A coordinated function of lncRNA HOTTIP and miRNA-196b underpinning leukemogenesis by targeting Fas signaling

MicroRNAs (miRNAs) may modulate more than 60% of human coding genes and act as negative regulators, while long non-coding RNAs (lncRNAs) regulate gene expression on multiple levels by interacting with chromatin, functional proteins, and RNAs such as mRNAs and microRNAs. However, the crosstalk between lncRNA HOTTIP and miRNAs in leukemogenesis remains elusive. Using combined integrated analyses of global miRNA expression profiling and state-of-the-art genomic analyses of chromatin such as ChIRP seq., (genome wide HOTTIP binding analysis), ChIP-seq., and ATAC-seq., we found that miRNA genes are directly controlled by HOTTIP. Specifically, the HOX cluster miRNAs (miR-196a, miR-196b, miR-10a and miR-10b), located cis & trans, were most dramatically regulated and significantly decreased in HOTTIP–/– AML cells. HOTTIP bound to the miR- 196b promoter, and HOTTIP deletion reduced chromatin accessibility and enrichment of active histone modifications at HOX cluster associated miRNAs in AML cells, while reactivation of HOTTIP restored miR gene expression and chromatin accessibility in the CTCF-boundary-attenuated AML cells. Inactivation of HOTTIP or miR-196b promotes apoptosis by altering the chromatin signature at the FAS promoter and increasing FAS expression. Transplantation of miR-196b knockdown MOLM13 cells in NSG mice increased overall survival compared to wild-type cells. Thus, HOTTIP remodels the chromatin architecture around miRNAs to promote their transcription, consequently repressing tumor suppressors and promoting leukemogenesis. Overall design: We have finished the small RNA-SEQ for WT and HOTTIP-KO MOLM13 leukemia cells微小RNA（miRNAs）可调控超过60%的人类编码基因，并作为负调控因子发挥作用；而长链非编码RNA（lncRNAs）则通过与染色质、功能蛋白以及mRNA、微小RNA等RNA分子相互作用，在多个层面调控基因表达。然而，长链非编码RNA HOTTIP与miRNAs在白血病发生过程中的相互调控机制仍尚不明确。本研究通过整合全球miRNA表达谱分析与当前最先进的染色质基因组分析技术（包括ChIRP-seq[全基因组HOTTIP结合位点分析]、ChIP-seq以及ATAC-seq），发现miRNA基因直接受HOTTIP调控。具体而言，位于顺式和反式调控区域的HOX簇miRNAs（miR-196a、miR-196b、miR-10a及miR-10b）在HOTTIP基因敲除的AML细胞中表达受到最显著的调控，且表达水平显著下调。HOTTIP可结合miR-196b的启动子区域；HOTTIP缺失会降低AML细胞中HOX簇相关miRNAs位点的染色质开放程度以及活性组蛋白修饰的富集水平，而在CTCF绝缘子边界减弱的AML细胞中重新激活HOTTIP，则可恢复miR基因的表达以及染色质开放程度。HOTTIP或miR-196b的失活可通过改变FAS基因启动子区域的染色质特征并上调FAS的表达，促进细胞凋亡。将miR-196b敲低的MOLM13细胞移植到NSG小鼠体内，可相较于野生型细胞显著延长小鼠的总生存期。综上，HOTTIP可重塑miRNAs位点周围的染色质结构以促进其转录，进而抑制抑癌基因的表达并促进白血病发生。实验整体设计：本研究完成了野生型（WT）与HOTTIP敲除（HOTTIP-KO）的MOLM13白血病细胞的小RNA测序（small RNA-SEQ）。