miR-16-5p enhances sensitivity to RG7388 through targeting PPM1D expression (WIP1) in Childhood Acute Lymphoblastic Leukemia.

Abstract

Aim: Given the encouraging results of the p53-Mdm2 inhibitor RG7388 in clinical trials and the vital function of miR-16-5p in suppressing cell proliferation, the aim of the present study was to investigate the combined impact of RG7388 and miR-16-5p overexpression on the childhood acute lymphoblastic leukemia (chALL). Methods: miRTarBase and miRDB, along with KEGG and STRING databases, were used to predict miR-16-5p target genes and explore protein-protein interaction networks, respectively. B- and T-lymphoblastic cell lines, in addition to patient primary cells, were treated with RG7388. Ectopic overexpression of miR-16-5p in Nalm6 cell line was induced through cell electroporation and transfection of microRNA mimics was confirmed by qRT-PCR. Cell viability was evaluated using the MTT assay. Western blot analyses were performed to evaluate the effects of RG7388 and miR-16-5p upregulation on the protein levels of p53 and its downstream target genes in chALL cells. Paired sample t-test was employed for statistical analyses. Results: MTT assay showed RG7388-induced cytotoxicity in wild-type p53 Nalm6 cell line and p53 functional patient primary cells. However, CCRF-CEM and p53 non-functional leukemic cells indicated drug resistance. Western blot analyses validated the bioinformatics results, confirming the downregulation of WIP1, p53 stabilization, as well as overexpression of p21^WAF1 and Mdm2 proteins in Nalm6 cells transfected with miR-16-5p. Moreover, enhanced sensitivity to RG7388 was observed in the transfected cells. Conclusion: This is the first study indicating the mechanistic importance of miR-16-5p overexpression in chALL and its inhibitory role in leukemia treatment when combined with the p53-Mdm2 antagonist, RG7388. These findings might be useful for researchers and clinicians to pave the way for better management of chALL.