Functional And Mechanism Study Of Hsa-miR-200a Regulating Erythroid Differentiation

Erythroid differentiation generates about 1011 red blood cells to fulfill daily requirements of transporting oxygen and carbon dioxide, maintaining acid-base balance in human body. Disorder of erythroid differentiation causes sickle cell anemia, thalassemia etc which seriously threaten human health. Thus, study the regulation of erythroid differentiation is rather vital. Previous studies indicated that erythroid differentiation is finely and precisely regulated at multiple levels:niche, chromatin structure, growth factors, transcription factors, noncoding RNAs et. al.MicroRNA (miRNA) has drawn researchers’ attention in recent years as a family of small noncoding RNA which regulate genes expression. Here, we analyzed miRNome data of different differentiated and developed erythroid cells to screen candidate miRNAs which may play a role in erythroid differentiation. The expression patterns of candidate miRNAs were examined in several other erythroid differentiation study models. Hsa-miR-200a was finally selected for further study for its consistent expression pattern in different erythroid differentiation models.Over expression of miR-200a inhibited globin genes, GATA-1 and KLF1 expression both in K562 and TF-1 cells. We further induce both control and miR-200a over expression TF-1 cells into erythroid differentiation. Persistent inhibition of globin genes expression at mRNA level and CD235a, CD71 at cell surface along induction, plus lower concentration of hematoglobin indicated that induced TF-1 erythroid differentiation is attenuated by miR-200a over expression. Transcriptome analyses of control and miR-200a over expression TF-1 cells further confirmed the affection caused by miR-200a over expression in TF-1 cells.Combined analyses of transcriptome data and miRNA targets predicting database was performed to screen out potential targets of miR-200a. Through a serial of screening stratages, PDCD4 and THRB were determined as potential targets of miR-200a. Dual-luciferase reporter assay and RISC-IP were performed and revealed that miR-200a is capable of binding at the 3’UTR of PDCD4 and THRB. Together with reduced expression of PDCD4 and THRB caused by miR-200a over expression in K562 and TF-1 cells, we concluded that PDCD4 and THRB are two targets of miR-200a. Functional studies of PDCD4 and THRB in K562 cells indicated that these two genes play a role in erythroid differentiation.Taken together, we began with miRNA-seq data; through data screening and functional studies finally proved that miR-200a regulate erythoid differentiation by targeting PDCD4...