| Objective:Chronic myelogenous leukemia(CML)is a malignant tumor resulting from clonal proliferation of bone marrow hematopoietic stem cells,characterized by excessive myeloid proliferation and positive Ph chromosome and/or BCR/ABL fusion genes.The BCR/ABL gene encodes a continuously activated BCR/ABL P210 fusion protein with activated tyrosine kinase activity that alters neutrophil proliferation,apoptosis,and migration characteristics and is a central link in the development of chronic myelogenous leukemia.Tyrosine kinase inhibitors can inhibit BCR/ABL tyrosine kinase at the cellular level,selectively inhibit proliferation and induce apoptosis of BCR/ABL positive cell lines.The advent of tyrosine kinase inhibitors has revolutionized the life of patients with chronic myelogenous leukemia.However,there are still some patients who may need treatment modification due to poor efficacy or disease progression.Interferon alpha was the best alternative to hematopoietic stem cell transplantation before tyrosine kinase inhibitors.It can regulate gene expression,but its exact anti-leukemia effect is not fully understood.In some clinical studies,interferon combined with tyrosine kinase inhibitors has enabled more patients to achieve treatment-free remission.In real-world experience,patients with tyrosine kinase resistance combined with interferon therapy have a certain reversal of drug resistance.The PI3K/AKT signaling pathway is one of the most common signaling pathways regulating tumor growth.It is activated by a variety of receptors.Its inhibitors have been shown to be effective against a variety of tumors.BCR/ABL protein can up-regulate a variety of signaling pathways,the most active of which is PI3K/AKT.PI3K can be divided into three types according to tissue distribution,among which type Ⅰ A is activated by BCR/ABL protein to activate AKT.In patients with tyrosine kinase inhibitor resistance,the PI3K/AKT pathway becomes a new target.Earlier studies have shown that interferon alpha and beta can regulate the downstream PI3K/AKT pathway activity by binding to interferon receptors,thereby affecting cell proliferation.This project aims to prove that interferon can regulate the expression of PI3K/AKT,and reverse the TKI drug resistance of chronic myeloid leukemia cells,and further explore the regulatory effect of interferon on PI3K/AKT signaling pathway in vivo and in vitro.Methods:First,K562 cell line(K562R)was induced slowly with imatinib solution,and the IC50 value of imatinib to K562 cells was measured by CCK-8 kit.Then,K562R cells were treated with imatinib,interferon and imatinib+interferon respectively,the relative expression levels of PI3K,AKT and BCR-ABL mRNA in each group were detected by Realtime PCR,and the protein expression levels of PI3K,AKT and BCR-ABL in each group were detected by Western Blot.CCK-8 assay was performed to detect the proliferation activity of each group of cells,and flow cytometry was performed to detect the apoptosis rate of each group of cells.The survival time and splenomegaly of mice in each group were compared.The infiltration of leukemia cells in the spleen of mice in each group was observed by HE staining,and the expression of target molecules in the spleen of mice in each group was compared by immunohistochemical staining.The expression levels of PI3K,AKT and BCR-ABL mRNA in K562R cells were detected by Realtime PCR,and the expression levels of BCR-ABL,PI3K and AKT protein were detected by Western Blot.CCK-8 assay was performed to detect the proliferation activity of each group of cells,and flow cytometry was performed to detect the apoptosis rate of each group of cells.Finally,the PI3K level of K562R cells was overexpressed by lentivirus,and the relative levels of PI3K,AKT and BCR-ABL in each group of cells were detected by Realtime PCR,and the protein expression levels of BCR-ABL,PI3K and AKT in each group of cells were detected by Western Blot.CCK-8 assay was performed to detect the proliferation activity of each group of cells,and flow cytometry was performed to detect the apoptosis rate of each group of cells.To verify that interferon reverses imatinib resistance in chronic myeloid leukemia by inhibiting PI3K/AKT signaling pathway.Results:The imatinib resistant K562 cell line K562R was successfully induced by imatinib solution.The IC50 value of imatinib was 221.82nM by CCK-8 assay.Compared with NC group,the relative mRNA level of BCR-ABL in imatinib combined with interferon group was significantly decreased,the relative mRNA level of BCR-ABL in interferon group was decreased,and there was no significant difference in the relative mRNA level of BCR-ABL in imatinib group.Compared with the NC group,the relative mRNA level of PI3K in the imatinib combined with interferon group was significantly decreased,the relative mRNA level of PI3K in the interferon group was decreased,and the relative mRNA level of PI3K in the imatinib group was not statistically different.Compared with NC group,the relative mRNA level of AKT in imatinib combined with interferon group was significantly decreased,the relative mRNA level of AKT in interferon group was decreased,and there was no significant difference in the relative mRNA level of AKT in imatinib group.Compared with NC group,the expression level of BCR-ABL protein in imatinib combined with interferon group was significantly decreased,the expression level of BCR-ABL protein in interferon group was decreased,and there was no significant difference in the expression level of BCR-ABL protein in imatinib group.Compared with NC group,the expression level of PI3K protein in imatinib combined with interferon group was significantly decreased,the expression level of PI3K protein in interferon group was decreased,and the expression level of PI3K protein in imatinib group was not statistically different.Compared with NC group,the expression level of AKT protein in imatinib combined with interferon group was significantly decreased,the expression level of AKT protein in interferon group was decreased,and the expression level of AKT protein in imatinib group was not statistically different.Compared with NC group,the proliferation ability of K562R cells in imatinib combined with interferon group was significantly decreased,the proliferation ability of K562R cells in interferon group was decreased,and there was no significant difference in the proliferation ability of K562R cells in imatinib group.Compared with NC group,the apoptosis rate of K562R cells in imatinib combined with interferon group was significantly increased,and the apoptosis rate of K562R cells in interferon group was increased,but there was no significant difference in the apoptosis rate of K562R cells in imatinib group.Compared with NC group,the survival time of mice in imatinib+interferon group was significantly prolonged,the survival time of mice in interferon group was prolonged,and there was no significant difference in the survival time of mice in imatinib group.Compared with the NC group,the size and weight of the spleen in the imatinib+interferon group were significantly decreased,the size and weight of the spleen in the interferon group were decreased,but there was no significant difference in the size and weight of the spleen in the imatinib group.HE staining showed that compared with the NC group,the number of leukemia cells in the spleen of mice in the imatinib+interferon group was significantly reduced,the number of leukemia cells in the spleen of mice in the interferon group was reduced,and the number of leukemia cells in the spleen of mice in the imatinib group was not significantly reduced.Immunohistochemical staining showed that compared with the NC group,the expression level of BCR-ABL protein in the imatinib combined with interferon group was significantly decreased,the expression level of BCR-ABL protein in the interferon group was decreased,and there was no significant difference in the expression level of BCR-ABL protein in the imatinib group.Compared with NC group,the expression level of PI3K molecule in imatinib combined with interferon group was significantly decreased,the expression level of PI3K molecule in interferon group was decreased,and the expression level of PI3K molecule in imatinib group was not statistically different.Compared with NC group,the expression level of AKT molecule in imatinib combined with interferon group was significantly decreased,the expression level of AKT molecule in interferon group was decreased,and the expression level of AKT molecule in imatinib group was not statistically different.Then shRNA was used to knock down the PI3K level of K562R cells.Compared with the shNC group,the relative mRNA levels of PI3K,AKT and BCR-ABL in the shPI3K group were significantly decreased.Compared with the shNC group,the expression levels of PI3K,AKT and BCR-ABL proteins in the shPI3K group were significantly decreased.Compared with the shNC group,the proliferation activity of K562R cells in the shPI3K group was significantly decreased.Compared with the shNC group,the apoptosis rate of K562R cells in the shPI3K group was significantly increased.Finally,lentivirus was used to over-express PI3K level in K562R cells.Compared with the Vector+saline group,the relative level of PI3K mRNA in the PI3K+saline group was significantly increased,the relative level of PI3K mRNA in the Vector+interferon group was significantly decreased,and there was no significant difference in the relative level of PI3K mRNA in the PI3K+interferon group.Compared with the Vector+saline group,the relative level of AKT mRNA in the PI3K+saline group was significantly increased,the relative level of AKT mRNA in the Vector+interferon group was significantly decreased,and there was no significant difference in the relative level of AKT mRNA in the PI3K+interferon group.Compared with the Vector+saline group,the relative level of BCR-ABL mRNA in the PI3K+saline group was significantly increased,the relative level of BCR-ABL mRNA in the Vector+interferon group was significantly decreased,and there was no significant difference in the relative level of BCR-ABL mRNA in the PI3K+interferon group.Compared with the Vector+saline group,the expression level of PI3K protein in the PI3K+saline group was significantly increased,the expression level of PI3K protein in the Vector+interferon group was significantly decreased,and there was no significant difference in the expression level of PI3K protein in the PI3K+interferon group.Compared with the Vector+saline group,the AKT protein expression level in the PI3K+saline group was significantly increased,the AKT protein expression level in the Vector+interferon group was significantly decreased,and the AKT protein expression level in the PI3K+interferon group was not significantly different.Compared with the Vector+saline group,the expression level of BCR-ABL protein in the PI3K+saline group was significantly increased,the expression level of BCR-ABL protein in the Vector+interferon group was significantly decreased,and the expression level of BCR-ABL protein in the PI3K+interferon group was not significantly different.Compared with the Vector+saline group,the cell proliferation activity of the PI3K+saline group was significantly increased,the cell proliferation activity of the Vector+interferon group was significantly decreased,and there was no significant difference in the cell proliferation activity of the PI3K+interferon group.Compared with the Vector+saline group,the apoptosis rate of the PI3K+saline group was significantly decreased,the apoptosis rate of the Vector+interferon group was significantly increased,and there was no significant difference in the apoptosis rate of the PI3K+interferon group.Conclusions:Interferon can reverse the drug resistance of imatinib in CML by inhibiting the PI3K/AKT signaling pathway. |
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