OBJECTIVE To investigate the effect of polyethylene imine glycol （PEI-PEG）/siRNA nanocomposites in the in vitro transfection of human gastric cancer SGC7901 cell lines and the down-regulation of gene expression of t...OBJECTIVE To investigate the effect of polyethylene imine glycol （PEI-PEG）/siRNA nanocomposites in the in vitro transfection of human gastric cancer SGC7901 cell lines and the down-regulation of gene expression of the adherence factor CD44v6. METHODS PEI-PEG/siRNA nanoparticles, in different N/P ratios, were synthesized and transfected into gastric cancer cells. Lipo2000/siRNA was used in the control group. The transfection efficiencies were observed under fluorescence microscope. The cytotoxicity of the nanoparticles was measured using the MTT assay （mononuclear cell direct cytotoxicity assay）, and the down-regulation effect of siRNA on CD44v6 gene was evaluated by Western blot. Based on the different N/P ratios, PEI-PEG/siRNA composites were synthesized and transfected into gastric cancer cells. Lipo2000/siRNA was used in the controls. The transfection efficiency was observed under fluorescence microscope. The cytotoxicity of the nanoparticles was measured using the MTT assay and the down-regulation effect of siRNA on CD44v6 gene was evaluated by Western blot. RESULTS After transfection, the transfection efficiency of the PEI-PEG/siRNA nanocomposites increased incrementally in N/P ratio value. The transfection efficiency improved with an increase in N/P ratio. When the N/P value was 15, fluorescence became more intense in the PEI-PEG/siRNA group than in the Lipo2000/siRNA group. At the same time, cell viability was （80.4 ± 5.6）% in the MTT reduction assay, which was similar to that in the Lipo2000/siRNA group. The results of Western blot analysis showed that the expression level of CD44v6 protein decreased to （59.7 ± 3.0）% after siRNA-CD44v6 was inhibited. CONCLUSION PEI-PEG could effectively form the nanocomposite in combination with siRNA, be transfected into the SGC7901 gastric cancer cell lines and inhibit CD44v6 protein expression. Moreover, as a genetic carrier, PEI-PEG copolymer has greater advantages, including high transfection e. ciency, less cytotoxicity and an easily alterable vector structure.展开更多
Following the publication of Liu et al.(2024),an error was identified in Figure 4B,in which the image representing the lung from the E529G group was inadvertently duplicated with the image of the lung from the WT grou...Following the publication of Liu et al.(2024),an error was identified in Figure 4B,in which the image representing the lung from the E529G group was inadvertently duplicated with the image of the lung from the WT group during figure preparation.展开更多
Objective Acute myeloid leukemia(AML)is a highly heterogeneous disease,and molecular events such as DNMT3A gene mutations are associated with poor prognosis in AML patients.Consequently,there is an urgent need for a n...Objective Acute myeloid leukemia(AML)is a highly heterogeneous disease,and molecular events such as DNMT3A gene mutations are associated with poor prognosis in AML patients.Consequently,there is an urgent need for a novel therapeutic approach for AML.Methods DNMT3A mRNA and protein expression were confirmed in DNMT3A-mutant AML cells via RT-qPCR and Western blotting.Cell proliferation and apoptosis were assessed via CCK-8 and Annexin V/PI staining,respectively.Flow cytometry was used to analyze surface antigens and CD44v6 CAR-T-cell transfection efficiency.CD44v6-directed CAR plasmids were constructed,and lentiviruses were packaged.Methylation-specific PCR was used to evaluate differences in promoter methylation,whereas ELISA was used to measure cytokine secretion.Results In this study,we found that the DNMT3A-mutant group presented significantly increased expression of CD44v6 on the cell surface.Methylation of the CD44 promoter region was lower in the mutant group than in the control group.CD44v6 CAR-T cells exhibited specific cytotoxicity against DNMT3A-mutant AML cells.Furthermore,pretreatment with low concentrations of decitabine significantly enhanced the killing effect of CD44v6 CAR-T cells on DNMT3A-mutant AML cells(P<0.05).Additionally,decitabine treatment upregulated the expression of CD44v6 on the surface of DNMT3A-mutant AML cells(P<0.05).Conclusion CD44v6 is a promising CAR-T-cell therapy target in AML patients with DNMT3A mutations.Notably,treatment with decitabine resulted in increased CD44v6 expression on the cell surface of DNMT3A-mutant AML cells.This increase in CD44v6 expression facilitates improved recognition and targeting by CD44v6 CAR-T cells.展开更多
基金This work was supported by the following funds： National Natural Science Foundation of China （No.30670951） Guangdong Provincial+5 种基金 Natural Science Foundation （No.06021322） Fund of Guangzhou Municipal Scientific Problem-Solving Program （No. 2003 Z 3-E0381） Fund of Guangdong Provincial Scientific Problem-Solving Program （No.2005 B31211002） Guangdong Provincial Government and Ministry of Education Project com- bining project initiation, study and research （No.2009B090300277）.
文摘OBJECTIVE To investigate the effect of polyethylene imine glycol （PEI-PEG）/siRNA nanocomposites in the in vitro transfection of human gastric cancer SGC7901 cell lines and the down-regulation of gene expression of the adherence factor CD44v6. METHODS PEI-PEG/siRNA nanoparticles, in different N/P ratios, were synthesized and transfected into gastric cancer cells. Lipo2000/siRNA was used in the control group. The transfection efficiencies were observed under fluorescence microscope. The cytotoxicity of the nanoparticles was measured using the MTT assay （mononuclear cell direct cytotoxicity assay）, and the down-regulation effect of siRNA on CD44v6 gene was evaluated by Western blot. Based on the different N/P ratios, PEI-PEG/siRNA composites were synthesized and transfected into gastric cancer cells. Lipo2000/siRNA was used in the controls. The transfection efficiency was observed under fluorescence microscope. The cytotoxicity of the nanoparticles was measured using the MTT assay and the down-regulation effect of siRNA on CD44v6 gene was evaluated by Western blot. RESULTS After transfection, the transfection efficiency of the PEI-PEG/siRNA nanocomposites increased incrementally in N/P ratio value. The transfection efficiency improved with an increase in N/P ratio. When the N/P value was 15, fluorescence became more intense in the PEI-PEG/siRNA group than in the Lipo2000/siRNA group. At the same time, cell viability was （80.4 ± 5.6）% in the MTT reduction assay, which was similar to that in the Lipo2000/siRNA group. The results of Western blot analysis showed that the expression level of CD44v6 protein decreased to （59.7 ± 3.0）% after siRNA-CD44v6 was inhibited. CONCLUSION PEI-PEG could effectively form the nanocomposite in combination with siRNA, be transfected into the SGC7901 gastric cancer cell lines and inhibit CD44v6 protein expression. Moreover, as a genetic carrier, PEI-PEG copolymer has greater advantages, including high transfection e. ciency, less cytotoxicity and an easily alterable vector structure.
文摘Following the publication of Liu et al.(2024),an error was identified in Figure 4B,in which the image representing the lung from the E529G group was inadvertently duplicated with the image of the lung from the WT group during figure preparation.
基金supported by the National Natural Science Foundation of China(No.82270177)Chen Xiaoping Foundation for the Development of Science and Technology of Hubei Province(CXPJJH122001-2221).
文摘Objective Acute myeloid leukemia(AML)is a highly heterogeneous disease,and molecular events such as DNMT3A gene mutations are associated with poor prognosis in AML patients.Consequently,there is an urgent need for a novel therapeutic approach for AML.Methods DNMT3A mRNA and protein expression were confirmed in DNMT3A-mutant AML cells via RT-qPCR and Western blotting.Cell proliferation and apoptosis were assessed via CCK-8 and Annexin V/PI staining,respectively.Flow cytometry was used to analyze surface antigens and CD44v6 CAR-T-cell transfection efficiency.CD44v6-directed CAR plasmids were constructed,and lentiviruses were packaged.Methylation-specific PCR was used to evaluate differences in promoter methylation,whereas ELISA was used to measure cytokine secretion.Results In this study,we found that the DNMT3A-mutant group presented significantly increased expression of CD44v6 on the cell surface.Methylation of the CD44 promoter region was lower in the mutant group than in the control group.CD44v6 CAR-T cells exhibited specific cytotoxicity against DNMT3A-mutant AML cells.Furthermore,pretreatment with low concentrations of decitabine significantly enhanced the killing effect of CD44v6 CAR-T cells on DNMT3A-mutant AML cells(P<0.05).Additionally,decitabine treatment upregulated the expression of CD44v6 on the surface of DNMT3A-mutant AML cells(P<0.05).Conclusion CD44v6 is a promising CAR-T-cell therapy target in AML patients with DNMT3A mutations.Notably,treatment with decitabine resulted in increased CD44v6 expression on the cell surface of DNMT3A-mutant AML cells.This increase in CD44v6 expression facilitates improved recognition and targeting by CD44v6 CAR-T cells.
