THE MIR-128A/LIN28A AXIS REGULATES MYELOMONOCYTIC DIFFERENTIATION IN ACUTE MYELOID LEUKEMIA
(Abstract release date: 05/19/16)
EHA Library. De Luca L. 06/09/16; 132444; E895
Dr. Luciana De Luca
Contributions
Contributions
Abstract
Abstract: E895
Type: Eposter Presentation
Background
Acute myeloid leukemia (AML) is a heterogeneous neoplastic disorder of hematopoietic progenitors. Lin28 is a conserved RNA-binding protein playing an important role in cancer stem cells. Ectopic expression of Lin28 reprograms hematopoietic progenitor cells from adult bone marrow (BM), endowing them to mediate multi-lineage reconstitution which resembles fetal hemotopoiesis. It has been reported the existence of reciprocal regulatory loops between Lin28 and mir-128. In particular, this miRNA is expressed in early hematopoietic progenitor cells preventing the differentiation of all hematopoietic lineages. Moreover, increased expression of mir-128 was associated with high-risk molecular features of AML.
Aims
Since Lin28 and its mir-128 seem to be important regulators of hematopoiesis, it could be interesting to study their involvement in the induction and maintenance of AML.
Methods
We analyzed, by qRT-PCR, Lin28A and mir-128 expression levels in 37 AML patients, AML cell lines and 13 healthy donors. Lin28 protein expression was evaluated by cytofluorimetric analysis in myeloid and lymphoid precursors of 10 BM healthy subjects and in 9 AML leukemic cells (LC). Overexpression of Lin28A or silencing of miR-128 were obtained in OCI-AML3 cell line for 24-48 h. Moreover, OCI-AML3 and ME1 cells were treated with PMA for 24, 48 and 72h. Cells were collected and used to perform cell cycle assay, apoptosis assay, caspase 3/7 assay, cytofluorimetric analysis of Lin28A, CD11b, CD45 and CD14, western blot for Lin28A and p21 and qRT-PCR for EGR2 and ZFP36.
Results
Real Time PCR data indicated a down-regulation of Lin28A in AML patients (p<0.001) and cell lines (p<0.05) as compared with controls. To confirm this data, we also analyzed its protein expression in AML LC compared with normal myeloid precursors, showing a significant down-regulation (p<0.01). Furthermore, our data showed an up-regulation of Lin28A in normal myeloid precursors with respect to lymphoid (p<0.001) and erithroid ones (p<0.01). The over-expression of Lin28A in OCI-AML3 induced myeloid differentiation; in fact, we observed a significant increased of CD11b (p<0.05), CD14 (p<0.05), EGR2 (p<0.05) and ZFP36 (p<0.01). We also observed a significant block of cell cycle in S phase (p<0.05), an increased expression of p21 protein and the induction of apoptosis (p<0.001). To confirm the involvement of Lin28A in myeloid differentiation of LC, we treated AML cells with PMA. As expected, we observed an increase of myeloid markers (CD11b p<0.05, CD14 p<0.05, EGR2 p<0.01, ZFP36 p<0.01) and also a significant overexpression of Lin28A (p<0.05). Moreover, PMA treatment confirmed a significant block of cell cycle in G2 phase (p<0.01), an increased expression of p21 and the induction of caspase-dependent apoptosis (p<0.01). Literature data indicate that miR-128 regulate Lin28A; in fact, we demonstrated by qRT-PCR its up-regulation in AML patients (p<0.05) and cell lines (p<0.01). Furthermore, its silencing in OCI-AML3 induced a significant increase of Lin28A, p21 and myeloid markers as EGR2 and ZFP36 (p<0.001), confirming their involvement in myeloid differentiation and in cell cycle arrest.
Conclusion
Our data indicate mir-128/Lin28A axis as a potential regulator of myeloid differentiation, suggesting its involvement in AML development.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, Myeloid differentiation
Type: Eposter Presentation
Background
Acute myeloid leukemia (AML) is a heterogeneous neoplastic disorder of hematopoietic progenitors. Lin28 is a conserved RNA-binding protein playing an important role in cancer stem cells. Ectopic expression of Lin28 reprograms hematopoietic progenitor cells from adult bone marrow (BM), endowing them to mediate multi-lineage reconstitution which resembles fetal hemotopoiesis. It has been reported the existence of reciprocal regulatory loops between Lin28 and mir-128. In particular, this miRNA is expressed in early hematopoietic progenitor cells preventing the differentiation of all hematopoietic lineages. Moreover, increased expression of mir-128 was associated with high-risk molecular features of AML.
Aims
Since Lin28 and its mir-128 seem to be important regulators of hematopoiesis, it could be interesting to study their involvement in the induction and maintenance of AML.
Methods
We analyzed, by qRT-PCR, Lin28A and mir-128 expression levels in 37 AML patients, AML cell lines and 13 healthy donors. Lin28 protein expression was evaluated by cytofluorimetric analysis in myeloid and lymphoid precursors of 10 BM healthy subjects and in 9 AML leukemic cells (LC). Overexpression of Lin28A or silencing of miR-128 were obtained in OCI-AML3 cell line for 24-48 h. Moreover, OCI-AML3 and ME1 cells were treated with PMA for 24, 48 and 72h. Cells were collected and used to perform cell cycle assay, apoptosis assay, caspase 3/7 assay, cytofluorimetric analysis of Lin28A, CD11b, CD45 and CD14, western blot for Lin28A and p21 and qRT-PCR for EGR2 and ZFP36.
Results
Real Time PCR data indicated a down-regulation of Lin28A in AML patients (p<0.001) and cell lines (p<0.05) as compared with controls. To confirm this data, we also analyzed its protein expression in AML LC compared with normal myeloid precursors, showing a significant down-regulation (p<0.01). Furthermore, our data showed an up-regulation of Lin28A in normal myeloid precursors with respect to lymphoid (p<0.001) and erithroid ones (p<0.01). The over-expression of Lin28A in OCI-AML3 induced myeloid differentiation; in fact, we observed a significant increased of CD11b (p<0.05), CD14 (p<0.05), EGR2 (p<0.05) and ZFP36 (p<0.01). We also observed a significant block of cell cycle in S phase (p<0.05), an increased expression of p21 protein and the induction of apoptosis (p<0.001). To confirm the involvement of Lin28A in myeloid differentiation of LC, we treated AML cells with PMA. As expected, we observed an increase of myeloid markers (CD11b p<0.05, CD14 p<0.05, EGR2 p<0.01, ZFP36 p<0.01) and also a significant overexpression of Lin28A (p<0.05). Moreover, PMA treatment confirmed a significant block of cell cycle in G2 phase (p<0.01), an increased expression of p21 and the induction of caspase-dependent apoptosis (p<0.01). Literature data indicate that miR-128 regulate Lin28A; in fact, we demonstrated by qRT-PCR its up-regulation in AML patients (p<0.05) and cell lines (p<0.01). Furthermore, its silencing in OCI-AML3 induced a significant increase of Lin28A, p21 and myeloid markers as EGR2 and ZFP36 (p<0.001), confirming their involvement in myeloid differentiation and in cell cycle arrest.
Conclusion
Our data indicate mir-128/Lin28A axis as a potential regulator of myeloid differentiation, suggesting its involvement in AML development.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, Myeloid differentiation
Abstract: E895
Type: Eposter Presentation
Background
Acute myeloid leukemia (AML) is a heterogeneous neoplastic disorder of hematopoietic progenitors. Lin28 is a conserved RNA-binding protein playing an important role in cancer stem cells. Ectopic expression of Lin28 reprograms hematopoietic progenitor cells from adult bone marrow (BM), endowing them to mediate multi-lineage reconstitution which resembles fetal hemotopoiesis. It has been reported the existence of reciprocal regulatory loops between Lin28 and mir-128. In particular, this miRNA is expressed in early hematopoietic progenitor cells preventing the differentiation of all hematopoietic lineages. Moreover, increased expression of mir-128 was associated with high-risk molecular features of AML.
Aims
Since Lin28 and its mir-128 seem to be important regulators of hematopoiesis, it could be interesting to study their involvement in the induction and maintenance of AML.
Methods
We analyzed, by qRT-PCR, Lin28A and mir-128 expression levels in 37 AML patients, AML cell lines and 13 healthy donors. Lin28 protein expression was evaluated by cytofluorimetric analysis in myeloid and lymphoid precursors of 10 BM healthy subjects and in 9 AML leukemic cells (LC). Overexpression of Lin28A or silencing of miR-128 were obtained in OCI-AML3 cell line for 24-48 h. Moreover, OCI-AML3 and ME1 cells were treated with PMA for 24, 48 and 72h. Cells were collected and used to perform cell cycle assay, apoptosis assay, caspase 3/7 assay, cytofluorimetric analysis of Lin28A, CD11b, CD45 and CD14, western blot for Lin28A and p21 and qRT-PCR for EGR2 and ZFP36.
Results
Real Time PCR data indicated a down-regulation of Lin28A in AML patients (p<0.001) and cell lines (p<0.05) as compared with controls. To confirm this data, we also analyzed its protein expression in AML LC compared with normal myeloid precursors, showing a significant down-regulation (p<0.01). Furthermore, our data showed an up-regulation of Lin28A in normal myeloid precursors with respect to lymphoid (p<0.001) and erithroid ones (p<0.01). The over-expression of Lin28A in OCI-AML3 induced myeloid differentiation; in fact, we observed a significant increased of CD11b (p<0.05), CD14 (p<0.05), EGR2 (p<0.05) and ZFP36 (p<0.01). We also observed a significant block of cell cycle in S phase (p<0.05), an increased expression of p21 protein and the induction of apoptosis (p<0.001). To confirm the involvement of Lin28A in myeloid differentiation of LC, we treated AML cells with PMA. As expected, we observed an increase of myeloid markers (CD11b p<0.05, CD14 p<0.05, EGR2 p<0.01, ZFP36 p<0.01) and also a significant overexpression of Lin28A (p<0.05). Moreover, PMA treatment confirmed a significant block of cell cycle in G2 phase (p<0.01), an increased expression of p21 and the induction of caspase-dependent apoptosis (p<0.01). Literature data indicate that miR-128 regulate Lin28A; in fact, we demonstrated by qRT-PCR its up-regulation in AML patients (p<0.05) and cell lines (p<0.01). Furthermore, its silencing in OCI-AML3 induced a significant increase of Lin28A, p21 and myeloid markers as EGR2 and ZFP36 (p<0.001), confirming their involvement in myeloid differentiation and in cell cycle arrest.
Conclusion
Our data indicate mir-128/Lin28A axis as a potential regulator of myeloid differentiation, suggesting its involvement in AML development.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, Myeloid differentiation
Type: Eposter Presentation
Background
Acute myeloid leukemia (AML) is a heterogeneous neoplastic disorder of hematopoietic progenitors. Lin28 is a conserved RNA-binding protein playing an important role in cancer stem cells. Ectopic expression of Lin28 reprograms hematopoietic progenitor cells from adult bone marrow (BM), endowing them to mediate multi-lineage reconstitution which resembles fetal hemotopoiesis. It has been reported the existence of reciprocal regulatory loops between Lin28 and mir-128. In particular, this miRNA is expressed in early hematopoietic progenitor cells preventing the differentiation of all hematopoietic lineages. Moreover, increased expression of mir-128 was associated with high-risk molecular features of AML.
Aims
Since Lin28 and its mir-128 seem to be important regulators of hematopoiesis, it could be interesting to study their involvement in the induction and maintenance of AML.
Methods
We analyzed, by qRT-PCR, Lin28A and mir-128 expression levels in 37 AML patients, AML cell lines and 13 healthy donors. Lin28 protein expression was evaluated by cytofluorimetric analysis in myeloid and lymphoid precursors of 10 BM healthy subjects and in 9 AML leukemic cells (LC). Overexpression of Lin28A or silencing of miR-128 were obtained in OCI-AML3 cell line for 24-48 h. Moreover, OCI-AML3 and ME1 cells were treated with PMA for 24, 48 and 72h. Cells were collected and used to perform cell cycle assay, apoptosis assay, caspase 3/7 assay, cytofluorimetric analysis of Lin28A, CD11b, CD45 and CD14, western blot for Lin28A and p21 and qRT-PCR for EGR2 and ZFP36.
Results
Real Time PCR data indicated a down-regulation of Lin28A in AML patients (p<0.001) and cell lines (p<0.05) as compared with controls. To confirm this data, we also analyzed its protein expression in AML LC compared with normal myeloid precursors, showing a significant down-regulation (p<0.01). Furthermore, our data showed an up-regulation of Lin28A in normal myeloid precursors with respect to lymphoid (p<0.001) and erithroid ones (p<0.01). The over-expression of Lin28A in OCI-AML3 induced myeloid differentiation; in fact, we observed a significant increased of CD11b (p<0.05), CD14 (p<0.05), EGR2 (p<0.05) and ZFP36 (p<0.01). We also observed a significant block of cell cycle in S phase (p<0.05), an increased expression of p21 protein and the induction of apoptosis (p<0.001). To confirm the involvement of Lin28A in myeloid differentiation of LC, we treated AML cells with PMA. As expected, we observed an increase of myeloid markers (CD11b p<0.05, CD14 p<0.05, EGR2 p<0.01, ZFP36 p<0.01) and also a significant overexpression of Lin28A (p<0.05). Moreover, PMA treatment confirmed a significant block of cell cycle in G2 phase (p<0.01), an increased expression of p21 and the induction of caspase-dependent apoptosis (p<0.01). Literature data indicate that miR-128 regulate Lin28A; in fact, we demonstrated by qRT-PCR its up-regulation in AML patients (p<0.05) and cell lines (p<0.01). Furthermore, its silencing in OCI-AML3 induced a significant increase of Lin28A, p21 and myeloid markers as EGR2 and ZFP36 (p<0.001), confirming their involvement in myeloid differentiation and in cell cycle arrest.
Conclusion
Our data indicate mir-128/Lin28A axis as a potential regulator of myeloid differentiation, suggesting its involvement in AML development.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, Myeloid differentiation
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