ABSTRACT
@#Objective To investigate the effects of long non-coding RNA(LncRNA) growth arrest specific transcript 5(GAS5) negatively regulating nucleophosmin 1(NPM1) on cisplatin(DDP) resistance of gastric cancer cells.Methods The normal human gastric mucosa cell line GES-1 and human gastric cancer cell lines BG3-823,MGC-803 and AGS were selected as the research objects,of which the level of LncRNA GAS5 in each cell was measured by qRT-PCR.The drug resistance of AGS cells to DDP(AGS/DDP) was induced,and the experiment was divided into control group,empty plasmid group(BC group),GAS5 nonsense interference group(pLJM-GAS5 NC group) and GAS5 overexpression group(pLJM-GAS5 group).MTT method was used to determine the effect of DDP on the proliferation of AGS and AGS/DDP cells;and the levels of NPM1,multidrug resistance 1(MDR1),excision repair cross complementation group 1(ERCC1),multidrug resistance-associated protein 1(MRP1) and N-cadherin in AGS and AGS/DDP cells were measured by Western blot.Results Compared with the normal gastric mucosa GES-1 cells,the level of LncRNA GAS5 in BG3-823 and AGS cells decreased significantly,and among them,the level of LncRNA GAS5 in AGS cells was the lowest,so AGS cells were used for the follow-up experiments.Compared with the control group,the level of LncRNA GAS5 in AGS cells of BC group and pLJM-GAS5 NC group decreased significantly,while the levels of NPM1,MDRl,ERCC1,MRP1 and N-cadherin increased significantly;compared with BC group and pLJM-GAS5 NC group,the level of LncRNA GAS5 in AGS/DDP cells of pLJM-GAS5 group increased significantly,while the levels of NPM1,MDR1,ERCC1,MRP1 and N-cadherin decreased significantly;after treatment with DDP of the same concentration(except 0 μmol/L),compared with the control group,the inhibition rate of AGS/DDP cell proliferation in BC group and pLJM-GAS5 NC group decreased significantly;compared with BC group and pLJM-GAS5 NC group,the inhibition rate of AGS/DDP cell proliferation in pLJM-GAS5group was significantly higher.The semi inhibitory concentration(IC_(50)) of DDP on AGS/DDP cells in pLJM-GAS5 group for 48 h was(65.38±5.04) μmol/L,which was significantly lower than(120.74±4.17) μmol/L and(120.24±4.29) μmol/L in BC group and pLJM-GAS5 NC group.Conclusion Up-regulating the level of LncRNA GAS5 in AGS/DDP cells can reverse the drug resistance of AGS/DDP cells,which may be related to the down-regulation of NPM1expression
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Objective To explore the protein acetylation/succinylation and histone 2AX (H2AX) expression levels in breast cancer, as well as their correlation. Methods By Western blotting and RT-PCR methods to detect the protein modification and H2AX expression levels in 11 breast cancer tissues and cells, as well as to explore the common regulation way of protein acetylation and succinylation by treatment of histone deacetylase inhibitors ; To study the relationship between H2AX expression level and protein modification level through the construction and over-expression of indicated plasmids. Results Compared with the adjacent normal tissues, there existed an increase protein acetylation/succinylation levels in breast cancer tissues, and the protein acetylation and succinylation were both regulated by histone deacetylase (HDAC) members. The H2AX mRNA and protein expression levels were increased both in breast cancer cell and tissues, its expression level and the expression and modification level of represented protein nucleophosmin 1(NPM1) showed a positive correlation. Conclusion The breast cancer possesses a characteristic of high protein acetylation/succinylation levels and high H2AX expression level, the H2AX expression level and the modification level of partial proteins in breast cancer have a positive correlation.
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Objective: To investigate the effect of nucleophosmin 1 (NPM1) mutant A on TGF-β1-induced K562 cell proliferation and AKT phosphorylation. Methods: K562 cells were infected with Ad5-NPM1 to create an NPM1 over-expression cell model. NPM1 levels were determined by ELISA and Western blot analysis. The levels of AKT and P-AKT were determined by Western blot. MTT was used to measure the proliferation of K562 cells. Results: NPM1 protein levels in K562 cells increased in an Ad5-NPM1-MOI-dependent manner. Cell proliferation and NPM1 levels in the supernatant were significantly increased in K562 cells infected with Ad5-NPM1-30 and Ad5-NPM1-100 compared to those infected with Ad5-vector-100 (P<0.01). Treatment with (10 ng/mL) TGF-β1 increased P-AKT levels, but not total AKT levels in K562 cells. TGF-β1-induced phosphorylation of AKT was significantly increased by infection of K562 cells with Ad5-NPM1-100. No significant differences were found in total AKT levels among all groups. TGF-β1 (10 ng/mL) treatment also in-creased the proliferation of K562 cells. TGF-β1-induced K562 cell proliferation was significantly increased by infection with Ad5-NPM1-100 (P<0.01). Conclusions: NPM1 improves TGF-β1-induced cell proliferation by up-regulating AKT phosphorylation levels.
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Objective To investigate the clinical characteristics and efficacy of acute myeloid leukemia (AML) with NPM1 and FLT3 mutations.Methods NPM1 and FLT3 mutations were detected in 67 patients with newly diagnosed AML by PCR-capillary electrophoresis.The relationship was analyzed between the mutations and efficacy.Results The incidence of NPM1 mutation was 10.4％ (7/67) in total AML patients and 26.1％ (6/23) in normal karyotypes AML patients.The incidence of FLT3-ITD mutation was 10.4％ (7/67) in total AML patients and 17.4％ (4/23) in normal karyotypes AML patients.The characteristics of 60 NPM1 wild type patients vs that of 7 NPMl mutation patients was as follow,platelet count (BPC) (54× 109/L vs.27.5 × 109/L,P ＜ 0.01),proportion of AML-M5 (57.1％ vs.27.3％,P ＜ 0.01),incidence of CD34+ (28.6％ vs.63.3％,P＜0.01),normal karyotypes (85.7％ vs.28.3％,P＜0.01),cases with particular fusion gene (0 vs.48.3％,P ＜ 0.01),incidence of FLt3-1TD-mutations positive (28.6％ vs.8.3％,P ＜ 0.01),and the differences were significant (P＜0.01).No statistic difference was found in white blood cell(WBC) counts,percentage of blasts in bone marrow,sex,median age and complete remission rate between the two groups (P ＞0.05).The WBC counts (26.9 × 109/L vs.8.1 × 109/L,P =0.013),percentage of blastsin in bone marrow (90％ vs.76％,P=0.014) in the FLT3-ITD mutationg positive patients were clearly higher than those in the FLT3-ITD negative patients.If not associated with FLT3-ITD mutations,mutant NPM1 appears to identify patients with improved response toward treatment.Conclusion It is necessary to detect NPM1 mutation and FLT3-ITD mutation in newly diagnosed AML patients,especially in patients with normal karyotype,which might help to molecular classification and treatment.
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Background: Acute promyelocytic leukemia (APL) with t (15;17) is a distinct category of acute myeloid leukemia (AML) and is reported to show better response to anthracyclin based chemotherapy. A favorable overall prognosis over other subtypes of AML has been reported for APL patients but still about 15% patients relapse. Methods: This study evaluated the presence of Famus like tyrosine kinase‑3 (FLT3) and nucleophosmin‑1 (NPM1) gene mutations in a cohort of 40 APL patients. Bone marrow/peripheral blood samples from patients at the time of diagnosis and follow‑up were processed for immunophenotyping, cytogenetic markers and isolation of DNA and RNA. Samples were screened for the presence of mutations in FLT3 and NPM1 genes using polymerase chain reaction followed by sequencing. Results: Frequency of FLT3/internal tandem duplication and FLT3/tyrosine kinase domain was found to be 25% and 7% respectively. We observed a high frequency of NPM1 mutation (45%) in the present population of APL patients.