IFIT2 Repress The Proliferation Of Chronic Myeloid Leukemia Cells K562 By Down-regulating BCR/ABL1

Objective:Although Tyrosine kinase inhibitor(TKI)is the first-line drug for chronic myeloid leukemia(CML)treatment,with the long-term use of clinical TKI drugs,more and more clinical drug resistance often occurs.Interferon alpha(IFN-alpha)is a third-generation drug for the treatment of CML.Its clinical efficacy has not been completely replaced by TKI drugs.According to the literature,the vast majority of patients with chronic phase of chronic granules receive a longer response after treatment with IFN-α and TKI.More interesting studies have shown that the combination of IFN-can accelerate the effectiveness of TKI drugs.So far,the molecular mechanism of IFN-α treatment of CML has not been comprehensively and systematically understood.Three cases of newly diagnosed CML patients and three control bone marrow samples were collected.After treatment with Trizol,RNA was extracted and the RNA quality was determined.The same amount of m RNA was taken for mixed CML specimens and iron deficiency control group.Perform RNA sequencing analysis.By analyzing the sequencing data,we found that the interferon-related signaling pathway was significantly inhibited in BCR-ABL1-positive patients,and interferon-induced expression genes such as interferon-induced tetrapeptide-repeat protein(IFIT)family members IFIT1,IFIT2,and IFIT3 were significantly down-regulated..Among them,IFIT2 has the effect of inhibiting the development of cancer in multiple solid tumors and hematological tumors,and is the most widely studied gene among the three.Our team published an article indicating that up-regulation of IFIT2 expression in human acute promyelocytic cell line(U937)inhibits cell growth with a G2/M phase.To explore the mechanism of action that IFIT2 may play in CML.The concrete research is divided into two parts: 1)Overexpression of IFIT2 in cell growth and cycle may cause changes;2)Exploring whether IFIT2 affects BCR-ABL1 signaling pathway to inhibit leukemia tumor cell proliferation.Method:1)RT-PCR was used to detect the level of IFIT2 transcription in bone marrow cells of 24 CML patients and 16 patients with iron deficiency.At the same time,6CML patients were tested for 3 months and 6 months after taking imatinib,and the bone marrow and clinical symptoms of chronic myeloid leukemia were improved,and the transcription levels change of IFIT2 and BCR-ABL1 in bone marrow cells was tested.2)human CML cell line K562 was selected as the research object.A K562 cell stably expressing the IFIT2 gene was constructed.Intracellular IFIT2 expression was detected by immunofluorescence and Western blotting.The expression of IFIT2 was overexpressed and the cell growth was observed.Cell cycle changes were detected by flow cytometry.3)We detected the change of BCR-ABL1 protein and PI3K-AKT-m TOR signaling pathway,change of cyclin P27 and its related proteins by Western blotting.Results:1)RT-PCR results showed that patients with iron deficiency anemia expressed IFIT2 significantly higher than slow-particle patients.After imatinib administration,the transcription levels of IFIT2 and BCR-ABL1 increased and decreased with the time of administration.2)In the exogenous overexpressing IFIT2 cells,the cell count results showed that the growth rate was significantly slowed down,and the cell cycle arrest was found in the G1 phase by flow cytometry;3)When IFIT2 was overexpressed,western blotting showed that BCR-ABL1 was significantly down-regulated.At the same time,the PI3K-AKT-m TOR signaling pathway has also been suppressed.c-Jun and p-c-Jun(ser63)were down-regulated,which is the cell cycle G1 phase regulatory proteins.IFIT2 promotes the accumulation of the cyclic negative regulatory protein p27 by degrading the scaffold protein cullin1 in the E3 ubiquitin ligase complex.Conclusion:1)In the BCR-ABL1-positive CML patients,the transcription level of IFIT2 in myeloid leukemia cells was significantly lower than that in iron deficiency.2)the effect of imatinib in the treatment of CML,down-regulation of BCR-ABL1 and up-regulation of the IFIT2 gene.3)Overexpression of IFIT2 can down-regulate c-Jun and up-regulate P27 cyclin to arrest cells in G1 phase,thereby inhibiting the proliferation of K562 cells.4)IFIT2 can inhibit the activity of BCR/ABL1-PI3K-AKT-m TOR signaling pathway to achieve the negative phase regulation of K562 cell cycle.