Construction Of Eukaryotic Expression Vector MicroRNA-144 And A Study About It Regulates Erythroid Differentiation Of K562 Cells

Hematopoiesis one of most important physiological process and the resulting number of fully differentiated red cells is precisely controlled to match physiologic and developmental need through the commitment of hematopoietic stem cells (HSCs) to erythroblasts and subsequently the proliferation and differentiation of erythroid progenitors to mature red cells. Erythroid differentiation is an important branch of it.MicroRNAs (miRNAs) are a class of newly found regulatory molecules that have been considered to be the common way of post-transcriptional level of multi-cellular regulating gene expression. With the increasing number and functional elucidating of miRNA, miRNA-mediated post-transcriptional regulation has been recognized as one of important component of the network that regulate eukaryotic gene expression, and synergistic action with the transcription factors, epigenetic regulators, co-regulating complex life activities. Among them, increasing attention have been attracted by miRNA regulating Erythroid Differentiation in K562 Cells. MiR-144 is one of them. However, the role and mechanism of miR-144 regulating Erythroid Differentiation of K562 Cells is not fully understood. This study was designed to detect that the role of miR-144 regulating erythroid differentiation of K562 Cells, and to further clarify the mechanism of miR-144 regulating Erythroid Differentiation; Constructed Eukaryotic Expression Vector MicroRNA-144 to find other targets of miR-144, and made preparations to construct K562 cells of expressing miR-144 stably and recombinant lentiviral vector of mir-144 Screening miR-144 lentiviral construct basis.In this study, K562 cells were treated with hemin to differentiate into mature red cells. Real-time PCR analysis was used to detect the changes of miR-144 expression during erythroid differentiation; K562 cells were transfected with oligonucleotides (mimic-144) and the influence of erythropoiesis was measured; miR-144 candidate target genes have been predicted by software. 3'UTR of these candidate genes containing miR-144 binding sites was cloned into downstream of pMIR-REPORTTM Luciferase, The target gene of miR-144 was identified by using bioinformatics analysis combined with Dual-luciferase reporter and Western blot analysis. The human miR-144 (hsa-miR-144) gene was cloned pmR-mCherry, real-time PCR analysis expression of mir144 in 293T and K562 cells. The major contents and results are as follows:1. During of K562 cells erythroid differentiation inducted by hemin, γ-globin expression was increased, indicating successful induction. MiR-144 was significantly increased during hemin-induced K562 erythroid differentiation(P<0.05),indicating mir-144 plays an important role of erythroid differentiation. The marker genes of erythroid differentiation, such as gamma-globin, CD235a, were all increased by enforced expression of miR-144. These results implied that over-expression of miR-144 promoted K562 erythroid differentiation.2.·We found that reporter gene containing 3'UTR of RB can be inhibited by miR-144, Site mutation experiments and western blot confirmed: RB was direct target of miR-144.3. Expression of RB increased slightly and then reduced obviously in K562 erythroid differentiation. Grey mean of RB/GAPDH minimum point: 12h 0.092±0.007(P<0.05 compared with 0h). MiR-144 played a positive role via targeting RB to decrease the control of cell cycle in the anaphase of K562 erythroid differentiation.4. We successfully constructed the eukaryotic expression vector of mir-144(pmR-mCherry-mir-144), identificated by restriction enzyme digestion and sequencing results consistent with published in GenBank. Real-time PCR analyzed that miR-144 over-expressed > 1500-fold in 293T cells (P <0.01) and > 150-fold in K562 cells (P <0.05). In summary, RB is a direct target gene of miR-144. miR-144 can decrease cell cycle by inhibiting expression of pRB in the anaphase of erythroid differentiation, and promote the erythroid differentiation;We constructed eukaryotic expression vector of miR-144 (pmR-mCherry-mir-144) to find other miR-144's target genes, and made preparation to construct K562 cells of expressing miR-144 stably.