The Effects Of Tyrosine Kinase Inhibitors On Ferritinophagy In Chronic Myeloid Leukemia And The Research On Related Mechanisms

Objective:To explore the effects of TKI on ferritinophagy in chronic myeloid leukemia.Methods:1.Vitro experiment1.1 Cell culture: chronic myeloid leukemia cell lines K562 and KCL22 were cultured in vitro and logarithmic growth phase cells were performed for subsequent experiments.1.2 CCK-8 assay was used to detect K562 and KCL22 cell activity.1.3 Western blot and real-time quantitative PCR were used to detect the ferritinophagy related factors FTH1,NCOA4’s mRNA and protein expression.1.4 The iron content detection kit was used to detect the content of iron ions in each group.2.Vivo experiment2.1 A total of 9 patients with chronic myeloid leukemia who were admitted to our hospital from December 2017 to March 2019 were analysed.General information,blood,BCR-ABL and bone marrow specimens before and after medicine were collected.2.2 Index2.2.1 Beckman AU5800 was used to analyze leukocyte and platelet counts.2.2.2 AB7500 real-time PCR and TaqMan probe were used to analyze BCR-ABL.2.2.3 Western blot was used to detect the ferritinophagy related factors FTH1,NCOA4’s mRNA and protein expression before and after TKI administration in CML patients.Results:1.Vitro experiments results:1.1 The proliferation inhibition rates of K562 and KCL22 cell lines treated with different concentrations of TKI for different time were shown in Fig.1-2 and Table 1-2.With the increase of TKI concentration and the prolongation of treatment time,the proliferation inhibition rates increased gradually(P<0.05).1.2 RT-qPCR and Western blot were used to analyze the ferritinophagy related factors’ mRNA and protein expression in chronic myeloid leukemia cell lines K562 and KCL22.The expression of ferritinophagy related factors’ protein and mRNA in K562 and KCL22 cell lines treated with TKI(0.5,1ug/ml)for 12 h,compared with the control group,the protein and mRNA of ferritinophagy related factors FTH1 and NCOA4 decreased,and the differences are statistically significant(P<0.05).(All the results are shown in Fig.3-6 and Tab 3-6)1.3 After 12 hours of different concentrations of TKI,K562 and KCL22 cell lines were measured and the relative expression of iron in the cells was measured.The results showed that the relative content of intracellular iron in K562 and KCL22 increased with the increase of TKI concentration(P <0.05).2.Vivo experiment results2.1 Compared with before medicine,the white cell count and platelet count were significantly lower in patients after medicine.The BCR-ABL level was significantly reduced in the patients after administration(see Table 7).2.2 Based on the Western Blot analysis of FTH1 and NCOA4 protein levels in patients with chronic myeloid leukemia before and after medicine,compared with before medicine,the expression of ferritinophagy related factors FTH1 and NCOA4 decreased,the difference between the groups was statistically significant(P<0.05).(All the results are shown in Fig 7,and Tab 8).Conclusion:TKI can promote ferritinophagy in CML.