Effects Of RPS27a On Proliferation And Apoptosis Of Chronic Myeloid Leukemia Cells The Synergistic Action Between IDH1^R132H Mutation Protein And AML1-ETO Fusion Protein

Objective:To explore the function of RPS27a in leukemia cells and find new targets for leukemia targeting therapy.Methods:(1) Real-time quantitative PCR (qRT-PCR) was applied to detect the mRNA level of RPS27a and Uba52in bone marrow mononuclear cells (BMMNCs) of15healthy controls and26patients with chronic myeloid leukemia (CML) including16chronic-phase CML and10advanced-phase CML. At the same time, the mRNA level of RPS27a in BMMNCs of12CML patients obtained complete cytogenetic response (CCyR) and20patients with acute leukemia (AL) was detected. qRT-PCR and Western blot were applied to detect the mRNA and protein level of RPS27a in Nalm6, NB4, HL60, K562, U937, Kasumi-1, SKNO-1and K562/G01cells.(2) RPS27a in K562and imatinib-resistant K562/G01cells was knockdown byshRNA interference, the stable cell clones were established, and efficiency of RPS27a knockdown in mRNA and protein level was confirmed by qRT-PCR and Western blot. The vitality of RPS27a knockdown K562and K562/G01cells was assessed by MTT assay. Cell cycle analysis was performed with a FACS Calibur flow cytometer. After K562-shl, K562-sh2, K562-scr, K562/G01-sh1, K562/G01-sh2and K562/G01-scr cells were incubated with different concentration of imatinib (0μM,0.5μM,1μM,2μM,4μM and8μM), the proliferation of cells was detected by MTT assay, and IC50of the cells to imatinib at48h and72h were calculated accordingly. After K562-shl, K562-sh2and K562-scr cells were incubated with0.5μM and1μM imatinib, and K562/G01-shl, K562/G01-sh2and K562/G01-scr cells incubated with2μM and4μM imatinib for24h and48h, respectively, cell apoptosis was analyzed with Annexin V-Alexa Fluor647-A/PI staining by a FACS Calibur flow cytometer. At the same time, cleaved-PARP, PARP, cleaved-caspase3and caspase3protein levels in above imatinib treated cells was detected by Western blot assay.(3) To elucidate the mechanism of RPS27a in K562and K562/G01cells, some key molecules (p-ERK, ERK, p-AKT, AKT, P21, BCL-2, BCL-XL and Bax) involved in proliferation, cell cycle and apoptosis pathways were investigated by Western blot assay in K562-sh1/sh2, K562-scr, K562/G01-shl/sh2and K562/G01-scr cells, respectively.(4) The IC50s of K562-shl, K562-sh2and K562-scr to histone deaceylase inhibitors suberoylanilide hydroxamic acid (SAHA) and cell cycle dependent chemtherapy drug VP16were detected by MTT assay. And the percent of apoptotic cells induced by different level of SAHA and VP16was analyzed with Annexin V-Alexa Fluor647-A/PI Apoptosis Analysis Kit by a FACS Calibur flow cytometer.Results:(1) qRT-PCR revealed a striking increase of RPS27a mRNA expression in bone marrow samples from CML-AP/BP and newly diagnosed AL patients than that from CML-CP and healthy donors (P<0.01). And the RPS27a mRNA level in CML patients obtained CCyR was reduced to the normal level (Compared with healthy control, P>0.05). While there is no difference in the mRNA level of Uba52between CML patients and healthy donors (P>0.05). In addition, RPS27a expression in all detected leukemia cell lines at an approximate level. Interestingly, it was observed that the expression level of RPS27a was high in K562cells and even higher in K562/G01cells (P<0.01).(2) Further analysis revealed that RPS27a knockdown by shRNA in both K562and K562G01cells inhibited the cell viability, induced cell cycle arrest at S and G2/M phases and increased cell apoptosis induced by imatinib (P<0.01). Combination of shRNA with imatinib treatment could lead to more cleaved PARP and cleaved caspase-3expression in RPS27a knockdown cells (P<0.01).(3) Further, it was found that phospho-ERK(p-ERK) and BCL-2were down-regulated and P21up-regulated in RPS27a knockdown cells (P<0.01).(4) Compared with K562-scr cells, the IC50s of K562-shl and K562-sh2to SAHA and VP16at24h and48h decreased (P<0.01). RPS27a slience significantly increased the percentage of apoptotic K562-shl and K562-sh2cells after incubation with1μM,2μM and5μM SAHA for24h and48h, and with2μM and5μM VP16for48h and72h compared with that of K562-scr cells (P<0.01).Conclusions:RPS27a promotes proliferation, regulates cell cycle progression and inhibits apoptosis of leukemia cells. It appears that drugs targeting RPS27a combining with tyrosine kinase inhibitor (TKI) may represent a novel therapy strategy in TKI resistant CML patients. Drugs targeting RPS27a combining with chemotherapy drugs may contribute to the outcome of leukemia treatment. Cell proliferation and differentiation are strictly regulated by intrinsic and extrinsic signals. Uncontrolled proliferation and impaired differentiation of hematopoietic cells lead to leukemogenesis. According to the "two hit"’hypothesis, most acute leukemias are the consequence of a collaboration of several types of mutations. The t(8;21)(q22; q22) translocation resulted in AML1-ETO fusion protein has been reported in acute myeloid leukemia (AML) subtype M2. IDH1mutation was firstly found in Glioma and then in AML and other tumors. In previous study, we found there were12%of AML1-ETO positive AML patients with IDH1mutation, and IDH1mut patients may have worse disease-free survival (DFS) than that of IDH1wild-type patients. It was supposed that AML1-ETO and IDH1mut might collaborate in leukemogenesis. There might be synergistic action between AML1-ETO and IDH1mut.Objective:To investigate whether there is synergistic action between AML1-ETO fusion protein and IDH1R132H mutation protein.Methods:pMSCV-IDH1R132H-cFlag-IRES-GFP (MH)、pMSCV-AML1/ETO-IRES-GFP (MA)、pMSCV-IDH1R132H-2A-AML1/ETO-IRES-GFP (M2A) and pMSCV-IDH1R132H-IRES-AML1/ETO-IRES-GFP (MIA) were constructed correctly via DNA recombination. pMSCV-IRES-GFP(MV), MA、MH. MIA and M2A were transduced into32D cells with the retroviral vector. The quantitative real time PCR and Western blot were applied to detected the mRNA and protein level of AMLl-ETO and IDH1R132H. The vitality of32D-MIA cells and32D-M2A cells incubated with different concentrations of IL-3was analyzed with MTT assay. The effect of IDH1R132H and AML1-ETO on the colony formation of32D cells was studied using methylcellulose semi-solid medium. Apoptosis of32D-MIA cells and32D-M2A cells incubated with different concentrations of IL-3was detected with Annexin V-Alexa Fluor647-A/PI Apoptosis Analysis Kit by a FACS Calibur flow cytometer.Results:There was no statistically significant difference in the vitality, colony formation and cell apoptosis induced by IL-3withdrawl between32D-MIA/M2A cells and that of any control group.Conclusions:There is no synergistic action between IDH1R132H mutation protein and AML1/ETO fusion protein. The IDH1R132H and AML1/ETO co-expression can not promote cell proliferation and inhibit apoptosis of32D cells.