Limb-Bud and Heart (LBH), Target of Mir-191, Is a Novel Positive Regulator of Erythroid Differentiation
Authors: Wang Wentian , Yang Yang , Duan Yongjuan , Zhao Huijuan , Hu Xiao 
Published: November 2019

Abstract
Many miRNAs have been characterized as important regulators through their targeted genes during mammalian erythroid differentiation. miR-191 is abundantly expressed in erythroblasts and has been reported to mainly regulate mouse erythroid enucleation through targeting on two genes, Riok3 and Mxi1. Recently, several studies with cancer cells have suggested that expression of miR-191 is under the control of hypoxia, posting the hypnosis that miR-191 might have more physiological regulatory function during erythropoiesis. Our initial work has shown reduced miR-191 level in K562 cell under hypoxia condition. We then found that enforced expression of miR-191 in human UCB-CD34+ cells impaired BFU-E colonies formation in CFU assay and delayed the terminal erythroid differentiation progress in an in vitro erythroid differentiation culture. These results revealed that miR-191 has broader negative impact on human erythroid differentiation, thus legitimate the search of new miR-191 target gene(s) with regulatory roles in erythroid differentiation.
To identify the new miR-191 target genes in erythroid cells, we first compared the microArray data of miR-191 and miR-191 sponge transducer K562 cell, then combined the list differentially expressed genes with an extensive search of miRNA target genes databases and human transcription factor databases, we then compile a list of 20 top candidates genes, including Cdk2,Cdk6, p53,p21 Rb1, Rebpb,Mxi1 ect. A target screening strategy combining qRT-PCR, 3'UTR report assay and miR-191 function rescue capacity assay led the identification of human Limb-bud and heart (LBH) coding gene, Lbh, as a novel miR-191 target gene.
LBH is a spatiotemporally expressed protein that marks the developing limbs and heart in early embryos. Aberrant gain-of-function and conditional knockout studies of LBH have implicated its roles in human congenital heart disease and breast cancer, respectively. Little is known, however, concerning its possible roles in hematopoiesis, including erythroid differentiation.
To explore the functional roles of LBH during human erythroid differentiation, we first compared its expression within human myeloid lineages, and found that expression of Lbh was almost 10 fold higher in BFU-E and CFU-E colonies. In the in vitro CD34+ erythroid differentiation culture, expression of Lbh increased almost 10 fold in early erythroblasts and continually increase throughout later stages. Next, we analyzed Lbh functions with in vitro human CD34+ erythroid culture and hemin induced K562 cells. While enforced expression of LBH only demonstrated a moderate to minor increase of erythroid colony formation and accelerated erythroid differentiation , knocking down LBH level with lentiviral expressed Lbh-shRNAs has a much severe erythroid phenotype, including comprehensive reduction of both BFU-E and CFU-E colony formation, severely impaired gamma- and beta-globin genes and protein expression. In both cases, expression of LBH was paralleled with that of key erythroid transcription factors, GATA-1, EKLF and TAL-1.
To pursue the molecular mechanism that LBH acts on human erythroid differentiation, we have searched the genes and signaling pathways that have most significantly changed by reduction of LBH. Microarray data analysis of the transcriptome of LBH knockdown K562 cell have identified JAK2/Stat as one of the signaling pathways. In comparison to the block of TGF and WNT signaling pathway, block of JAK2 pathway through AZD, specifically reduced LBH level and STAT5 phosphorylation. On the other hand, knockdown of LBH in both K562 and HEL cell significantly reduced both JAK2 and STAT5a phosphorylation level. More detail work is undertaken and we would like add the most updated data at the conference.
In all, our studies has identified LBH as a miR-191 target gene , more importantly, our work suggested that LBH is a novel positive regulator of human erythroid differentiation. We have also evidence suggesting a reciprocally regulation between JAK2/Stat5 pathway and LBH, which might raises its potential functions in pathological erythropoiesis.

Disclosures
No relevant conflicts of interest to declare.