The Proliferation And Methylation Regulated By β-arrestin1in Leukemia-initiating Cells From Children Acute Lymphoblastic Leukemia

Objective: In our previous study, high β-arrestin1expression wasfound in leukemia-initiating cells (LIC) of children acute lymphoblasticleukemia (ALL). Here, we further investigate the potential role ofβ-arrestin1(βArr1) in LIC proliferative capacity in vitro and in vivo, toexplore the role of β-arrestin1on DNA methylation transferase (DNMTs),HOXa9, PTEN, P15gene methylation and expression, to explore thesynergistic role of β-arrestin1and methylation transferase inhibitors inALL-LICs proliferation and leukemogenesis. The ultimate aim of this studyis to explore the epigenetic mechanisms of β-arrestin1in ALL-LICs cellmethylation, thus to provide experimental basis for the new targets ofchildhood ALL diagnosis and therapy.Methods: LICs labeled with CD34+CD38-CD19+were isolated andidentified from bone marrow of childhood ALL patients. β-arrestin1expression was knockdown by using recombined lentiviral system. And theestablished si-βArr1ALL-LICs cell and NOD/SCID mice were identified with RT-PCR, Western blot. ALL-LICs proliferative capacity was detectedby using clone formation and survival rate in vitro and in vivo. DNMTsactivity was detected by ELISA assay. The gene and protein expression ofmultiple genes were measured by fluorescent quantitative RT-PCR andWestern blot. The methylation level was detected by usingmethylation-specific PCR. The methyltransferase inhibitors (5-Aza) wereapplied in ALL-LICs and NOD/SCID mice knocked downβ-arrestin1.Results: LICs and NOD/SCID mice model knockdown β-Arr1weresuccessfully established. The clone forming ability in ALL-LICs cells, andthe leukemogenesis and infiltration in NOD/SCID leukemia mice weresignificantly reduced when β-arrestin1inhibited. The depressed enzymeactivity and expression of DNMTs, the downregulation of PTEN and P15gene methylation levels and increased their mRNA expression wereobserved in ALL-LICs cells and NOD/SCID leukemia mice whenβ-arrestin1knocked down. The5-Aza could repress proliferation capacity,delay leukemogenesis and reduce leukemic cell infiltration, synergicallywith siβArr1.Conclusion: The expression of β-arrestin1was significantly increasedin ALL-LICs cells. Inhibition of β-arrestin1could reduce the cellproliferation, leukemogenesis, DNMTs activity and expression of LICs, thusregulate PTEN gene methylation levels and expression. The methylationinhibitors could synergically inhibit ALL-LICs proliferation and leukemogenesis with siβArr1, suggesting the potential clinically usage ofmethylation inhibitors in ALL treatment, correlated with the synergistic roleofβ-arrestin1.