| Chemotherapy is the major therapy method in leukemia.Multi-drug resistance (MDR) and disease relapse are challenging clinical problems in the treatment of leukemia.Relapsed disease is frequently refractory to chemotherapy and exhibits multiple drug resistance.Multiple drug resistance usaully caused by the expression of gene MDR1(multi-drug resistantce protein 1 gene). MDR1 is activated during emerging resistance to anti-neoplastic drugs,resulting in over-expression of P-glycoprotein (P-gp) which is a 170–190 kDa transmembrane glycoprotein that belongs to the ATP-binding cassette superfamily and acts as a multi-drug transporter to expel agents.MicroRNAs (miRNAs) are a family of non-coding 17- to 24-nucleotide RNA duplexes that post-transcriptionally regulate gene expression by inhibiting mRNA translation or by mRNA degradation. miRNAs function in the regulation of a range of physiological responses including stem cell differentiation, haematopoiesis,cardiac and skeletal muscle development and the immune response.MiRNA also can act as oncogene and tumor supressor. However,the mechanism invovled in miRNA in leukemia multi-drug resistance and relapse remains unclear. Therefore,it is important to identify the mechanism by which cancer cells develop resistance and leukemia relapse.In this study,we successfully established the chronic myeloid leukemia resistance cell line by concentration gradient streadily increasing method and one-step drug selection,then we used miRNA microarray and qRT-PCR approaches to investigate the expression of miRNAs in three leukemia cell lines with different degrees of resistance to doxorubicin (DOX) compared with their parent cell line, K562. We found that the expresion of miR-214﹑miR-188-3p and miR-20a was upregulated and miR-338-5p﹑miR-455-3p﹑miR-15a﹑miR-27a and miR-331-5p were downregulated.Further qRT-PCR validated that the expression of miR-331-5p and miR-27a was inversely correlated with the expression of a drug resistant factor, P-glycoprotein (P-gp), in leukemia cell lines with gradually increasing resistance. The development of drug resistance is regulated by the expression of the P-gp. Transfection of the K562 and HL60 DOX-resistant cells with miR-331-5p and miR-27a, separately or in combination,resulted in the increased sensitivity of cells to DOX,suggesting that correction of altered expression of miRNAs may be used for therapeutic strategies to overcome leukemia cell resistance.In addition, we also studied the miRNAs associated with drug resistance in clinical samples. Importantly,miR-331-5p and miR-27a were expressed at lower levels in a panel of relapse patients compared with primary patients at diagnosis, further illustrating that leukemia relapse might be a consequence of deregulation of miR-331-5p and miR-27a.Furthermore,we carried out a genome-wide microRNA (miRNA) microarray analysis to determine the miRNA expression profiles and relapse-specific miRNA patterns in a panel of matched diagnosis-relapse or complete remission childhood ALL samples.we found miR-27a and miR-223 were downregulated in relapse samples and miR-223 could predict ALL relapse as a biomarker, patients with high miR-223 expression at diagnosis had a higher RFS rate (87.6%)compared with patients in the low expressional group, which had 63.3% of RFS.We experimantalily determined the target gene of miR-27a and miR-223 and found that Bmi-1 could be another target of miR-27a and E2F1 directly regulated by miR-223 in leukemia cells.Our studies suggest that miR-27a and miR-331-5p invovled in the new mechanism of leukemia multidrug resistance.We report the miRNA is associated with leukemia relapse and miR-223 can be a predictor of relapse at the first time. The results here provide important insight into the biology of relapsed childhood ALL. These studyies may be useful for further studies of MDR prediction in patients and might provide a strong rationale for the development of miRNA-based therapeutic strategies aiming to overcome drug-resistant and relapse leukaemia. |
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