Structural And Functional Analysis Of NcRNA-SPIU And The Role Of P19INK4D In Acute Promyelocytic Leukemia

Acute promyelocytic leukemia(APL) is a distinct subtype of acute myeloid leukemia, and characterized by a balanced reciprocal translocation between chromosomes 15 and 17, which results in the PML/RAR? fusion gene and oncoprotein. PML/RAR? interferes with its target genes, eventually leading to a differentiation block at the promyelocytic stage and a hyperproliferation of blocked promyelocytes; both are the hallmarks of tumors. Thus, APL represents a remarkable cancer model.The dysregulation of transcription factors plays a key role in the pathogenesis of APL. PU.1 is a key transcriptional regulator in hemotapoiesis, and the precise expression of PU.1 is critical for maintaining normal hemotapoiesis. Besides transcriptional and epigenetic regulation, the non coding RNA is crucial in gene regulation. The effect of non coding RNA on PU.1 is much less studied. MicroArray detects known genes with designed probes. RNA sequencing, with high sensitivity, detects every expressed RNAs, including non coding RNAs. Using the RNA sequencing technology, we found that there was a RNA(SPIU) transcripted from the upstream of PU.1 gene in NB4 cells. We identified the full length structure of SPIU with 5’RACE and 3’RACE. The protein coding potential prediction found that there was no long open reading frame in SPIU. SPIU and PU.1 were coexpressed in different hematopoietic cell lines. The further findings demonstrated that PU.1 was positively regulated by long non coding RNA – SPIU, in not only NB4 cells but also U937 myeloid cells. Overexpression of SPIU activated the promoter of PU.1 gene. These results demonstrated that PU.1 was positively regulated by neighbouring long non coding RNA- SPIU. Knockdown of SPIU not only decreased the expression of PU.1 targeted genes, but also attenuated the differentiation of NB4 cells. Then we found that the expression of SPIU was repressed by PML/RAR? and the repression could be restored by ATRA in APL cells. Thus, we found that a long non coding RNA transcripted from the upstream of PU.1 gene, and identified its structure. PU.1 was positively regulated by SPIU. In APL cells, SPIU was repressed by PML/RAR?, which may be involved in the dysregulation of PU.1 and contribute to the pathogenesis of APL. Collectively, we demonstrated a novel mechanism of PU.1 gene regulation, and its disturbance may contributed to the pathogenesis of APL.In APL, PML/RARa inhibits differentiation and promotes proliferation by interfering with its target genes. It has been reported that CKIs are involved in differentiation process, but the involvement is cell-type-specific. We found that expression of p19INK4 D in APL is significantly lower than that of normal promyelocytes and mononuclearcytes. Forced expression of p19INK4 D induced G0/G1 phase arrest and a partial granulocytic differentiation in NB4 cells. Our results demonstrate that p19INK4 D is a direct link between cell growth and differentiation. Furthermore, we demonstrated that PML/RAR? repressed p19INK4 D expression through binding to the ER8 site on the p19INK4 D promoter. Knockdown of p19INK4 D expression impaired the differentiation of NB4 cells and promoted cell growth. The inhibition of p19INK4 D was restored by ATRA and p19INK4 D is direct ATRA-responsive gene. Up-regulation of p19INK4 D is required for APL cells differentiation and is involved in G0/G1 phase arrest. Taken together, our findings show that suppression of p19INK4 D by PML/RAR? disrupts both cell growth and differentiation process, which contributes to the pathogenesis of APL.