Establishment Of Drug-resistant KG1a Cells And Its Small Extracellular Vesicles Reduce The Sensitivity Of Recipient Cells

The azanucleoside decitabine（5-aza-2’-deoxycytidine,DAC）,a DNA hypomethylating agent,has been used as a low-toxic chemotherapy reagent for patients with myelodysplastic syndrome（MDS）and acute myeloid leukemia（AML）who cannot tolerate intensive chemotherapy.Compared with cytarabine and other cytidine analogs that do not possess DNA hypomethylation characteristics,decitabine has better curative effect and no obvious damage to Natural killer（NK）cells.A large number of studies have been carried out on drug resistance and recurrence of patients with AML and MDS.However,there has been less research on whether decitabine resistant cells can affect recipient cells to develop resistance.Small extracellular vesicles（sEVs）are microvesicles with a diameter of 40-150nm which secreted from cells.sEVs can stably deliver functional molecules such as key proteins or micro RNAs（miRNAs）.Therefore,sEVs are an important medium for cell-cell communication and play an important role in the development and resistance of tumor.In this thesis,the decitabine-resistant KG1a cells（KG1a-DAC）were constructed by induction with different gradients of decitabine.IC50 of KG1a and KG1a-DAC was detected by CCK8.Cell proliferation and cell viability of KG1a and KG1a-DAC were detected by flow cytometry and CCK8.Results showed that KG1a-DAC were successfully constructed（the IC50 of KG1a-DAC to decitabine is more than 10-fold as KG1a）.In this research,KG1a and KG1a-DAC were used to study whether KG1a-DAC affects the decitabine sensitivity of KG1a.The main experimental results are as follows:（1）KG1a-DAC or its conditioned medium can reduce the sensitivity of KG1a cells to decitabine.（2）Cell proliferation and viability of KG1a after co-cultured with sEVs derived from KG1a-DAC were detected.Results show that sEVs from KG1a-DAC can reduce the sensitivity of KG1a to decitabine.（3）Expression of ENT1,DCK and CDA,the decitabine metabolism related proteins,and the expression of P15^INK4B in KG1a before and after co-cultured with KG1a-DAC were detected.Results show that decreased expression of P15^INK4B in KG1a but the expression of ENT1,DCK and CDA remains unchanged.Results also showed expression of P15^INK4B was decreased in KG1a after co-cultured with sEVs derived from KG1a-DAC.（4）Three databases（miRDB,miRWalk,Targetscan）were utilized and predicted that miR-323b-5p binds to P15^INK4B.The miR-323b-5p inhibitor was transiently transfected into KG1a and then co-cultured with KG1a-DAC.Expression of P15^INK4B in KG1a was detected by western blot.Results indicated that miR-323b-5p bind to P15^INK4B.（5）The miR-323b-5p inhibitor was transiently transfected into KG1a and then co-cultured with KG1a-DAC.Cell viability of KG1a treated with decitabine were then detected by CCK8.Results proved that miR-323b-5p inhibitor blocks the sensitivity of KG1a to decitabine induced by KG1a-DAC.In summary,this paper found that KG1a-DAC can delivered sEVs containing miR-323b-5p to inhibit expression of P15^INK4B in KG1a and reduced the sensitivity of KG1a to decitabine.