Antisense Long Non-Coding RNA in the LEF1 Locus Regulates Sense LEF1 Expression in Leukemic Cell Line KG1

Aberrant regulation of the WNT signaling pathway is a signature in numerous human cancers. Lymphoid enhancer-binding factor-1 (LEF1) is an important transcription factor downstream of this pathway. LEF1 over-expression induces AML in mice and plays a critical role in hematopoietic cell differentiation (Petropoulos et al JME 2008). Reduction of LEF1 expression through the progression of myelodysplastic syndrome has been reported and further supports the relevance of this gene in the disease pathogenesis (Pellagatti et al Br J Haematol. 2009).

Our previous work using microarray technology revealed a decreased expression of a long non-coding RNA antisense to LEF1 (LEF1-AS) in MDS patients (Baratti et al BMC Medical Genomics 2010). Mounting evidence suggests that long non-coding transcripts play important roles in the epigenetic regulation of coding genes. In this context it is not surprising that long non-coding RNAs are emerging as key players in disease development and progression. Non-coding expression overlapping coding genes is very common and several examples of local regulation have been described in the literature.

Here we investigate for the first time the role of LEF1 antisense long non-coding in hematopoiesis and demonstrated its contribution in the regulation of the LEF1 locus in a leukemic cell line.

To explore a possible role of LEF1-AS in differentiation, we evaluated the expression pattern of LEF1-AS through erythroid cell differentiation using qRT-PCR. CD34+ HSC cells from 6 healthy donors were induced to differentiate into erythrocytes by addition of erythropoietin during 12 days. We observed that LEF1-AS is modulated during erythroid differentiation. It was significantly down-regulated during the first stages of differentiation from CD34+ HSC to erythroblast (from collection day 6 to day 8 after addition of erythropoietin, 78% mean reduction, P<0.0001) and it was up-regulated at the end-point of collection, day 12 (not significant). Lef1 coding gene displayed a similar expression pattern, consistent with previous reports of Lef1 expression during erythroid maturation (Edmaier et al Leukemia 2014).

To explore a possible regulatory role of LEF1-AS, we cloned and over-expressed the transcript in KG1 CD34+ leukemia cell line. Transient over-expression of Lef1-AS led to a significant up-regulation of Lef1 gene (22% increase, P<0.05). We also observed an increase in cell viability (19% increase P<0.05), measured by MTT, which is consistent with the up-regulation of LEF1, a pro-proliferative and anti-apoptotic transcription factor.

Our preliminary results from over-expressing LEF1-AS in CD34+ HSCs suggest a similar regulatory effect of LEF1-AS upon its coding counterpart, LEF1. Since aberrant expression of LEF1 is known to disrupt normal differentiation of CD34+ cells, LEF1-AS could potentially affect differentiation through the modulation of LEF1 coding gene. Our results reveal LEF1-AS transcript as a novel player in hematopoiesis and hematologic malignancy.