Study Of Gene Expression Profile Of K562Cells With Imatinib-resistance After EphB4Gene Silence

Backgroud:Chronic myeloid leukemia (chronic myeloid leukemia, CML) is a malignant clonal proliferative disease that arises from the hematopoietic stem cells (HSC), accounting for15%of adult leukemias. However, disease progression is inevitablewhen there is no treatment intervention.At present, treatments like hydroxyurea, interferon, tyrosine kinase inhibitor (TKI) such as imatinib (IM)have achieved relatively good efficacy through intervention of disease progression. But primary or secondary resistance is still a treatment obstacle formany CML patients, especially patients at accelerated phase(AP) or blast crisis(BC). Recently IM resistance rates was reported as high as24%[1], which has become a major problem for the clinicians. Study about IM resistance mechanisms has currently become a hot spot, which mainly focus on BCR/ABL gene amplificationor overexpression,point mutations of BCR/ABL gene, and BCR/ABL independent mechanismsthathas attracted more and more attention. In addition tosome BCR/ABL independent mechanisms related to a variety of compensatory signal cascadelike excessive Scr family kinase activation[2], the expression of multidrug resistance gene[3],the hematopoietic microenvironment has also been thought to contribute to IM resistance,which is rarely reported in current literatures. Therefore, it is necessary to thoroughly explore the molecular changes and the abnormal pathways involved in drug-resistance process in order to providing laboratory basis for new therapeutic targets and reversal of drug-resistance.Eph receptors whose ligandEphrin express at bone marrow stromal cells comprise the largest family ofmembrane receptor tyrosine kinases, whichexpress in hematopoietic stem and progenitor cells. Eph receptors are divided into A and Bsubtypes whose correspondence ligand EphrinA and EphrinB are also expressed on cell membranes. Tyrosine phosphorylation of both Eph receptors and Ephrin ligands simultaneously happens at cell surface when receptor and ligand binds to each other, which induces bi-directional signal transductions and plays a key role in maintaining bone marrow homeostasis.Recently, Studies about the role of Eph receptors in tumors has become a research hotspot, their interaction with the stromal cellsof the tumor microenvironment, leading to increased resistance of cells, playsimportant roles incell adhesionmediated drug-resistance[4]. Eph receptors are highly expressed in a varietyof tumor cells, which couldpromote tumor development and drug-resistance[5],the detailed mechanism of which is largely unknown. HoweverEph receptors have been taken as therapeutic target by some scholars in order to achieve suppression oftumor neovascularization andgrowth[6]Among other Eph receptors, EphB4has been studied for more details, which could affect angiogenesis, cell migration, axon guidance and bone homeostasis by inducing some relative signal cascades. And it has been demonstrated to be related with tumorigenesis and progression of a variety of tumors such as breast cancer, liver cancer, gastric cancer, prostate cancer, lung cancer, bladder cancer and ovarian cancer [4-7]. Furthermore, it could also plays in tumors with dual roles of both oncogene and tumor suppressor, for most studies suggest that high expression of EphB4promotes tumor cell proliferation, migration, and down-regulation of its expression could inhibit tumor growth, enhance its sensitivity to chemotherapydrugs and promote apoptosis of tumor cells;Whereas in some other tumor contexts, EphB4plays the role of tumor suppressor gene [8]. Themechanism for such contradiction is still largely unknown, which may be related to different tumor contexts and signaling pathways.Studies about EphB4in hematological malignancies have rarely been reported yet. Momoko Suzuki et.al reported thatEphB4was related to drug-resistance of acute lymphoblastic leukemia (ALL) withPh chromosome [12].According to existing reports about EphB4, we speculate that EphB4may be closely related to the development of CML and its drug-resistance.K562cell line, as a model ofCML,holds not only the characteristics ofacute leukemia cells, but also the unique nature of CML, whichnow serves as preferred cell model for studies of mechanisms about drug resistance.Our previous study has found that K562-resistant cells (K562-R) bears higher EphB4expression compared to wild-type K562cells and downregulation of EphB4with specificsiRNAscould enhancethe sensitivity of K562-R cells to imatinib, the involved mechanisms of which are not clear.In this study, we detected key molecular changes after EphB4gene silencing by gene chip technology, which has the advantages of high throughput, rapid, sensitive, and accurate detection, allowing for the simultaneous determination of tens of thousands of genes expression level and relative mutual relations. Then functions of these genes,theirmutual relationshipsand related signaling pathways wereanalyzed through modern biological information technique in order to further explore relative mechanisms aboutEphB4mediated drug resistance.Objective:K562cell line resistant to imatinib(K562-R), and K562-R cells withstable low expression of EphB4(K562-R-shRNA-EphB4) established in our previous works were chosen as research models. Differentially expressed genes in two cell lines were determined by using Affymetrix microarray expression profiling in order to analyzerelative genes functions and construct the regulation networksof signal pathways. Then real time fluorescent quantitative PCR was performed for further validation of selected genes expression based on microarray results.Methods:1、Determination of stability of K562-R-shRNA-EphB4cells were determined by positive rate of GFPmore than50%, K562-R-shRNA-EphB4cells and K562-R cells were cultivated by the conventional method.2、Cells at logarithmic growth phase were lysed byTrizolfor extraction of total RNA. The qualifiedtotal RNA were used for chip test, and the qualified remaining RNA were stored in-80refrigerator.3、Based ona total of5.5ug RNA as templates, the first chain of cDN A containing T7promoterwas formed by reverse transcription byoligo (dT)primers with T7. Then the first chain cDNA as a template induces the second strand DNA synthesis and final formation of double-stranded DNA. After purification of this double-stranded DNA, biotin labeled cRNA was synthesizedbased on this double-stranded DNA templates. Thenstability and fragmentation of cRNA were constructed after its purification. Hybridization was performed at GeneChip Human Gene1.0ST Array human genome microarray in the Affymetrix chip detection system context. Hybridizationlasted for16h at45℃followed by elution and staining, and results were got by microarrayscanning.4、Gene expression difference in two cell lines defined as the value of gene expression difference (foldchange) reaching2times were analyzed. And GO analysis of different gene expression and construction of relative signal pathway network were performed by softwares like DAVID and pSTIING.5、Based on the microarray results,differently expressed genes and important gene candidates involved in relative signal pathways were selected. Then primersfor candidate genes and reference gene like GAPDH were designed by GeneBankand then synthesizedfor further experiments.6、cDNAwas synthesized by reverse transcription of the total RNA. Differently expressed genes were chosen forreal time fluorescence quantitative PCR detection in order to further validate the microarray results. Specificity of the PCR products was determined by melting curve analysis. And the mRNA gene relative expression amount was analyzed. Delta CT=(gene CT values minus internal control gene CT value), and then statistical analysis were performed after exchange of CT value into2-Act.7、Statistical analysis:SPSS13.0software was used for quantitative PCR data analysis.Thevariance homogeneity of the mRNA expression level value of Vavl, Racl, NF-kB, MAP3K7IP2, PPP1R12Agenes in two cell lines was determined, and the data value was descripted as mean±standarddeviation.Two independent samples t-test were used for comparison of two sets of measurement data withP<0.05defined a statistically significant difference.Results:1、Cell line with stable low expression of EphB4was determined by detection of K562-R-shRNA-EphB4cells with positive rate of GFP more than80%.2、A total of760differentially expressed genes were detected by Microarray screening, among which641genes with their demonstrated names. After GO ontology functional analysis of differentially expressed genes performed byPANTHER database,641genes were classified into11types, includingBindingmoleculars (33.3%), catalytic activitymoleculars (25.7%), transcription regulatorymoleculars(10.5%), receptormoleculars (9%).3、Interference of EphB4expression inducedupregulation of243genes and downregulation of398genes. Further analysis of differentially expressed genes mediated biological processes using DAVID software showed that upregulated genes involved in processes like cell adhesion, immune response, negative regulation of apoptosis, positive regulation of macromolecule metabolism, negative regulation of programmed cell death, and positive regulation of cell differentiation.,anddownregulated genes involved in processes like protein transport, angiogenesis, cell morphology, enzyme-linked receptor protein signaling pathway, and receptor tyrosine kinase mediated signaling pathways.4、KEGG pathway analysis:Signaling pathway analysis related to all differentially expressed genes by using the DAVID online software and KEGG database showed that differentially expressed genes involved in signaling pathways including hematopoietic cells generation signaling pathways, the ECM receptor interaction signaling pathways, focal adhesion plaque adhesion signaling pathways, cancer signaling pathways, cytokine receptor interaction mediated signaling pathways, and cytoskeletal regulation signaling pathways.5、Network mapping of all differentially expressed genes at mRNA regulatory level was constructed by pSTIING Software, which showed40genes were at the location of the network nodes and EphB4in the location of the network-centric that could regulate expression ofa variety of genes like PPP1R12A, VAV1, Rac1APC, the KIT is, PSMB9, MAP3K7IP2, and NFκB1genes.6、Based on results of bioinformatics analysis, part of the differentially expressed genes were selectedfor real-time quantitative PCR analysis, the results of which suggested that Vav1, and NF-kB and MAP3K7IP2PPP1R12A in the knockout strains showed lower expression compared with that in K562-R, and the differences were statistically significant (t values were-47.419,-5.707,-2.924,-11.442respectively, P value were<0.001,0.005,0.043,<0.001respectively). Rac1expression showed no significant difference between two cell lines(t=-1.887, P=0.134).All datas from real-time PCR are consistent with the microarray results.Conclusion:1、Gene chip is a powerful tool fordetermination of gene expression with the capacity of high-throughput and fast detection.2、Silence of EphB4expressionbyRNA interference in K562-R cells leaded tochanges of many genesexpression and signaling pathways involved in cell apoptosis, adhesion,proliferation, metabolism, as well as focal adhesion, cytoskeletal regulation and tumorigenesis.3、Network mappingshowed EphB4was at the centerof the map, suggesting that EphB4mediated drug-resistance may be related with genes like PPP1R12A, VAV1, Rac1, APC, KIT, PSMB9, MAP3K7IP2, and NFκB1.4、Since KEGG analysis showed both PPP1R2A and VAV1are involved in signaling pathway related to local adhesion and cytoskeleton regulation, we hyothesized that EphB4probably regulates cell adhesion through regulating activities of PP1R2A and VAV1, which contributes to cell adhesion mediated IM resistance of Chronic Myeloid Leukemia.5、Fluorescent quantitative PCR showed differentially gene expression was consistent withthe microarray results, suggesting microarraytechnique a reliable and accurate tool.