Defective Expression of ANKHD1 Transcript Variant 3 in Myelodysplastic Cells and Its Role in Apoptosis.
Authors: Duarte Adriana S.S. , Traina Fabiola , Costa Fernando F. , Saad Sara T.O. 
Published: April 2020

Abstract
Ineffective erythropoiesis is a common feature of myelodysplastic syndromes (MDS). GATA-1, a transcription factor essential for erythroid maturation was found to be upregulated in normal hematopoietic cells after erythroid differentiation, but not in differentiating MDS erythroblasts. ANKHD1 transcript variant 3, also named PP2500, (NM_024668), is a recently described gene that contains a GATA-1 binding site at its promoter region, and has been shown to be upregulated during erythroid differentiation of normal hematopoietic cells. We then sought to evaluate the expression of PP2500 in normal and MDS cells, the expression of GATA-1 and PP2500 in CD34+ normal and MDS cells during erythroid differentiation, as well as PP2500 functional consequences in terms of apoptosis. Bone marrow samples were obtained from 16 patients with a diagnosis of low-risk MDS (13 RA and 3 RARS) and 5 normal donors, and submitted to RNA extraction. For erythroid differentiation, bone marrow samples from one low-risk MDS patient and one normal donor were submitted to CD34+ selection using MIDI-MACS immunoaffinity columns and cells were plated on plastic culture dishes in methylcellulose medium with appropriate growth factors for 6 days. BFU-E, CFU-E and proerythroblasts were then cultured in alpha MEM for another 8 days. At days 6 and 14, cells were collected and submitted to RNA extraction and apoptosis analysis. Posttranscriptional PP2500 gene silencing was performed in Jurkat cells, through electroporation, using small interfering RNA (SMARTpool siRNA duplexes-Dharmacon). Forty-eight hours after transfection, cells were submitted to RNA extraction and apoptosis analysis. The expression level of mRNA was detected by real time RT-PCR. Endogenous controls used were GAPDH and b-actin. The relative quantification value of gene expression was calculated using 2−DDCT. Apoptosis was evaluated using annexin V-FITC/PI staining; erythroblast differentiation was verified with transferring-receptor/glycophorin-A and FACS analysis. Reduced expression of PP2500 was observed in low-risk MDS cells (RA/RARS) when compared with normal hematopoietic cells (mean ± SD; 0.15 ± 0.18 vs 1.07 ± 0.53, respectively; P=0.0004). Erythroid differentiation of CD34+ normal cells resulted in upregulation of PP2500 and GATA-1: 12-fold increase on day 14 compared to day 6, for both mRNAs. However, differentiating MDS erythroblasts failed to show a noteworthy increment in PP2500 and GATA-1 expressions: 1.5-fold increase for GATA-1 and 2-fold increase for PP2500, on day 14 compared to day 6. After erythroid differentiation, apoptotic cells accounted for 5.6% of cultured normal marrow samples on day 6 and 2.5% on day 14, whereas MDS cultures contained 4.8% apoptotic cells on day 6 and 9.5% on day 14. Posttranscriptional PP2500 gene silencing resulted in a 2-fold increase in apoptotic cells compared to control electroporated cells (27% vs 14% annexin V+/PI−, respectively). In conclusion, reduced expression of PP2500 mRNA was found in low-risk MDS cells compared with normal hematopoietic cells and MDS erythroblasts showed a defective GATA1 and PP2500 expression pattern during erythroid differentiation associated with an increased apoptosis. In addition, PP2500 gene silencing resulted in increasing apoptosis. These results suggest that PP2500 may have an anti-apoptotic function and a defective expression in MDS cells, which may be implicated in the pathophysiological process of MDS.