PROMOTER METHYLATION IS ASSOCIATED WITH miR-34b REDUCED EXPRESSION AND ITS RESTORATION DO NOT REVERSE CYTARABINE RESISTANCE PHENOTYPE IN AML, IN VITRO

INTRODUCTION AND OBJECTIVES: Acute myeloid leukemia (AML) is a hematological neoplasm highly heterogeneous. The standard chemotherapy treatment is based on the association of one anthracycline with cytarabine (ara-C). However, patients’ response to chemotherapy is reduced due to treatment resistance phenomenon, which contributes to AML poor overall survival. Deregulation of microRNAs expression is associated with resistance, tumor progression and relapse. miR-34b inhibits the translation of factors involved with proliferation and apoptosis regulation, being considered a tumor suppressor microRNA. miR-34b exogenous expression sensitizes solid tumor’s cells to chemo- and radiotherapy. However, there is no literature report evaluating miR-34b potential to reverse chemoresistance or to potentialize synthetic compounds effects in AML. In this context, besides the study of chemoresistance, our group also performs in vitro testing of synthetic compounds’ antitumoral effects. LQB-223 is a synthetic compound that has antitumor effect on different tumor cell lines, including AML. Therefore, the study aim is to evaluate miR-34b expression levels in AML cell lines with different resistance phenotypes along with the effect of miR-34b overexpression in treatment response in the chemoresistant cell line, HL-60R. MATERIAL AND METHODS: U937, HL-60 and HL-60R (ara-C chemoresistant cell line developed by our group) cell lines were selected to develop the study. Methylation specific PCR (MSP) was used to evaluate the methylation status of miR-34b/c promoter in the selected cell lines. Cell lines were treated with the synthetic compound LQB-223 to analyze miR-34b expression by quantitative PCR (qRT-PCR). Furthermore, miR-34b was overexpressed in HL-60R cell line by transfection of miRNA mimics with Lipofectamine reagent. The effect of miR-34b transient overexpression associated with ara-C or LQB-223 was evaluate by trypan blue exclusion and MTT assays, and the following flow cytometry assays annexin V, cell cycle distribution and DNA fragmentation. RESULTS AND CONCLUSION: Our data demonstrated that AML cell lines U937, HL-60 and HL-60R express low levels of miR-34b and miR-34b/c promoter methylated. Treatment with LQB-223 does not modulate miR-34b expression in AML cell lines. We performed cytotoxicity assays to evaluate the combined effect of miR-34b exogenous expression with ara-C or LQB-223 treatments in HL-60R cell line. Our results demonstrated that there was no reduction of cell viability, no changes in cell cycle distribution and no induction of cell death by the overexpression of miR-34b alone or in combination with ara-C and LQB-223 treatments. In conclusion, the AML cell lines have low miR-34b expression due to methylation in the miR-34b/c gene promoter. Furthermore, miR-34b does not participate in LQB-223 mechanism of action. Finally, miR34b restoration did not reverse cytarabine resistance or potentialize LQB-223 antitumoral effect.