MicroRNA-181b Inhibits ATRA Induced Differentiation in the APL Cell Line NB-4 Via Negative Regulation of the Tumorsupressor RASSF1A

MicroRNAs (miRNAs) are small non-coding RNAs of 19–25 nucleotides that are involved in the regulation of critical cell processes such as apoptosis, cell proliferation and differentiation. However, little is known about the role of miRNAs in AML and granulopoiesis. In this study, we analysed the expression of miRNAs in the acute promyelocytic leukemia cell line NB-4 during all-trans-retinoic acid (ATRA) treatment by using a miRNA microarray platform (Exiqon) and quantitative PCR analysis. We identified several ATRA regulated microRNAs. Among them we found microRNA-181b as negatively regulated by ATRA. MicroRNA-181b is known to be overexpressed in several cancers such as thyroid papillary carcinoma, colorectal cancer, acute lymphocytic leukemia (ALL) and also acute promyelocytic leukemia (APL). To determine the role of microRNA-181b in the formation and maintenance of the APL-blast phenotype, we performed LNA knock down and transient overexpression experiments. Here we could show in quantitative PCR analysis an inhibitory effect of microRNA-181b on the transcription of the ATRA induced granulocytic differentiation markers C/EBPα and C/EBPβ. Furthermore in FACS analysis we could observe a reduction of the ATRA stimulated formation of the granulocytic surface marker CD11b under microRNA-181b overexpression conditions. To identify the mechanism how microRNA-181b affects ATRA induced differentiation we performed data base analysis to identify putative targets of microRNA-181b. One promising candidate is the tumorsuppressor RASSF1A. RASSF1A was already shown to be involved in proliferation and differentiation by regulation of cell cycle processes. As first group we were able to show in this study the ATRA induced upregulation of RASSF1A in NB-4 cells by quantitative PCR analysis. To demonstrate the negative effect of microRNA-181b on RASSF1A we analysed RASSF1A protein level under LNA knock down and overexpression conditions of microRNA-181b. Here we could observe an upregulation of RASSF1A protein level when microRNA-181b was downregulated. And in addition, a higher amount of microRNA-181b elicits the reduction of RASSF1A protein level. The regulatory effect of RASSF1A in the ATRA induced differentiation of NB-4 cells we confirmed by using siRNA against RASSF1A. In this experiment the FACS analysis showed a reduction of ATRA induced CD11b expression under siRNA mediated knock down of RASS1A. In conclusion, these findings support our hypothesis that microRNA-181b can act as oncogenic microRNA and thereby promotes the leukemic phenotype of APL-blasts by negative regulation of the tumorsuppressor RASSF1A.