显降低。本课题拟对microRNA-320b参与MM发病的机制进行研究，并验证其作用的靶基因，为探索以microRNA及其靶基因为新靶点的生物治疗提供理论和实验基础。

本项目为了研究1号染色体短臂的遗传学异常特点，在266例新诊断MM，90例复发MM，8例继发性浆细胞白血病和8例MGUS中通过制作四色荧光原位杂交探针检测了1p12-1p32的异常。结果如下：1）MM患者1p异常呈现多样性，复杂性，多为大片段缺失，且缺失片段大小和预后无明显关系; 1p缺失患者预后不良，1p32.3为1p位点的关键区域，适合常规检测。 2）检测了275例初治MM患者del(13q14), del(17p), amp(1q21), IgH translocation, t(11;14), t(4;14), and t(14;16) 和12p13 位点在患者中的特点和预后情况。发现12p13缺失在初治MM患者中缺失率为10.5%，且12p13缺失和ISS III, BM 浆细胞比例超过50%, 肾损害，del(17p)，IGH 异和t(4;14)相关。del(12p) 的患者PFS和OS较差, 甚至在硼替佐米治疗的患者中。 多因素分析显示del(12p13) 是PFS 和OS的独立预后因素。此外，del(12p13) 联合高β2-MG, 高LDH和骨损害能进一步筛选出高危患者，证实del(12p13)能作为评估骨髓瘤患者预后不良的指标。3）检测了117例初治MM, 11例复发，16例缓解患者mir-15a和16-1的表达情况。发现miR-15a 和16-1 在初治MM患者中低表达，并且和疾病进展和不良预后相关，此外， miR-15a联合高β2-MG和高危遗传学异常能进一步界定出高危患者。4）收集了29例初治多发性骨髓瘤（MM）患者和6正常人骨髓138阳性细胞，检测了microRNA320的表达，初步结果显示MM患者中microRNA320的表达低于正常人（P=0.037）。将microRNA320 的前体序列导入病毒载体: pEZX-MR03中，感染293T细胞，收集病毒液，感染h929和MM1S细胞系。 5）转染320后MM1R细胞增殖速度减慢（P=0.005)。6）收集40个初治MM样本DNA送亚硫酸氢盐测序。 7）荧光素酶基因报告系统验证320靶向PTEN基因的证据。8）培养两名青年医师（藏美蓉博士，万柏林硕士，周丽丽硕士）。9）期间发表了3篇SCI文章，两篇中文论著，两篇综述，成功立项3项江西省科技厅课题，2项江西省卫生厅课题。