MicroRNA-mediated Chemosensitivity Regulation Of Human Leukemia Cells To Hydroxyurea

Leukemia is a malignant neoplasm of the blood-forming organs comprising four major types,among which chronic myeloid leukemia（CML）accounts for over 15%of all adult leukemia cases.Currently,tyrosine kinase inhibitors（TKIs）are routinely used as the firstline treatment for CML.Nevertheless,cell cycle-specific chemotherapeutic drug hydroxyurea（HU）,a standard treatment for CML before TKIs,is still valuable for the early control of CML,as well as for the treatment of patients with special conditions（such as women in pregnancy）due to its safety,ready availability,and relatively low cost.Resistance to drugs is a common obstacle during cancer chemotherapy and treating CML with HU is no exception.MicroRNAs（miRNAs）are a class of small non-coding regulatory RNAs with potent functions in the regulation of chemosensitivity of many cancer types.Up till now,there is no comprehensive identification of miRNAs that are responsible for the regulation of chemosensitivity of CML cells to HU.Therefore,uncovering their roles and functional mechanisms will provide valuable information for the effective long-term treatment of CML with HU.ObjectiveIn this study,a strategy of combining high-throughput sequencing screening and bioinformatic analysis for functional predictions was used to identify miRNAs that are responsible for the regulation of the chemosensitivity of human K562 CML cells to HU.Four representative miRNAs with remarkable up-regulation were chosen for further studies of their functions and possible mechanisms on regulating the sensitivity of K562 cells to HU treatment.Methods（1）miRNA libraries of K562 cell treated or not treated with HU were constructed and subjected to high-throughput sequencing and bioinformatic analyses to identify miRNAs that were significantly up-or down-regulated upon HU treatment.（2）Real-time qPCR analyses were performed to verify the differentially expressed miRNAs screened by high-throughput sequencing,and four of the most up-regulated miRNAs were selected for further studies.（3）RNA sequencing data of LAML patients were retrieved from the TCGA database.Single-gene GSEA method was performed to analyze possible regulatory mechanisms of the four miRNAs on leukemia cells.（4）Inducible K562 stable cell lines over-expressing each of the four candidate miRNAs were constructed by using a Tet-on inducible lentiviral expression system.（5）Real-time qPCR analyses were used to detect the expression levels of each of the four candidate miRNAs in the K562 parental cells and the four stable K562 cell lines treated or not treated with HU.（6）The effects of each candidate miRNA on the proliferation of K562 cells and the chemosensitivity of K562 cells to HU were examined by live cell counting.（7）The effects of each candidate miRNA on the cell cycle regulation of K562 cells treated or not treated with HU were examined by PI staining and flow cytometry.（8）The effects of each candidate miRNA on the apoptosis of K562 cells treated or not treated with HU were examined by sub-G1 phase cell ratio analysis,caspase 3 activity assay and western blot of apoptosis marker protein cleaved PARP1.（9）The effects of each candidate miRNA on the regulation of erythroid differentiation of K562 cells induced by HU were examined by O-Tolidine staining and real-time qPCR analyses of the erythroid differentiation-related protein markers.（10）Target Scan software was used to predict the potential targets of each candidate miRNA.High-confidence targets shared by the four miRNAs were selected and the changes of their mRNA levels upon HU treatment and/or miRNA over-expression were examined by real-time qPCR.Results（1）The expression levels of several miRNAs in K562 cells were significantly changed upon HU treatment,among which miR-22,miR-27a,miR-224 and miR-374a were the most significantly up-regulated miRNAs with a change fold of 3.4,3.4,2.1 and 3.5,respectively.（2）Single-gene GSEA analyses suggested that miR-22/-27a might be involved in the regulation of drug response,miR-22/-27a/-374a might be involved in the apoptotic pathway,miR-22/-224/-374a might be involved in heme metabolism,and all four miRNAs might be involved in the regulation of cell cycle related pathways in leukemia cells.（3）Real-time qPCR analyses confirmed that all four candidate miRNAs in K562 cells that were screened by high-throughput sequencing were also significantly up-regulated when treated with IC50 dosage of HU.（4）Real-time qPCR analyses confirmed that all four inducible K562 stable cell lines were able to achieve successful miRNA overexpression upon doxycycline（DOX）treatment.（5）Living cell counting showed that overexpression of miR-22/-27a/-224/-374a could inhibit the proliferation of K562 cells and decrease the sensitivity of K562 cells to HUmediated cytotoxicity.（6）Cell cycle analyses showed that HU treatment could induce S phase arrest in K562 cells,and overexpression of miR-22/-27a/-224/-374a could induce G0/G1 phase arrest in K562 cells.（7）Significant up-regulation of the sub-G1 phase cell proportion,Caspase 3 activity and the expression level of apoptosis marker protein cleaved PARP1 were observed in K562 cells treated with HU,and these changes were reduced to a much lesser extent when overexpressing each of the four candidate miRNAs.（8）O-Tolidine staining showed that HU treatment could induce erythroid differentiation of K562 cells,and overexpression of each of the four candidate miRNAs could further promote HU-mediated erythroid differentiation.ConclusionsBy combining high-throughput sequencing screening and bioinformatic analysis for functional predictions,we have successfully identified miRNAs that may modulate chemosensitivity to HU in human CML cells.Experimental validations have suggested that overexpression of miR-22/-27a/-224/-374a could induce G0/G1 phase arrest in K562 cells,which could help the cells escape the killing effect of HU in S phase.Overexpression of miR-22/-27a/-224/-374a could significantly inhibit HU-mediated apoptosis,including subG1 phase proliferation,up-regulation of Caspase 3 activity and up-regulation of cleaved PARP1 expression.Overexpression of miR-22/-27a/-224/-374a could also promote the erythroid differentiation mediated by HU.These observations suggest dual roles of these miRNAs in leukemogenesis:reducing the sensitivity of K562 cells to HU-mediated cytotoxicity and enhancing the sensitivity of K562 cells to HU-mediated cell differentiation.