Functional Screen Analysis Reveals MiR-3142 As Central Regulator In Chemoresistance And Proliferation Through Activation Of The PTEN-AKT Pathway In CML

Background and Objective: Micro RNAs(miRNAs)are endogenous,~ 22-nucleotidelength small RNA molecules that negatively regulate the gene expression by directly targeting the 3?-untranslated region(3?-UTR)of m RNAs.MiRNAs regulate the expression of a wide variety of target genes and aberrant expression of miRNAs functions as tumor suppressors or oncogenes according to the role of their target genes.Increasingly,miRNAs are involved in modulating cancer cell behavior,including cell proliferation and apoptosis,cell cycle and differentiation.Dysregulated miRNA expression is a common feature of solid and hematopoietic malignancies.Chronic myeloid leukemia(CML)is a clonal disease of hematopoietic stem cells(HSCs)that occurs as a result of a reciprocal translocation between chromosomes 9 and 22 This rearrangement gives rise to the Philadelphia(Ph)chromosome that,at the molecular level,contains a genomic fusion between the BCR gene from chromosome 22 and the ABL1 gene from chromosome 9.ABL1 encodes the Abelson tyrosine kinase,and its enforced expression from the BCR-ABL1 gene fusion drives the pathogenesis of Ph+ leukemias through phosphorylation of a wide range of substrates that regulate cell proliferation,differentiation,migration,survival,and DNA repair.Doxorubicin is one of the most widely used chemotherapy agents.While its normal tissue toxicity may complicate its therapeutic use,the most serious problem with doxorubicin the development of drug resistance.The mechanisms of drug resistance that relate to prevention of tumor cell death and poor survival in patients include increased rates of drug efflux,DNA damage repair,alterations in the tumor microenvironment and in drugmetabolism,the emergence of cancer stem cells,mutations of drug targets,and cell death inhibition.Nevertheless,miRNA expression in chemoresistant CML is not widely investigated and the mechanisms that underlie aberrant miRNAs expression are not well understood.Methods: 1.First,the K562-resistant ADR cell line K562 / ADR was established by gradually increasing the concentration of ADR from a low concentration.Then,miRNA arrays,which were used to identify miRNAs involved in Adriamycin resistance,were performed for K562 group and K562/ADR group(n=3/group)by Exiqon(Kang Chen,China)using the miRCURY?Hy3?/Hy5? power labelling kit and the miRCURY? LNA Array(v.10.0;757 human miRs).The expression values are log2(Hy3/Hy5)ratios.Unsupervised hierarchical clustering of miRNAs was performed.2.The expression levels of miRNA in cells were identified by real-time quantitative RT-PCR(q RT-PCR).A total of 36 CML patients from the First Affiliated Hospital of Dalian Medical University(Dalian,China)were enrolled in the study.The diagnosis of CML was based on cytomorphology,cytochemistry,multiparameter flow cytometry,immunology,molecular genetics and cytogenetics.Written informed consent was obtained from all of the patients and the study were approved by and the institutional human ethical committee.Peripheral blood mononuclear cells(PBMCs)were isolated from patient blood by Ficoll-Hypaque solution according to manufacturer's instructions(Stem Cell Technologies,Inc.,Vancouver,BC,Canada).The membrane expression of P-gp was studied with flow cytometry.Furthermore,the PBMCs were divided into two groups,CML(without MDR)and CML/MDR(multidrug resistance).Then,the expression levels of miRNA in patient samples were identified by real-time quantitative RT-PCR(q RT-PCR).Based the expression levels of miRNA in cells and patient samples,further confirmed the differential expression of miRNA.3.To investigate the potential targets of miR-3142 several well-developed miRNA algorithms were employed,such as Target Scan,Pic Tar and miRNA.org.A pmir GLO Dual-Luciferase miRNA target Expression Vector was used for 3? UTR Luciferase assays(Promega,Madison,WI,USA).The plasmid p MIR-REPORT-PTEN wt or p MIR-REPORT-PTEN mut was transfected into HEK-293 cells cells together with miR-3142 mimics or the control.Luciferase and Renilla signals were measured 48 h after transfection using a Dual-Luciferase? Reporter Assay Kit(Promega)according to the manufacturer's protocol.Renilla was used as a transfection control.4.The expression levels of PTEN m RNA and miR-3142 in patient samples were identified by real-time quantitative RT-PCR(q RT-PCR).Moreover,the correlation between miR-3142 expression and PTEN expression was analyzed.5.K562 cells were transfected with miR-3142 mimic,negative control(NC),respectively.K562/ADR cells were transfected with Anti3142 or control.Then,the expression levels of protein and m RNA of PTEN were identified by real-time quantitative RT-PCR(q RT-PCR)and Western Blot.6.CCK8 cell cytotoxicity assay,flow cytometry,Western Blot,CCK8 cell proliferation assay and colony formation were used to verify the changes of drug sensitivity,apoptosis,proliferation and colony-forming capacity after ectopic expression of miR-3142 in K562 cells.7.CCK8 cell cytotoxicity assay,flow cytometry,Western Blot,CCK8 cell proliferation assay and colony formation were used to verify the changes of drug sensitivity,apoptosis,proliferation and colony-forming capacity after knockdown of miR-3142 in K562/ADR cells.8.Western Blot was used to examine the influence of miR-3142 on PI3K/Akt signaling. 9.The changes of drug sensitivity,apoptosis,proliferation and colony-forming capacity were examined after miR-3142-transfected K562 cells were treated with Akt sh RNA or PI3 K inhibitor LY294002.10.Western Blot was used to examine the expression of PI3K/Akt signaling related molecules after reexpression of PTEN in miR-3142-transduced cells.11.CCK8 cell cytotoxicity assay,flow cytometry,Western Blot,CCK8 cell proliferation assay and colony formation were used to verify the changes of drug sensitivity,apoptosis,proliferation and colony-forming capacity after silencing PTEN in Anti3142-transduced cells.12.Western Blot was used to examine the expression of PI3K/Akt signaling related molecules after silencing PTEN in Anti3142-transduced cells.13.CCK8 cell cytotoxicity assay,flow cytometry,Western Blot,CCK8 cell proliferation assay and colony formation were used to verify the changes of drug sensitivity,apoptosis,proliferation and colony-forming capacity after silencing PTEN in Anti3142-transduced cells.14.Vivo experiment was applied to confirm the effect of miR-3142 on CML cell chemosensitivity and cell growth.Results: 1.miRNAs were dysregulated in resistant compared with adriamycin(ADR)-sensitive parental cells in CML by miRNA arrays.Among these candidate miRNAs,miR-3142 were expressed at significantly higher levels in ADR-resistant CML cells.The expression levels of miR-3142 were further assessed by real-time quantitative RT-PCR(q RT-PCR).miR-3142 was significantly downregulated in K562 and KU812 cells as compared with K562/ADR,KU812/ ADR cells.Then,the expression levels of miR-3142 were analyzed in 36 CML patients.miR-3142 were significantly downregulated in CML patients compared with that in CML/MDR patients.These data suggested that alterations of miR-3142 may be involved in the chemoresistance of CML.2.By searching the Target Scan,Pic Tar and miRNA.org,we identified PTEN as a theoretical target gene of miR-3142.A dual-luciferase reporting system showed that miR-3142 transfection imposed a reduction in the luciferase activities.The suppressive effects of miR-3142 on luciferase activities were completely deprived by introduction of nucleotide mutations in PTEN-3?-UTR.The m RNA level of PTEN was higher in CML and K562 cells than in CML/MDR and K562/ADR cells.Moreover,there was a strong negative correlation between Log2-transformed miR-3142 expression and Log2-transformed PTEN expression in CML patients.The results revealed that PTEN was a target of miR-3142.3.CCK8 cell cytotoxicity assay,flow cytometry,Western Blot,CCK8 cell proliferation assay,colony formation results showed that transfection of miR-3142 in K562 cells could increase cell viability and reduced cell apoptosis.Furthermore,the proliferation rate and colony-forming capacity of miR-3142 expressing cells were significantly increased.4.CCK8 cell cytotoxicity assay,flow cytometry,Western Blot,CCK8 cell proliferation assay,colony formation results showed that knockdown of miR-3142 in K562/ADR cells could reduce cell viability and increase cell apoptosis.Furthermore,the proliferation rate and colony-forming capacity were significantly decreased.5.Ectopic expression of miR-3142 could lead to activation of PI3K/Akt pathway,whereas knockdown of miR-3142 inhibited the activation of PI3K/Akt pathway.Akt sh RNA/LY294002 abrogated miR-3142-activated Akt and inhibited miR-3142-induced ADR resistance,as indicated by a decrease in cell viability and an increase in apoptosis.In addition,Akt sh RNA/LY294002 abrogated miR-3142-induced enhanced cell proliferation,as indicated by a decrease in colony-forming ability and proliferation rate.6.Compared with the control group,reexpression of PTEN could inhibit cell viability,increase apoptosis rates and reduce cell proliferation of K562 cells induced by miR-3142.7.Silencing PTEN in Anti3142-transduced cells could promote cell viability,suppress apoptosis rates and promote cell proliferation.8.Restoration of PTEN decreased Akt activation induced by miR-3142.Conversely,silencing PTEN in K562/ADR-Anti3142 cells exhibited opposite effects.9.Tumors grow at a slower rate in Anti3142 cells than control cells.Significantly,the combined Anti3142 and ADR treatment markedly restricted the tumor growth to low volumes.In addition,decreases in weights of tumors excised from animals of the Anti3142 group were also observed as compared with those of the control group.These results indicated that downregulation of miR-3142 inhibit tumor growth and enhance ADR sensitivity in vivo.Conclusion: 1.miR-3142 expression was significantly upregulated in K562/ADR cells and CML/MDR patients compared with K562 cell and CML patients.2.Alteration of miR-3142 seemed to be associated with chemoresistance,cell apoptosis,proliferation rate and colony-forming capacity in CML.3.PTEN was a target gene of miR-3142.4.miR-3142 regulated the chemoresistance and proliferation by simultaneously activating PI3K/Akt pathway in CML.5.miR-3142 regulated the chemoresistance and proliferation through activation of the PTEN-PI3K/Akt pathway in CML.