MicroRNA调控人视网膜母细胞瘤肿瘤干细胞多药耐药性的机制研究

Cancer stem cells (CSCs) are believed as the root of tumor recurrence, metastasis and tumor multidrug resistance， however, the mechanism of multidrug resistance mediated by CSCs in retinoblastoma (RB)is still unclear. In this study ,we sort CSCs in the RB line Y79 by fluorescence-activated cell sorting (FACS), using an antibody against the stem cell marker ATP-binding cassette, subfamily G, member 2 (ABCG2), and the sorted CSCs are characteried for stemness and multidrug resistance . We intend to identify the CSCs-specific microRNA by using solexa sequencing and northern blot . Then we evaluate the expression of the candidate microRNA in tissue and cellular level respectively by realtime PCR and locked in situ hybridization. whereafter, the target gene of the candidate microRNA will be identified by in silico and in vitro , and its fucntion on multidrug resistance will be investigated by overexpression and knock-down of the candidate microRNA . Through this study, the mechanism underlying microRNA mediated multidrug resistance in retinoblastoma will be intensively investigated. It is the key to clarify the mechanism of multidrug resistance in RB and to define a new target of RB invention provide the scientific basis.

肿瘤干细胞（CSCs）被认为是肿瘤复发、转移与多药耐药性产生的根源之一，但是在视网膜母细胞瘤（RB）中调控肿瘤干细胞多药耐药性的分子机制尚不明确。本项目拟以耐药标志ABCG2为标记,采用流式细胞术分选RB细胞系Y79中CSCs，验证其干细胞特性和多药耐药性；其次通过Solexa测序、northern blot等方法筛选、鉴定CSCs特异性microRNA；继而采用realtimePCR、锁定原位杂交等方法分别于组织水平和细胞水平研究候选microRNA与RB肿瘤耐药性的相关性；最后联合运用生物信息学及分子生物学实验鉴定候选microRNA的靶基因，通过过表达和抑制实验研究候选microRNA的功能。项目预期将建立RB中CSCs的多药耐药的microRNA调控机制，对于揭示RB多药耐药性的分子机制具有重要意义，并将为RB靶向治疗提供新的科学依据。

肿瘤干细胞（CSCs）被认为是肿瘤复发、转移与多药耐药性产生的根源之一，但是在视网膜母细胞瘤（RB）中调控肿瘤干细胞多药耐药性的分子机制尚不明确。本项目以耐药标志ABCG2为标记,采用流式细胞术分选RB细胞系CSCs，验证了其干细胞特性和多药耐药性；其次通过基因芯片、鉴定CSCs特异性miR-22-5P、miR-125b；继而采用realtimePCR、westernblot等方法分别于组织水平和细胞水平，证实了miR-22-5P、miR-125b与RB肿瘤生长、侵袭和耐药性的相关性；最后联合运用生物信息学及分子生物学实验鉴定miR-22-5P、miR-125b的靶基因分别为Erbb3基因和DRAM2基因，通过过表达和抑制实验研究miR-22-5P、miR-125b的功能。miR-22-5P在RBCSCs表达增多后，肿瘤细胞增殖力下降，肿瘤细胞凋亡增多，且耐药蛋白表达下降。miR-22-5P在RB是一抑癌因子，其表达增多可使RB耐药性下降，其可能是通过Erbb3基因来发挥其抑制肿瘤细胞增殖、转移的作用。miR-125b在RB细胞中表达增多后，肿瘤细胞增殖力和迁移力提高，肿瘤细胞凋亡下降。miR-125b在视网膜母细胞瘤是一致癌因子，其可能是通过DRAM2基因来发挥其促进肿瘤细胞增殖、转移的作用。项目对于揭示RB多药耐药性的分子机制具有重要意义，并将为RB靶向治疗提供新的科学依据。