The Effect Of MicroRNA-10b On The Differentiation Block Of NPM1Mutated Acute Myeloid Leukemia

PART Ⅰ The effect of nucleophosmin (NPM1) mutations onthe differentiation of acute myeloid leukemic cellsNucleophosmin (NPM1) mutations play an important part in theleukemogenesis, whereas its role in the differentiation block of leukemiccells is largely unknown. To explore the effect of NPM1mutations on thedifferentiation of leukemic cells in vitro, the expression plasmidpEGFPC1-NPM1-mA carrying NPM1mutation A (NPM1-mA) wastransfected into THP-1cells, and the cells with stable expression ofNPM1-mA protein (THP-1mA) were constructed. The cells transfected withpEGFPC1-NPM1-wt (THP-1wt)、pEGFP-C1(THP-1C1) and the untreatedcells (THP-1) were used as control. RT-PCR、Western blot and ICC wereperformed to verify the mRNA and protein expression of NPM1-mA in thestable cell lines. After induction with Phorbol-12-myristate-13-acetate(PMA), the morphological changes of cells were observed under lightmicroscope following Wright-Giemsa staining; the surface marker CD14was analyzed by flow cytometry. Compared with the control groups, there was no obvious changes in the cell morphology and the percentage ofadherent cells in THP-1mA group was reduced significantly after PMAtreatment for72h (P<0.05); Meanwhile, there was a lower expression ofCD14in THP-1mA group (P<0.01). Our data indicate that NPM1mutationsblock differentiation of leukemic cells in vitro. PART Ⅱ MicroRNA-10b blocks the differentiation ofleukemic cells through regulating the expressionof zinc finger protein KLF4We aimed to investigate the effect of microRNA-10b (miR-10b) on thedifferentiation of leukemic cells through regulating the expression of zincfinger protein Krüppel-like factor4(KLF4). Dual-Luciferase ReporterAssay and Western blot were performed to identify KLF4as a direct andfunctional target of has-miR-10b. The expression of miR-10b and KLF4inleukemic cell lines at different differentiated status was detected by qPCRand Western blot respectively. Leukemia cell line HL60were induced with1,25-dihydroxy-vitamin D3(1,25D3) to differentiate along the monocyticlineage. The expression of miR-10b and KLF4was detected during 1,25D3-induced differentiation of HL60. The synthesized has-miR-10bmimics were transfected into HL60cells. The morphological changes ofcells treated with1,25D3were observed under light microscope followingWright-Giemsa staining, and the monocyte surface marker CD14wasanalyzed by flow cytometry. KLF4was a functional target gene ofhas-miR-10b. miR-10b was detected at the highest levels of expression inless differentiated status (such as KG-1a) and at the lowest in more matureU937and THP-1cells (P<0.01), while the KLF4exhibited an oppositeexpression pattern. miR-10b was decreased in a time-dependent manner inHL60cells during induction with1,25D3, whereas the KLF4was increased.Enforced expression of miR-10b in HL60cells inhibited the1,25D3-inducedmorphological changes and the expression of CD14(P<0.05). Our dataindicate that miR-10b suppresses monocytic differentiation of HL60cellsvia targeting KLF4. PART Ⅲ miR-10b is up-regulated and blocks thedifferentiation of NPMc+AML cellsThe NPM1mutation can induce a myeloproliferative disorder, however,evidence indicates that other lesions are necessary for the development of AML. Epigenetic regulation of miRNAs contributes to the leukemogenesis.To explore if miR-10b dysregulation could be involved in the differentiationblock of NPMc+AML, the expression of miR-10b and KLF4in THP-1mAand OCI/AML3was detected by qPCR and Western blot respectively. Thesynthesized has-miR-10b inhibitor was transfected into OCI/AML3cells.The KLF4protein levels were detected and the surface marker CD14wasanalyzed. The expression of miR-10b was increased in the THP-1mA(P<0.05), meanwhile, the KLF4was reduced. miR-10b was detected at thehighest levels in OCI/AML3bears NPM1gene mutation-A (P<0.01).Knockdown of miR-10b in OCI/AML3boosted the expression of KLF4andincreased the percentage of CD14-positive cells (P<0.05). miR-10b isdysregulated and responsible for the block of monocytic differentiation ofNPMc+AML.