Role of miR-30a-5p and miR-26a-5p in induction of terminal differentiation in human K562 erythroleukemia cells

Abstract

Background & Aim Background Plasmodium vivax (Pv) infections affect ∼300 million people every year. However, studies into this parasite species are hampered by the inability to set up a continuous, long-term in vitro blood stage culture for Pv. Establishing an in vitro “red blood cell matrix” that would allow continuous access to reticulocytes would facilitate the establishment of continuous, long-term in vitro Pv blood stage cultures and ultimately the study of parasite vulnerabilities that can be exploited in therapeutic approaches. Our study aims to establish culture conditions that allow for the differentiation of K562 cells into enucleated erythroid cells, serving as a constant source of reticulocytes for continuous, long-term Pv blood stage cultures. Methods, Results & Conclusion Materials and Methods Since the K562 cell line (K562) does not express the Duffy blood group antigen receptor (Fy), essential for Pv invasion, the Duffy FyB receptor was stably introduced into the K562 by lentiviral gene transfer. In addition, we combined different factors, which were known to enhance the terminal erythroid differentiation of K562 (knockdown of miR-30a-5p and -26a-5p, co-cultivation with macrophages, EPO, transferrin, insulin and mithramycin A). Effects were analysed by quantification of total cell numbers, surface markers and Giemsa staining. Hb expression was detected by Benzidin staining and PCR. Results K562 cells transduced with anti-miR-26a-5p and -30a-5p (C26) and stimulated with the induction cocktail showed a significantly increase in the amount of enucleated reticulocytes as compared to cells transduced with the empty vector. This is reflected in a stronger down-regulation of CD45 and up-regulation of CD71 as well as in the number of enucleated reticulocytes and the increase in hemoglobin production. CD235a expression was already higher in clone C26 prior to stimulation with the induction cocktail as compared to unstimulated K562 empty cells. The Duffy receptor expression remained stable in stimulated cells of the clone C26, while it was downregulated in K562 empty vector cells over the stimulation period. Conclusions The data show that knockdown of miR-26a-5p and -30a-5p are necessary to induce terminal differentiation of K562 cells. Further optimisation of culture conditions to enhance the yield of RBC blasts from K562 is necessary, as is the confirmation of functionality and susceptibility to Pv infection of these cells.