Effects Of Lentivirus Mediated ShRNA Downregulates STMN1 Gene On Human Myeloid Leukemia Cells K562 And Drug-resistant Cells

Stathmin is a ubiquitous, high conserved phosphoprotein,19kDa also known as oncoprotein 18, p18, p19, metablastin, pp20. It is encoded by the human STMN1 gene located at chromosome 1p36.1. Stathmin is a microtubule-destabilizing protein by interacting with tubulin that regulate the dynamics of microtubule, spindle formation and cell cycle. It plays a critical role in cell mitosis, differentiation, apoptosis and motility. Dysfunction of Stathmin could lead to constant microtubule assembly and uncontrolled cell cycle, which then result in continuous abnormal cell growth-tumor formation. High levels of expression of Stathmin has been observed in a wide variety of human cancers. Stathmin is important in tumorigenesis and in keeping tumor cells malignant phenotype.The newly developed technology of RNA interference may be an effective way to study gene function. It is a kind of endogenous gene-silencing mechanism. Inhibition of oncogene expression in malignant cells could induce cell apoptosis, restraint proliferation and metastasis.In our study, altered protein expressions between K562 and adriamycin-resistant K562/A02 cells were identified by 2D-DIGE. Differentially expressed proteins were analyzed by MALDI-TOF/TOF, and protein database searching. The expression of Stathmin was up-regulated in K562/A02 cells.Secondly, the differentially expressed proteins were verified by Western blotting assay and real-time fluorescent quantitative PCR. We detected the expression of Stathmin in the normal control group and the clinical classification was confirmed by the MIC primary, remission and relapse patients. Meanwhile, real-time fluorescent quantitative PCR was used to analysis STMN1 gene expression, in order to discuss the relationship between Stathmin and leukemia.Finally, RNA interference helps downregulat the expression of STMN1 gene on human myeloid leukemia cells, K562 and its drug-resistant cells, and investigated the role of Stathmin in K562 and its drug-resistant cells proliferation, apoptosis and drug-resistance.Proteomics analysis on K562 and its resistant cell line K562/A02To compare the protein expression profiles between human myeloid leukemia K562 cells and adriamycin-resistant K562/A02 cells and to explore novel proteins related to resistance in myeloid leukemia.Methods:Total celluar proteins extracted from K562 and adriamycin-resistant K562/A02 cells were separated by two dimensional differences in gel electrophoresis. Differentially expressed proteins were analyzed by MALDI-TOF/TOF, and protein database searching. The differentially expressed proteins were verified by Western blot assay and real-time fluorescent quantitative PCR.Results:Eight significantly different protein expression spots were screened, among which two protein spots were identified to be down-regulated and six up-regulated in adriamycin-resistant K562/A02 cells. The identified proteins were metabolic enzymes, transcription proteins, and proteins related with signal transduction, celluar proliferation, apoptosis. The results of Western blot were similar to those of 2D-PAGE. Real-time fluorescent quantitative PCR analysis showed that there was no correlation between the protein expression changes and mRNA levels of Stathmin.Research on the expression of Stathmin in acute leukemiaTo investigate the expressin of Stathmin protein and mRNA in acute leukemia and its significance in order to provide a new biological marker for diagnosis of acute leukemia and minimal residual leukemia.Methods:Peripheral blood/bone marrow samples were collected from seventy-six cases of acute leukemia patients and were examined by real-time fluorescent quantitative PCR and Western blot (including 25 cases of normal). Results:The expression of Stathmin protein and mRNA in acute leukemia primary was higher than that of normal(P<0.05), while Stathmin was not expressed in normal population and mRNA was expressed in a low level. The level was expressed even higher in the case of recurrence. But there was not significant difference between acute myeloblastic leukemia and acute lymphocyte leukemia(P> 0.05).Downregulation of STMN1 gene on K562 and its resistant cell lines by RNA interferenceKnocked down STMN1 gene on K562 and its resistant cell lines by RNA interference, then detected the inhibitory efficiency.Method:Constructed the eukaryotic expression vectors. Then K562 and its resistant cell lines were transfected with lentivirus mediated shRNA. And detected the inhibitory efficiency on K562 and its resistant cell lines by Western blot and quantitative PCR.Results:Constructed the eukaryotic expression vectors successfully. Lentivirus mediated shRNA could knock down the transcription of STMN1 gene on K562 and its resistant cell lines.Effects of lentivirus mediated shRNA on downregulating Stathmin on human myeloid leukemia cells K562 and drug-resistant cells.To study the RNA interference in inhibiting STMN1 gene on the proliferation and apoptosis of human myeloid leukemia cells K562 and drug-resistant cells.Method:1. MTT and clonogenic assays were used to access the cell growth inhibition.2. Flow cytometry was used to detect the changes of cell cycle.3. Annexin V-FITC/PI double staining analysis was used to access apoptosis.4. Caspase 9, PARP, bax, p53, P-gp and GSTлwere detected by Western blot.5.Fluorescent quantitative PCR was used to detect mdr-1 mRNA.6. MTT was used to access the resistance of K562/G01 and K562/A02 cells by inhibiting the Stathmin expression.Results:1. Inhibition of Stathmin expression obviously suppressed K562, K562/G01 and K562/A02 cells proliferation and markedly induced them to apoptosis. 2. Inhibition of Stathmin expression could lead to cells G2/M phase blocking.3. Inhibition of Stathmin expression could down-regulate the mdr-1 mRNA and MDR protein expression on K562/G01 cell and K562/A02 cell.5. Inhibition of Stathmin expression could partialy reverse the resistance of K562/G01 and K562/A02 cells by decreased of MDR expression.Conclusion:1.2D-DIGE is a useful approach to investigate different protein expressions related to resistance. Stathmin may be involved in drug resistance and could provide a novel clue for the resistant mechanisms in leukemia.2. Stathmin was overexpressed in acute leukemia, and may provide a new biological marker for the diagnosis of acute leukemia and minimal residual leukemia.3. RNA interference could knock down the transcription of STMN1 gene on K562 and its resistant cell lines. At the same time, transfected cells proliferation was suppressed and apoptosis was increased. And it could partialy reverse resistance of K562/G01 and K562/A02 cells by decrease of MDR expression.