Mechanistic Study Of Long Noncoding RNA RUNXOR In Acute Myeloid Leukemia

Background and Objective：Acute myelogenous leukemia (AML) is the most common hematopoieticmalignancy that primarilyaffects adults. The RUNX1gene, also known as AML1, isa Runt-related transcription factor that regulates critical processes in hematopoiesisand defines the definitive hematopoietic stem cells. Located on chromosomal21, theRUNX1gene is involved in many forms of chromosomal translocations in leukemia.For example, t(8,21) is one of the most common chromosomal translocations foundin acute myeloid leukemia, where it results in a fusion protein between RUNX1andETO. However, the specific molecular mechanism underlying the chromosomaltranslocation and mutation in AML is poorly defined. We have recently identified anovel RUNX1overlapping long non-coding RNA (LncRNA), called RUNXOR, inthe RUNX1gene locus. RUNXOR is transcribed from a upstream promoter andoverlapps with RUNX1.We thus propose to use RT-PCR to mapping and characterize this LncRNA;Use luciferase reporter to identify its promoter region; Determing the LncRNAdifferential expression between normal and leukemic samples by RT-qPCR. We areparticularly interested in examining whether the LncRNA regulates the geneexpression of RUNX1and its fusion gene, like RUNX1-ETO, through an epigeneticmechanism.Methods：Several epigenetic technologies available in the Lab were used, including3C(chromatin conformation capture), RAT(Reverse associate trap assay), RIP（RNAimmunoprecipitation）and ChIP (chromatin immunoprecipitation), we will examine how the LncRNA epigenetically regulates its target genes, including the interactionof LncRNA with its target DNA sequence, the formation of the intra-andinter-chromosomal looping, and the LncRNA-mediated recruitment of chromatinmodifying factors, like histone H3-K27methylase EZH2. We will test whether thesechromatin interactions are correlated with the gene expression in leukemic cells.Results：We have identified a novel RUNX1overlapping long non-coding RNA(RUNXOR), it transcripted by a putative promoter which located3.8kb upstream ofRUNX1’s distal promoter, the promoter can drive luciferase-GFP reporter gene inhemogenic cell line. The RT-qPCR result revealed that RUNXOR expressed muchhigher in AML patients’ bone marrow than normal bone marrow, and can beupgraded by Ara-C treatment in K562cell line; The ChIP result indicated thatRUNXOR can bind to RUNX1promoter and recruit H3K27methylase EZH2, mayrole as an RUNX1suppressor; RAT data implied that RUNXOR may role as atranslocation helper by scaffold translocation associated genes, meanwhile it maymediate the formation of RUNX1intra-chromasome looping by scaffold RUNX1promoters and its enhancer.Conclusion：The RUNXOR is a novel LncRNA, span more than216kb cross the RUNX1gene locus, it share the same transcribe orientation of RUNX1gene. The differentialexpression between normal bone marrow and AML patients’ bone marrow impliedit’s may function in leukemogenesis. It’s can epigenetically regulate RUNX1gene bybingding to its promoter and recruiting the chromatin factor EZH2. Further more,RUNXOR may involed in RUNX1assaociated translocation by scaffold the EVI1,ETO and RUNX1’s translocation breakpoint region, which provide the accessibilityof two long range aparted genes to fuse.Given RUNXOR is a novel LncRNA just be found, most of its function and mechanism need to be clarified by subsequent research. Finally, the illustration of itsfunctions and mechanisms may provide a novel gene therapy target for AMLtreatment.