The effects of siRNA-mediated inhibition of E2A-PBX1 on EB-1 and Wnt16b expression in the 697 pre-B leukemia cell line.
OBJECTIVE
A common non-random translocation in childhood pre-B acute lymphoblastic leukemia (ALL) is t(1;19)(q23;p13), usually resulting in the expression of the chimeric gene E2A-PBX1. The role of this fusion gene during leukemogenesis is not yet fully understood; one approach to investigate its function is to selectively deplete the E2A-PBX1 protein and examine the consequences.


METHODS
We tested the efficacy of anti-E2A-PBX1 siRNA in the 697 pre-B leukemia cell line. Transfection was monitored by fluorescence microscopy and FACS, while E2A-PBX1 mRNA expression was measured using real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis. The reduction of the level of the corresponding fusion protein was assessed by western blot analysis and the expression of putative downstream target genes was detected by SYBR Green PCR.


RESULTS
We demonstrated efficient downregulation induced by anti-E2A-PBX1 siRNA in 697 t(1;19)-positive leukemia cells. In particular, E2A-PBX1 silencing affected the EB-1 gene, which encodes for a protein that could contribute to the transformed phenotype of pre-B ALL. The detected EB-1 expression was reduced to 25% of the normal expression level in non-transfected 697 cells. Furthermore, the significant decrease in Wnt16b mRNA levels (but not of the Wnt16a isoform of the Wnt16 gene), observed following depletion of the fusion gene, confirms the hypothesis that Wnt16b is a target of E2A-PBX1. The siRNA inhibition was followed by an increase in apoptosis and similar results were obtained in two other ALL cell lines, one with and one without the t(1;19) translocation.


CONCLUSIONS
Targeted-E2A-PBX1 inhibition leads to reduced expression of the EB-1 and Wnt16b genes; aberrant expression of these genes may be a key step in leukemogenesis in t(1;19)-positive pre-B leukemia.
Casagrande
Giovanna
G
Laboratory of Hematology and Oncology, Department of Pediatrics, University of Padova, Italy.
te Kronnie
Geertruy
G
Basso
Giuseppe
G
D017209:Apoptosis; D045744:Cell Line, Tumor; D002878:Chromosomes, Human, Pair 1; D002888:Chromosomes, Human, Pair 19; D017931:DNA Primers; D015972:Gene Expression Regulation, Neoplastic; D006023:Glycoproteins / Q000235:genetics*; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000235:genetics*; D015452:Precursor B-Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D012334:RNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D034741:RNA, Small Interfering / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014178:Translocation, Genetic; D051153:Wnt Proteins / Q000235:genetics*
D017931:DNA Primers; D006023:Glycoproteins; C500985:MAPRE1 protein, human; D008869:Microtubule-Associated Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D034741:RNA, Small Interfering; C417262:WNT16 protein, human; D051153:Wnt Proteins