MiR-221 Sensitizes Chronic Myeloid Leukemia To Imatinib By Targeting STAT5

Background and purposeChronic myeloid leukemia(CML)is a myeloproliferative disorder characterized by the Philadelphia chromosome and the BCR–ABL fusion gene.BCR–ABL exhibits high tyrosine kinase activity and activates different signaling pathways,such as the MAPK,STAT5,and PI3K/Akt pathways,and ultimately promotes cell proliferation and inhibits apoptosis in CML cells.Recently,the wide use of tyrosine kinase inhibitors(TKIs)has significantly improved the prognosis and the life expectancy of CML patients,approximately 15–20% of patients develop drug intolerance or resistance.Drug-resistance remains a major obstacle towards achieving treatment-free remission.Previous reports have revealed numerous factors that may lead to imatinib resistance,including mutations in the ABL kinase domain(KD),BCR–ABL overexpression and amplification,and clonal evolution.which seriously affects the treatment effect of CML patients.Hence,it is urgent to look for new therapeutic targets to conquer TKI resistance.In recent years,mi RNAs have been widely considered as key players in numerous types of cancer progression and chemoresistance,including CML.Dysregulated mi R-221 expression has been implicated in CML progression.Ihle et al have reported that mi RNA-221 can induce cells apoptosis via the KIT/AKT pathway in gastrointestinal stromal tumors.And Dentelli et al have found that mi R-221 can regulate breast cancer cell proliferation by target STAT5.However,the underlying mechanisms whereby mi R-221 regulates chemoresistance,and crosstalk between mi R-221 and the STAT5 pathway in CML are still not completely understood.Based on the above research background,we detect the expression of mi R-221 and STAT5 in K562 cells and IM-resistant K562/G cells.And we investigate the key roles of mi R-221 and STAT5,as well the crosstalk of them in IM resistance of CML,both in vitro and in vivo,based on samples from CML patients,IM-sensitive and IM resistant CML cell models.We can understand the IM resistance mechanism of CML and explore the potential roles of mi R-221-STAT5 axis and mi R-221 in CML deeply,provide scientific data for the new therapeutic targets of CML.Methods(1)K562/G cells(IM-resistant K562 cells)were established by the concentration gradient method.(2)Cell transfection was performed using the Lipofectamine 2000 reagent(Invitrogen,Carlsbad,CA,USA)according to the manufacturer's protocols.The transfection efficiencies of mi RNA and RNA interference were detected by quantitative real-time,reverse transcriptase polymerase chain reaction(q RT-PCR)and western blot experiments at 48 h or 72 h post-transfection.(3)qRT-PCR was performed to detect the mi RNA and m RNA expression levels.U6 small nuclear RNA(sn RNA)and GAPDH m RNA levels were used for normalization purposes.Western blot was used to detect the expression levels of target proteins,and?-actin expression was measured to normalize the expression levels of target proteins.(4)An apoptosis antibody array(Ray Biotech,Inc.,Norcross,GA)was used to explore the effects of mi R-221 overexpression on the apoptotic protein profile.An Ma Pix Microarray Scanner was used to scan the fluorescent signals(Molecular Devices,Sunnyvale,CA,USA),and AAH-APO-G1 analysis software was used for the data analysis.(5)To study cell proliferation,cell viability was measured using the CCK-8Kit.Apoptosis was assessed using the Annexin V/PI Double Staining Kit(Beyotime Biotech,Shanghai,China)and analysis by flow cytometry(FCM).(6)For mi R-221 target validation,two luciferase reporter vectors(PGL3-STAT5B-3'UTR-wt and PGL3-STAT5B-3 ' UTR-mut)and mi R-221 mimics or mi R-NC were cotransfected into K562/G cells.After 48 h,the Firefly and Renilla luciferase activities were analyzed.(7)Thirty-five CML samples were collected from CML patients in chronic phase who received chemotherapy.The thirty-one normal controls were collected from healthy individuals.Mi R-221 expression was confirmed by q RT-PCR analysis.Results(1)The results of qRT-PCR confirmed that mi R-221 was significantly downregulated in IM-resistant CML cells and patients with treatment failure when compare with IM-sensitive CML cells(P<0.001)and CML patients with optimal responses to IM(P<0.001).(2)Upregulation of mi R-221 significantly increased the sensitivity of K562/G cells to IM.Mi R-221 upregulation significantly inhibited K562/G cell proliferation(P<0.5).Mi R-221 overexpression obviously increased apoptosis in untreated K562/G cells(P<0.01)and in K562/G cells pretreated with IM(P<0.001).(3)The effects of mi R-221 overexpression on the apoptotic protein profile were analyzed by We also found obvious differences in the expression levels of 27apoptosis-related proteins.The increased pro-apoptotic protein Caspase3(P<0.01),Bax(P<0.5),and the decreased Bcl-2(P<0.5)were observed in mi R-221 overexpression group.Similarly,western blot analysis showed that the levels of Bax,Caspase3,and C-PARP were increased,while Bcl-2 was decreased when mi R-221 was overexpressed.(4)STAT5B was identified as a target of mi R-221.The two luciferase reporter system results revealed that expression from the STAT5B-wt luciferase reporter activity was reduced by the addition of mi R-221 mimics.However,the luciferase activity of PGL3-STAT5B-3'UTR-mut was not significantly different when co-transfected with mi R-221 mimics or mi R-NC(P<0.01).(5)Mi R-221 induced CML cell apoptosis and suppressed cell proliferation by targeting STAT5 B.The data demonstrated that suppression of STAT5 B increased the percentage of cells undergoing apoptosis,compared to si RNA-NC-transfected cells,with or without IM treatment(P<0.5).We found that Bax expression increased,whereas Bcl-2expression decreased in STAT5 B si RNA transfected cells compared with si RNA-NC-transfected cells(P<0.5).Similarly,STAT5 B knockdown significantly inhibited cell growth(P<0.5).ConclusionTaken together,these data indicate that mi R-221 might act as a key regulator of CML chemosensitivity.Our present study can provide a scientific basis for a new therapeutic target,namely the mi R-221–STAT5 axis,which may be important in potential target applications and in the upstream effects on the ABL KD mutation.However,the detailed molecular mechanism of mi R-221 in CML is still uncertain and should be explored further in future research.