Acute lymphoblastic leukemia is a disease characterized by a genetic or chromosomal imbalance that occurs in the lymphoid precursor cells of the bone marrow, primarily affecting children, and also in elderly people.

The objective of this study is to find differentially expressed miRNAs in Jurkat cell cultures treated with wild-type asparaginase and two variants. As the variants have greater affinity and specificity, it is assumed that this is related to a possible lower side effect. For these reasons, we want to explore the differences in expression of some miRNAs.

We used the Jurkat cell line (E6-1), and two mutated asparaginases were constructed (de Lima et al, 2024). Then, three asparaginases (WT, EC25 and ECA89) were used as chemotherapy in culture experiments. Asparaginase exposure was evaluated at three different times (24, 48, and 72 hours) using three variants at two different concentrations (5 and 10U). After exposure, the supernatant and the cell pellet were separated and submitted to biochemical analysis to evaluate glucose and lactate levels, while total RNA was extracted (TRizol TM) from the cells pellet to perform reverse transcription (cDNA), allowing us to analyze some microRNAs expression levels by qPCR. The exogenous cel-miR-39-3p was added to the RNA to normalize the expression data. First of all, a curve with different concentrations of the exogenous cel-miR-39-3p was performed to determine the concentration to be add to the RNAs before the cDNA synthesis (TaqMan ™ Advanced miRNA cDNA Synthesis Kit).

Cellular assays in Jurkat cells showed that both wild-type asparaginase and the variants reduced cell viability at similar rates, with only a significant difference compared to the control (cells without asparaginase), and the exposure time (24, 48 and 72h) did not showed significant results. In the supernatant it was possible to observe differences in lactate and glucose concentrations after 48 and 72h of exposure.

Cell feasibility analyses showed the reduction in cell viability, that the asparaginase variants kill as much as the wild type, which is good, since the goal of obtaining these variants is only that they be less toxic in terms of side effects, due to their increased specificity (data not shown). With the supernatant results it was possible to observe the Warburg effect across the different treatment types, with variations depending on the type of asparaginase used and the concentrations applied. In conclusion, we obtained RNA from cell cultures and observed metabolic differences between the conditions and variants tested.