Figure 5.

MiR-181a promoted MK hematopoiesis. (a) Flow cytometry results showed that the percentage of CD41/CD61-positive cells in miR-181 stable K562 cells increased to 21.6% at 24 h and 63.3% at 36 h after TPA induction when compared with GFP control cells (5.3% and 46.8%, respectively). (b) Flow cytometry results showed that the percentage of miR-181a-K562 cells undergoing endomitosis after TPA treatment increased. (c) Transiently transfecting the designated inhibitor effectively downregulated the expression of miR-181 and upregulated Lin28 when compared with the controls as assessed by qRT-PCR and western blotting. Relative quantitation (RQ) of western blotting was computed by dividing the density values of Lin28 blots to that of α-tubulin blots, which was measured by the software GelQuantNET (http://biochemlabsolutions.com/GelQuantNET.html). The result shows that the miR-181a inhibitor is capable of upregulating the expression of the Lin28 protein 2.3-fold over the control (0.86/0.23). (d) The miR-181a inhibitor consequently retarded the transition of the MK lineage as evidenced by the decline of CD41/CD61-positive rate when compared with the control (64.6% versus 83.8%). (e) Lin28 is the direct target of miR-181a for driving K562 cells toward MK differentiation, and its overexpression abolished the effect of miR-181a on the process of MK differentiation. (f) Morphological changes of miR-181a-HPCs and control cells after CC220 induction toward MK differentiation. MiR-181a-HPCs showed early adhesion and larger nuclei than the control cells as observed by day 5 and 8. (CD41/CD61-positive rate is computed by adding the rates of CD41+, CD41+/CD61+, and CD61+). The error bars represent S.D. t-Tests were used for statistical analysis; ^*indicates P<0.05 and ^**indicates P<0.01. RQ stands for relative quantitation and NC means non-target control