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| Effects of Rapamycin on Proliferation Apoptosis and PD-1 / PD-L1 Signaling Pathway of JAK2 V617F Positive HEL Cells |
| QI Lin, et al |
| Chengde Medical University, Hebei Chengde 067000, China |
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Abstract Objective: To explore the effect of Rapamycin (Rapa) on proliferation and apoptosis of JAK2 V617F positive HEL cells and the expression of programmed death receptor 1 and its ligand (PD-1/PD-L1). Methods: Human erythroleukemia HEL cells with JAK2 V617F mutation positive were cultured in vitro. Rapa with concentrations of 10nm, 50nm and 100nm were added respectively. The control group was established. The inhibition rate of cell proliferation was detected by CCK-8. Caspase 3 / 7 activity detection kit was used to detect the caspase 3/7 activity of cells in each group, Transwell chamber was used to observe the cell migration ability of each group, flow cytometry was used to detect the apoptosis and PD-1 / PD-L1 expression of cells in each group, real-time fluorescence quantitative polymerase chain reaction was used to detect the mRNA changes of JAK2, PD-1 and PD-L1 in cells in each group, and Western blot was used to detect the expression of mammalian rapamycin target protein (mTOR) in each group. Five healthy volunteers were selected. Their lymphocytes were isolated and co cultured with HEL cells. Different concentrations of Rapa were added. The number of Treg cells in each group was detected by flow cytometry. Results: Results of CCK-8 showed that:The inhibition rates of cell proliferation at 72h with different final concentrations of Rapa (10 nM, 50 nM, 100 nM) were (33.33±4.6)%, (49.12±3.72)%, (55.16±4.14)%(P<0.05), respectively. Rapa treated HEL cells at concentrations of 50nM and 100nM after 24h,Caspase 3/7 activity was significantly increased compared with control(P<0.05).The results of flow cytometry showed that the apoptotic rate increased significantly compared with the control.PD-L1 expression was significantly decreased compared with the control(P<0.05).The mRNA expression of JAK2 and PD-L1 was significantly downregulated compared with the control (P<0.01).Healthy volunteer lymphocytes co-cultured with HEL cells for 24h showed an increase in Treg cells(P<0.05).Rapa could reduce Treg cells in a dose-dependent manner.Western blot showed that Rapa was able to inhibit mTOR expression in a dose-dependent manner. Conclusion: Rapa may interfere with the JAK2 pathway by affecting mTOR, which leads to the inhibition of Hel cell proliferation, the reduction of PD-L1 expression, and the suppression of Treg cells.
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2022, Vol. 28 
