Investigating Apoptotic Effect through Blocking miR-181b and miR-222 Using LNA-anti-miRNA in HL-60 Cell Line: Strategies to Improve Hematopoietic Stem Cell Transplantation

Abstract

Background: Several genes that control the commitment and differentiation of hematopoietic stem cells are regulated by miRNAs. Hematologic cancers like acute myeloid leukemia (AML) have been found to express miRNAs abnormally. Objective: In this current study, we assessed the apoptotic effect of miR-181b and miR-222 blockage, which can influence the expression of WT1, CEBPA, and C-KIT genes in an HL-60 cell line. Methods: Relative gene expression was observed by the SYBR Green Real-Time PCR method. By transfecting the HL-60 cell line with locked nucleic acid (LNA)-anti-miRNA, miRNA expression was suppressed. MTT assay was used to determine the viability of transfected cells, and PE Annexin V apoptosis detection kit I was used to evaluate the apoptosis. Results: After LNA transfection, the results showed a reduction in the expression of miR-181b and miR222. The flow cytometry data showed the apoptosis reduction by the inhibition of miR-181b and apoptosis increase by the inhibition of miR-222. We also found that miR-222 inhibition dramatically reduced c-KIT level, however, miR-181b blockage was associated with up-regulated of C-KIT expression. Moreover, the LNA-modified miR-222 could up-regulate BAX and down-regulate Bcl-2, whereas, after the transfection of the LNA-anti-miR-181b, BAX expression levels were significantly lower on average. Conclusion: We concluded that the inhibiting of miR-222 and increasing miR-181b could help to control AML disease. MiR-222 could be a possible prognostic biomarker in patients who had hematopoietic stemcell transplantation (HSCT) due to its higher expression in HSCT patients who got a graft-versus-host disease (GVHD).