利用CRISPR/Cas9靶向表观遗传学修饰技术研究IGF2在白血病干细胞中基因印记异常的机制

Leukemic stem cells (LSCs) play an important role in leukemia initiation, progression, relapse, and resistance to chemo-therapies. Insulin-like growth factor-II (IGF2) is an imprinting gene, with the paternal allele expressed and the maternal allele silenced in normal tissues. In leukemic stem cells, however, IGF2 exhibits loss of imprinting (LOI), with the two parental alleles aberrantly expressed. Through its receptor IGF1R, the overexpressed IGF2 activates the PI3K/AKT and MAPK signal cascades in the self-renewal of LSCs. However, the specific factors that drive IGF2 LOI in leukemia stem cells remain to be defined...Using a CRISPR/Cas9-promoter immunoprecipitation (CasIP) approach, we recently have identified miR483 that interacts with the IGF2 promoter and regulates IGF2 imprinting. In this study, we propose to utilize Cas-IP to identify unknown components in the IGF2 promoter chromatin complex, including long noncoding RNAs (lncRNAs) and microRNAs. In addition, we will synthesize an “IGF2 imprinting insulator”(S2I),consisting of dCas9(inactivated Cas9), Krab suppressor domain, and ICR(imprinting control region)-specific sgRNAs. With the aid of sgRNAs, dCas9 binds to the ICR, where the Krab domain functions as an “insulator” to block the access of the IGF2 promoter to the H19 downstream enhancer, leading to correction of aberrant IGF2 LOI in leukemia stem cells. ..We will specifically address the following challenges: 1)What are the molecular components in IGF2 promoter that induce aberrant imprinting? 2) Is the epigenetic modifier S2I able to silence IGF2 gene and affect self-renewal and tumorigenicity of leukemia stem cells?3) What are the epigenetics mechanism of aberrant imprinting in leukemia stem cells. This study will characterize pathological role of IGF2 LOI in leukemic stem cells. In addition, the Cas9 system may provide a new strategy to develop epigenetic antitumor reagents to target leukemia stem cells.

白血病干细胞（Leukemic Stem Cells， LSCs）是导致白血病发生、发展及治疗后复发、耐药的根源。类胰岛素生长因子II（IGF2）是最早发现的印迹基因。本课题组前期研究发现，IGF2在白血病干细胞中遗传印迹丢失（LOI），而在非干性白血病中印记正常（MOI）。IGF2 LOI后，过表达的IGF2通过IGF1R受体，激活PI3K/AKT、MAPK信号通路，调控白血病干细胞的无限增殖及分化。本课题拟采用首创的“dCas9-靶向启动子共沉淀技术”寻找IGF2 LOI的启动分子及调控环节，并采用“dCas9-靶向IGF2印记技术”对白血病的IGF2 印记异常进行纠正，以明确其在白血病干细胞调控中的作用；并进一步探索dCas9-靶向IGF2印记子纠正白血病干细胞IGF2 LOI的表观遗传学分子机制，为白血病干细胞表观遗传学分子靶向新药的研发提供理论依据。

白血病干细胞（Leukemic Stem Cells， LSCs）具有自我更新能力，并参与白血病的进展、复发及耐药。类胰岛素生长因子II（IGF2）是最早发现的印迹基因。本课题组前期研究发现，IGF2在白血病干细胞中遗传印迹丢失（LOI）而在非白血病干细胞中印记正常（MOI）。IGF2 LOI后，过表达的IGF2通过IGF1R受体，激活PI3K/AKT、MAPK信号通路，调控白血病干细胞的自我更新。本课题用首创的“dCas9-靶向启动子共沉淀技术”寻找IGF2 LOI的启动分子及调控环节，并采用“dCas9-靶向IGF2印记技术”对白血病的IGF2 印记异常进行纠正，但未能证实其能具有调控白血病干细胞自我更新的能力，也未能改变白血病干细胞的干性。课题衍生探索了干细胞与lcnRNA以及肿瘤干细胞与GRN等分子的表观遗传学分子机制，为后续研究提供理论了一定的理论依据和参考。