Abstract:
Objective To determine the effect of rapamycin(Rapa) on JAK2, ABCA3, and the immune checkpoint PD-1/PD-L1 in exosomes derived from JAK2 V617F positive HEL cells.
Methods Human erythroleukemia HEL cells (JAK2 V617F mutation-positive) were cultured in vitro, and rapamycin was added at concentrations of 10, 50, and 100 nmol/L. The control group was established and cell proliferation inhibition rate was detected by CCK-8. Based on the inhibition rate of cell proliferation, cells intervened with 10 and 50 nmol/L Rapa were selected. Exosomes were extracted using a kit and identified by Western blot and flow cytometry. JAK2, ABCA3, and PD-1/PD-L1 mRNA changes in exosomes were detected by fluorescent quantitative PCR. The expression of exosome PD-1/PD-L1 protein was determined by flow cytometry.
Results The exosomes extracted with the exosome kit all expressed characteristic CD9, CD63, and CD81 proteins, which were consistent with the general characteristics of exosomes. JAK2 mRNA can be detected in exosomes from HEL cells; Rapa reduced exosome production by HEL cells, and dose-dependently decreased gene expression of JAK2, ABCA3, and PD-L1 in exosomes. In addition, Rapa inhibited exosomal PD-L1 protein expression and had no significant effect on PD-1 protein expression.
Conclusion HEL cells may transmit JAK2 mutant gene signals via exosomes. Rapa can reduce the production of exosomes and inhibits JAK2 mutated signals delivered by exosomes and suppresses PD-L1 protein expression.
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