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Objective To investigate the effect of knockdown or overexpression of G6PD on proliferation, growth and migration of human hepatocellular carcinoma cell PLC/PRF/5. Methods Lentivirus-mediated knock-down or overexpression of G6PD was achieved in human hepatocellular carcinoma cell line PLC/PRF/5. RT-PCR and Western blotting assay were used to detect the overexpression or knockdown of G6PD.Cell proliferation and mi-gration curves were recorded by real-time cell analysis system(RTCA),the cell proportion in the DNA replication phase can be directly displayed with EDU experiment,cell growth ability was detected by colony forming assay. Results The doubling time of cells in G6PD knockdown group was longer than that of the control group,and the cell growth rate decreased significantly,the proportion of cells in proliferative phase(43.2%)was lower than that in the control group,but the rates colony formation and migration were significantly decreased(P<0.05,respective-ly),and the migration curves separated apparently.While no significant differences in proliferation,growth and mi-gration of PLC/PRF/5 cells were found between the over-expressed strain and the control group. Conclusion The reduction of G6PD expression in HCC cells inhibits the proliferation and growth of HCC,which may lay a foun-dation for the further study of the pathogenesis and treatment of HCC.
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Objective To explore the potential impact of low concentration of metformin on the morphology and function of mitochondria of HepG2 cells. Methods HepG2 cells in experimental group and control group were treated with or without low concentration of metformin (1mM/L), respectively. The cells were incubated for 12h in the incubator with constant temperature and humidity as well as 1% oxygen. Orange Mitoview was used to stain the mitochondria to detect the effects of the drug on its morphology and quantity. Transmission electron microscope was utilized to observe the effect of metformin on the ultrastructure of mitochondria. The mitochondrial respiratory chain complex I activity in HepG2 cell was detected by Complex I Enzyme Activity Dipstick Assay Kit (DAK). Results Orange Mitoview staining showed that low concentration of metformin had little effect on the morphology and number of mitochondria of cells in experimental group , and the difference between control and experimental group was not statistically significant (P > 0.05). In addition, the result was further determined by transmission electron microscopy. However, DAK analysis showed that complex I activity of cells in experimental group was significantly lower than that in control group. Conclusion Under Hypoxia conditions, low concentration of metformin had no significant effect on the morphology and number of mitochondria of HepG2 cells, but it significantly reduces the activity of mitochondria of HepG2 cells.
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<p><b>OBJECTIVE</b>To investigate the effect of PIK3CA/siRNA chitosan nanoparticle on the invasiveness of gastric carcinoma and the potential value of PIK3CA/siRNA chitosan nanoparticle in suppressing the metastasis of gastric carcinoma.</p><p><b>METHODS</b>Gastric cancer cells were treated with PIK3CA/siRNA nanoparticle (with a diameter of 350 nm), and the efficiency of PIK3CA gene interference was evaluated using Western blotting and real-time PCR. The changes of the invasive capacity of the treated cells was assessed with Transwell assay.</p><p><b>RESULTS</b>PIK3CA/siRNA chitosan nanoparticle efficiently lowered the expression level of PIK3CA and significantly decreased the invasion of BGC823 cells.</p><p><b>CONCLUSION</b>PIK3CA gene interference mediated by PIK3CA/siRNA chitosan nanoparticle can decrease the invasive capacity of gastric cancer cells in vitro.</p>
Subject(s)
Humans , Cell Line, Tumor , Cell Proliferation , Chitosan , Class I Phosphatidylinositol 3-Kinases , Nanoparticles , Phosphatidylinositol 3-Kinases , Genetics , RNA, Small Interfering , Real-Time Polymerase Chain Reaction , Stomach Neoplasms , PathologyABSTRACT
Objective To explore the effect of metformin on hepatoma cells senescence and the underlying mechanism.Methods Cell proliferation,cycle and apoptosis were examined by MTS and flow cytometry assay in response to different concentrations of metformin(0,0.01,0.1,1,10 and 50mmol/L).Senescence-associated β-galactosidase (SA-β-ga1) staining and senescence marker Dec1 protein levels were used to evaluate the effect of metformin on hepatoma cells senescence.In addition,protein expression of p-AMPK,p-ACC and AMPK was detected by Western blot analysis.Results Metformin suppressed proliferation of HepG2 cells in a dose-dependent manner.High concentrations of metformin (10 and 50mmol/L) promoted cell apoptosis,while lower doses of metformin (0.01,0.1 and 1mmol/L) led to enlarged and flatten senescent morphology and increased SA-β-ga1 positive cells.Moreover,cell cycle was blocked in G0/G1 phase and protein levels of senescent marker Dec1,p-AMPK and p-ACC were significantly enhanced,whereas AMPK protein expression was almost unchanged.Conclusion We showed here that high dose of metformin promotes HepG2 cells apoptosis,but low doses of metformin induce cellular senescence,which may be related to the activation of AMPK signaling.These data will provide vital evidence for improving the outcome of comprehensive treatment in HCC patients by driving hepatoma cells to undergo senescence.
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Objective To compare transfection properties of two different fluorescently labeled PIK3CA siR-NA and to screen out PIK3CA siRNA with high transfection efficiency and strong anti-quenching ability.Methods Two different fluorescently labeled PIK3CA siRNA was transfected into gastric cancer cell BGC-823 by Lipofectamine 2000.The distribution and quenching of fluorescence were observed by inverted fluorescence microscope.Image J software was used to analyze their transfection efficiency.Real-time quantitative PCR was performed to detect the effect of different fluorescently labeled PIK3CA siRNA on PIK3CA mRNA expression.Results The transfection efficiency and anti-quenching ability of two different fluorescently labeled PIK3CA siRNA were different under the same transfection conditions.The transfection efficiency showed no significant differences between Cy3 or FAM labeled PIK3CA siRNA and negative control siRNA(P ＞ 0.05),but the transfection efficiency of Cy3 labeled PIK3CA siRNA and negative control siRNA was significantly higher than the FAM labeled (P ＜ 0.05).Inhibitory efficacy of target mRNA expression induced by Cy3 labeled PIK3CA siRNA was significantly higher than that of FAM labeled PIK3CA siRNA.Conclusion Cy3 labeled PIK3CA siRNA could act as a good tracer and provide an important evidence for further construction of Cy3 labeled PIK3CA siRNA nanoparticle.
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Objective To explore the effect of dendritic cells (DC) primed by total RNA extracted from human colon cancer Lovo cell on specific cytotoxicity of cytokine-induced killer cells (CIK) in vitro.Methods Cord blood mononuclear cells extracted by Ficoll density gradient centrifuge were induced into CIK and DC cells separately, and their Immunophenotype was detected by Flow cytometer. Trizol harvested total RNA from colon cancer cell Lovo and the RNAs were loaded to DCs obtained from cord blood as tumor anti gens. Effectors were grouped accordingly as CIK cells co-cultured with DCs transfected with Lovo RNA, CIK cells co- cultured with unloaded DCs and CIK cells. Targets was Lovo cells. In vitro cytotoxicity of CHK cells was extured with DCs loaded with Lovo RNA(76.49%±4.21%), DC + CIK group was lower(53.84% ± 2.15%),and CIK cells group possessed the lowest cytotoxicity(32.20% ± 3.07%), showing statistic significance( P ＜0.05). Conclusion Extraction of total RNA from tumor cells is simple and easy for clinical implementation.Total RNAs acted as antigen to pulse DCs can strengthen the specific cytotoxicity of CIK cells, which will have good prospects for clinical application.