Investigation Of EIF4E Expression And Its Regulation In Chronic Myelogenous Leukemia Cells

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder of the pluripotent hematopoietic stem cell characterized by the presence of a specific chomosomal translocation t(9;22) (q34;q11) resulting in the production of a 210 kD fusion protein termed Bcr-Abl with elevated tyrosine kinase activity which has been shown to be central to the pathogenesis of CML. CML can be divided into three phases-chronic phase (CP), accelerated phase (AP) and blast crisis (BC) according to its development.The molecular mechanisms responsible for transition to CML-BC remain poorly understood, recently, it seems that eukaryotic translation initiation factor-4E(eIF4E), a key translation factor and a promoter of nucleocytoplasmic transport of specific transcripts might play a role during CML development. eIF4E is overexpressed in a variety of malignant cells and tissues, however, the study of eIF4E in CML has mainly focused on its inhibition protein 4E-BP with little attention on its own expression and corresponding regulation mechanism. BCR/ABL increases heterogeneous nuclear ribonucleoprotein K(hnRNP K) expression in a dose- and kinase-dependent manner through MAPKERK1/2 activation,Both BCR/ABL and hnRNP K expression are quite high in CML-BC cells, meanwhile, overexpression of hnRNP K induced malignant phenotype by increasing eIF4E mRNA level.Thus, in this experiment,we first performed an investigation on eIF4E and hnRNP K mRNA expression in CML patients and K562 cells, and their correlation , then STI571, a specific inhibitor of BCR-ABL kinase, was applied to study the possible regulation of BCR-ABL oncoprotein on eIF4E mRNA in K562 cells, at last, gene silencing was taken to know better of the relation between eIF4E and a downstream molecule of BCR/ABL-hnRNP K with hnRNP K shRNA, as well as hnRNP K function in K562 cells, which might helps further investigation of CML-BC transition and potencial molecular therapeutic targets. This study is divided into three parts; the methods and results are as follows:1.RT-PCR was taken to analyze eIF4E mRNA in 25 bone marrow (BM) samples (10CML-BC, 8CML-CP, 1CML-AP, 3 CML-BC-R, 3 anaemia control) as well as hnRNP K mRNA in 9 CML patients for the following analysis of its correlation with eIF4E mRNA. eIF4E mRNA in CML-BC (P<0.05) and CML-BC-R group(P<0.05) but not CML-CP group (P>0.05) are significantly higher than control group. There is positive linear correlation between mRNA expression of hnRNP K and eIF4E gene (r=0.79, P<0.01). 2.RT-PCR was taken to analyze eIF4E mRNA in K562 cells after treatments with 0.0μmol/L, 0.5μmol/L, 1.0μmol/L, 2.5μmol/L, 5.0μmol/L STI571 respectively for 24h. STI571 could slightly down-regulate eIF4E mRNA expression, there are might others pathways participating in its regulation;3.RT-PCR was taken to analyze eIF4E mRNA in K562 cells after hnRNP K shRNA retrovirus infection, hnRNP K shRNA retrovirus was packaged with pSuper retro hnRNP K shRNA in PT67 after its identification with enzyme digestion and sequencing analysis, then FCM, MTT assay, wright staining, RT-PCR were used to analyze its effects on cell cycle and cell apoptosis, cell proliferation, cell morphology, hnRNP K and eIF4E mRNA in K562 cells respectively. The sequence of the inserted fragment in pSuper retro hnRNP K shRNA was correct. Compared with the control K562 cells, hnRNP K shRNA treated group showed significantly more cells at G2/M phase (P<0.05), growth inhibition and more cells with≥2 nucleus (P<0.05) as well as 55.22% and 55.27% decrease of hnRNP K and eIF4E mRNA respectively.Conclusion:1. eIF4E mRNA is significantly higher in CML-BC, there are potisive correlation between eIF4E and hnRNP K mRNA expression in CML; 2. BCR-ABL might paticipate but not very powerful in the regulation of eIF4E mRNA expression, suggesting that there are pathways contributing to its elevation in CML-BC;3. hnRNP K interference blocks K562 cells at G2/M phase, inhibits cell growth, and down-regulates both eIF4E and hnRNP K mRNA expression, increases cells with≥2 nucleus, suggesting that hnRNP K might play a role during cell cycle progression , in cell proliferlation as well as the regulation of eIF4E expression in K562 cells.In a word, CML-BC cells have high expression of eIF4E mRNA, which might be influenced by BCR-ABL and hnRNP K protein, meanwhile, hnRNP K plays a role in the cell cycle progression, cell proliferation, cell division in K562 cells. This investigation helps further understanding of the role of hnRNP K and eIF4E and the corresponding mechanism during CML development.