Knockdown of miR-128a induces Lin28a expression and reverts myeloid differentiation blockage in acute myeloid leukemia

Abstract

Lin28A is a highly conserved RNA-binding protein that concurs to control the balance between stemness and differentiation in several tissue lineages. Here, we report the role of miR-128a/Lin28A axis in blocking cell differentiation in acute myeloid leukemia (AML), a genetically heterogeneous disease characterized by abnormally controlled proliferation of myeloid progenitor cells accompanied by partial or total inability to undergo terminal differentiation. First, we found Lin28A underexpressed in blast cells from AML patients and AML cell lines as compared with CD34+ normal precursors. In vitro transfection of Lin28A in NPM1-mutated OCI-AML3 cell line significantly triggered cell-cycle arrest and myeloid differentiation, with increased expression of macrophage associate genes (EGR2, ZFP36 and ANXA1). Furthermore, miR-128a, a negative regulator of Lin28A, was found overexpressed in AML cells compared with normal precursors, especially in acute promyelocytic leukemia (APL) and in 'AML with maturation' (according to 2016 WHO classification of myeloid neoplasms and acute leukemia). Its forced overexpression by lentiviral infection in OCI-AML3 downregulated Lin28A with ensuing repression of macrophage-oriented differentiation. Finally, knockdown of miR-128a in OCI-AML3 and in APL/AML leukemic cells (by transfection and lentiviral infection, respectively) induced myeloid cell differentiation and increased expression of Lin28A, EGR2, ZFP36 and ANXA1, reverting myeloid differentiation blockage. In conclusion, our findings revealed a new mechanism for AML differentiation blockage, suggesting new strategies for AML therapy based upon miR-128a inhibition.

Figure 1

Lin28A expression in leukemic blasts from AML patients. (a) qRT-PCR of Lin28A in 13 healthy controls, 38 AML patients and 7 AML cell lines (OCI-AML3, KG-1, Kasumi-1, NB4, CMK, ME-1 and MOLM-14); ABL1 was used for normalization. Relative values were calculated on the basis of the ΔCp method. Results are shown as mean±S.E.M. (b) Expression of Lin28A mRNA in AML patients stratified for morphologic features (with minimal differentiation, n=2; without maturation, n=9; with maturation including: n=3 with maturation, n=10 acute myelomonocytic leukemia, n=2 acute monoblastic/monocytic leukemia; APL, n=3; secondary AML, n=3) was compared with 13 healthy controls. Results are shown as mean±S.E.M. (c) Expression of Lin28A mRNA in AML patients with specific mutations (NPM1, n=9; FLT3, n=8; PML/RARα, n=3 or with other alterations, n=26) was compared with 13 healthy controls. Results are shown as mean±S.E.M. (d) Percentage of Lin28A+ cells in 11 BM healthy controls and 9 AML patients, by cytofluorimetric analysis. (e) Percentage of Lin28A+ cells in normal myeloid (CD34+ CD45+ CD33+), erythroid (CD34+ CD45+ CD71+) and lymphoid (CD34+ CD45+ CD19+) precursors, by cytofluorimetric analysis. Statistically significant analyses are indicated by asterisks: *P<0.05, **P<0.01 and ***P<0.001

Figure 2

Overexpression of Lin28A in OCI-AML3 cell line. (a) Western blotting (WB) analysis of Lin28A, p21 and β-actin in OCI-AML3 after 24 and 48 h of transfection with pcDNA3.3_Ctrl or pcDNA3.3_Lin28A plasmids. (b) Absolute OD values of (a) were normalized to β-actin and shown as mean±S.D. from two independent experiments. (c) Representative cytofluorimetric analysis of percentage Lin28A+ cells in OCI-AML3 after 24 and 48 h of transfection with pcDNA3.3_Ctrl or pcDNA3.3_Lin28A plasmids. (d) Percentage of Lin28A+ OCI-AML3 cells after 24 and 48 h of transfection with pcDNA3.3_Ctrl or pcDNA3.3_Lin28A plasmids, by cytofluorimetric analysis. (e) Representative cytofluorimetric analysis of CD11b+ cells in OCI-AML3 after 24 and 48 h of transfection with pcDNA3.3_Ctrl or pcDNA3.3_Lin28A plasmids. (f) Percentage of CD11b+ OCI-AML3 cells after 24 and 48 h of transfection with pcDNA3.3_Ctrl or pcDNA3.3_Lin28A plasmids. (g) Representative cytofluorimetric analysis of CD14+ cells in OCI-AML3 after 24 and 48 h of transfection with pcDNA3.3_Ctrl or pcDNA3.3_Lin28A plasmids. (h) Percentage of CD14+ OCI-AML3 cells after 24 and 48 h of transfection with pcDNA3.3_Ctrl or pcDNA3.3_Lin28A plasmids, by cytofluorimetric analysis. (i) Cell-cycle analysis in OCI-AML3 after 24 and 48 h of transfection with pcDNA3.3_Ctrl or pcDNA3.3_Lin28A plasmids. (j–l) qRT-PCR of EGR2 (j) ZFP36 (k) and ANXA1 (l) in OCI-AML3 after 24 and 48 h of transfection with pcDNA3.3_Ctrl or pcDNA3.3_Lin28A plasmids. The bar graphs represented mean±S.D. from three independent experiments. Statistically significant analyses are indicated by asterisks: *P<0.05, **P<0.01 and ***P<0.001

Figure 3

Lin28A upregulation during macrophage-like differentiation in AML cell lines. (a and b) Percentage of CD11b+ and CD14+ cells in ME-1 (a) and OCI-AML3 (b) after 24, 48 and 72 h of treatment with PMA, by cytofluorimetric analysis. (c) Percentage of CD11b+ and CD11c+ cells of MOLM-14 after 24, 48 and 72 h of treatment with ATRA, by cytofluorimetric analysis. (d–f) qRT-PCR of EGR2, ZFP36 and ANXA1 in ME-1 (d), OCI-AML3 (e) and MOLM-14 (f) after 24, 48 and 72 h of treatment with PMA or ATRA. (g–i) Western blotting (WB) analysis of Lin28A, p21 and β-actin in ME-1 (g), OCI-AML3 (h) and MOLM-14 (i) after 24, 48 and 72 h of treatment with PMA. (j) Percentage of Lin28A+ ME-1, OCI-AML3 and MOLM-14 cells after 24, 48 and 72 h of treatment with PMA or ATRA, by cytofluorimetric analysis. (k) Cell-cycle analysis in ME-1, OCI-AML3 and MOLM-14 cells after 24, 48 and 72 h of treatment with PMA or ATRA. The line and bar graphs represented mean±S.D. from three independent experiments. Statistically significant analyses are indicated by asterisks: *P<0.05, **P<0.01 and ***P<0.001

Figure 4

MiR-128a expression in leukemic blasts from AML patients and its inhibition in OCI-AML3 cell line. (a) qRT-PCR of MiR-128a in 10 healthy controls, 35 AML patients and 6 AML cell lines (OCI-AML3, KG-1, Kasumi-1, NB4, CMK, ME-1 and MOLM-14); RNU44 was used for normalization. Relative values were calculated on the basis of the ΔCp method. Results are shown as mean±S.E.M. (b) Expression of miR-128a in AML patients stratified for morphologic features (with minimal differentiation, n=1; without maturation, n=8; with maturation including: n=3 with maturation, n=9 acute myelomonocytic leukemia, n=2 acute monoblastic/monocytic leukemia; APL, n=3; secondary AML, n=3) was compared with 10 healthy controls. Results are shown as mean±S.E.M. (c) Expression of miR-128a in AML patients with specific mutations (NPM1, n=8, FLT3, n=9 or with other alterations, n=26) was compared with 10 healthy controls. Results are shown as mean±S.E.M. (d) qRT-PCR of miR-128a in OCI-AML3, ME-1 and MOLM-14 cells after 24, 48 and 72 h of treatment with PMA or ATRA. The bar graphs represented mean±S.D. from three independent experiments. (e and f) qRT-PCR of miR-128a (e) and ZFP36 (f) in OCI-AML3 after 24 and 48 h of scramble or anti-miR-128a transfection. The bar graphs represented mean±S.D. from three independent experiments. (g) Western blotting (WB) analysis of Lin28A, p21 and β-actin in OCI-AML3 after 24 and 48 h of transfection with scramble or anti-miR-128a. Statistically significant analyses are indicated by asterisks: *P<0.05, **P<0.01 and ***P<0.001

Figure 5

MiR-128a overexpression in OCI-AML3 cell line. (a and b) qRT-PCR of miR-128a (a) and Lin28A (b) in OCI-AML3 infected with pLKO.1_scr or pLKO.1_miR-128a after 24, 48 and 72 h of PMA treatment. (c and d) Representative histogram plots of CD11b+ (c) and CD14+ cells (d) in OCI-AML3 infected with pLKO.1_scr or pLKO.1_miR-128a after 24, 48 and 72 h of PMA treatment. (e) Percentage of CD11b+ and CD14+ OCI-AML3 cells infected with pLKO.1_scr or pLKO.1_miR-128a after 24, 48 and 72 h of PMA treatment, by cytofluorimetric analysis. (f) May–Grünwald Giemsa staining of OCI-AML3 infected with pLKO.1_scr or pLKO.1_miR-128a after 24, 48 and 72 h of PMA treatment. (g) Colony-forming assay of OCI-AML3 after infection with pLKO.1_scr or pLKO.1_miR-128a. Colonies were observed at day 14 of the semisolid culture under × 20 magnification. (h) Count of CFU-M colonies. The line and bar graphs represented mean±S.D. from three independent experiments. Statistically significant analyses are indicated by asterisks: *P<0.05 and **P<0.01

Figure 6

Inhibition of miR-128a in leukemic cells from AML patients. (a and b) Percentage of CD11b+ (a) and CD14+ cells (b) from three AML patients (AML1, AML2 and AML3) infected with Lenti-GFP or Lenti-miRZip-128a after 3 days of macrophage-like induction culture, by cytofluorimetric analysis. (c) qRT-PCR of miR-128a in AML patients infected with Lenti-GFP or Lenti-miRZip-128a. (d) qRT-PCR of Lin28A, EGR2, ZFP36 and ANXA1 in AML patients infected with Lenti-GFP or Lenti-miRZip-128a after 3 days of macrophage-like induction culture. Statistically significant analyses are indicated by asterisks: *P<0.05, **P<0.01 and ***P<0.001