Ros Of MiR-149-SP1 Axis In Imatinib Resistance Of Chronic Myeloid Leukemia

Background:Chronic myeloid leukemia(CML)is a malignant disease that occurs in the bone marrow hematopoietic system,with a global distribution.The median age of onset in China is younger than that in the West.It is mainly caused by the formation of characteristic BCR-ABL fusion oncogene and BCR-ABL fusion protein with strong tyrosine kinase activity.At present,tyrosine kinase inhibitor TKIs,has achieved very good clinical efficacy,but it also faces some new challenges.Because of the single target of chronic myeloid cell therapy drugs and the formation of drug resistance,chronic myeloid leukemia is once again faced with treatment without drugs.Therefore,it is of great significance to further explore the drug resistance mechanism of TKIs and find new targets.MiR-149 is found to be a tumor suppressor gene in many tumors.This study focuses on the role of miR-149 in chronic myeloid leukemia and its relationship with drug resistance of TKIs.Purpose:This study mainly includes clinical specimens and CML cell lines,and focuses on screening the differential drug resistance-related miRNAs from in vivo and in vitro experiments.After that,further functional experiments in vitro were carried out in CML cell lines,and through the combined application of a variety of techniques,the mechanism of drug resistance of TKIs was studied more deeply from the aspects of gene,protein and so on,and the potential therapeutic effect of miRNAs could become a new therapeutic target for TKIs drug resistance.Method: 1 According to the relevant clinical guidelines,patients with chronic myeloid leukemia were divided by the different responses to IM into drug-resistant and non-drug-resistant patients.Clinical specimens of patients were collected,and then qRT-PCR was carried out,and the data were collated and analyzed.2 Drug-resistant’ cell lines were constructed by IM concentration gradient induction method,and the drug resistance ratios of K562 and K562 G cell lines were detected by CCK8 method.The drug resistance multiple of K562 and K562 G cell lines was calculated by IC50,which laid the foundation for the follow-up experiment.3 In the drug-resistant cell line K562 G,the stable overexpression of miR-149 gene and the corresponding negative control cell lines,miR-149/OE and miR-149/NC,were constructed by lentivirus transfection.Apoptosis microarray was also used to screen differential apoptotic proteins after transfection.Subcellular localization and pathway enrichment of differential proteins were analyzed by GO and KEGG.4 By using RNA interference(RNAi)technique,the cells were divided into K562 and K562 G group,miR-149/OE and miR-149/NC group,miR149 inhibitor and Inhibitor NC group,siRNA SP1 group and siRNA NC group.The function of the cells was analyzed in vitro by real-time quantitative PCR(qRT-PCR),Western blotting(WB)and flow cytometry.5 Apply the principle of statistics to describe and analyze the data,combined with bioinformatics technology to summarize and mine the data.Result: 1 Part of clinical specimen:The expression of miR-149 in CML-resistant group(the best response)was significantly higher than that in Drug-resistant group(treatment failure)and healthy control group(P=0.000 < 0.001).There was a significant negative correlation between the relative expression of miR-149 and the relative expression of BCR-ABL fusion gene in CML patients(correlation coefficient was-0.607,P<0.05).2 Cell experiment part: the IC50 of the successfully constructed IM-resistant cell line K562 G was about 50 times higher than that of IM-sensitive K562 cells.qRT-PCR results showed that the expression of miR-149 in IM-resistant cell line K562 G was significantly lower than that in IM-sensitive cell line K562(P < 0.00001< 0.001).The overexpression of miR-149 and the action of IM drugs can induce apoptosis in synergistic way.3 Apoptosis chip results showed that 22 differential proteins were screened in miR-149/OE and miR-149/NC,and GO and KEGG suggested that these differential proteins were highly enriched in the pathways of apoptosis,necrosis and drug resistance.4 SP1 was predicted to be the target gene of miR-149,and there was a significant negative correlation between the expression of miR-149 and SP1 in CML cell line.Knocking down SP1 can affect the cell cycle,block the cell cycle in G 1phase and decrease the S phase significantly,which is consistent with the effect of overexpression of miR-149 in K562 G.Conclusion: MiR-149-SP1 axis plays an inhibitory role in CML by inducing apoptosis and cell cycle arrest.Combined with IM has a more obvious inhibitory effect on proliferation than either alone,and may become a potential target for CML therapy.The study on the mechanism of drug resistance of CML provides a new idea for clinical treatment.