Effects Of Nuclephomin Gene Inhibited By RNA Interference On Biologic Characteristics Of Leukemic Cells

Leukemia is a heterogenicity disease, caused by clone proliferation of hematopoietic stem cell. The main treatment measure of leukemia is chemotherapy targeting leukemic cells, but there are some inevitable problem, such as relapse, metastasis, drug-resistance and so on. Targeting treatment of leukemia at molecular level is the present research hotspot.Neclephomin (NPM) is a multifunctional protein, mainly located in the granular zone of nucleolus. NPM takes part in lots of crucial molecule event in the control of cell proliferation, including ribosome biogenesis and centrosome duplication. Meanwhile, NPM can control apoptosis through regulating interferon regulatory factors(IRF-1). Abnormal expression of NPM gene could induce cell malignant proliferation and cell apoptosis, which is close correlated with tumorigenesis. Overexpression of NPM gene was found in many kinds of solid tumor. NPM was uesed as a tumor marker of malignant tumor, such as hepatocellular carcinoma. Kondo et al reported that NPM was expressed highly in leukemic cells. However the molecule mechanisms of NPM involved in the development of leukemia is not clear yet. RNA interference(RNAi) is a new gene therapy technology, which can effectively and specifically inhibit gene expression. Silence of some genes or fusion genes in leukemia can inhibit cell proliferation, induce cell apoptosis and enhance drug sensitivity to chemotherapeutic agents. Therefore, RNAi can be used as a new approach for leukemia gene therapy.To investigate the effects of NPM inhibition on proliferation and apoptosis of leukemic cells. K562 cells are chosen as research object and RNA interference are applied to suppress the exepression of NPM gene.Main results and conclusions as follow:1. Detecting the mRNA and protein expressions of NPM in leukemic cellsThe results of RT-PCR revealed that NPM mRNA was overexpressed in K562 cells. Further, FQ-PCR was applied to detect the quantity of NPM mRNA. The results showed the copies of NPM mRNA in K562 cells was 7.3 times higher than in normal blood cells. The results of immunohistochemistry confirmed that leukemic cells highly expressed NPM protein.2. Inhibiting the expression of NPM gene in leukemic cellsFirstly, pGenesil-1/NPM vector was constructed successfully and transfected into K562 cells by Lipofectamin 2000. Then, the expression of NPM mRNA in transfected cells was detected by RT-PCR and FQ-PCR. The protein expression of NPM was detected by immunohistochemistry. NPM expression was inhibited by 70%, and no marked difference was observed in control group.3. Observing the effects of NPM inhibition on biologic characteristics of leukemic cellsThe proliferation ability of transfected K562 cells are tested by MTT, colony formation assay and flow cytometry. Those results revealed the ability of cell growth and proliferation potentiality were markedly decreased, and most of leukemic cells were arrested in G1 phase. Furthermore, the activities of caspase-3 and caspase-8 were detected by spectrophotometry, and the increase of two protease activities was observed in transfected cells. It suggest that NPM inhibition could promote leukemic cells apoptosis through the Fas/FasL pathway.