RNA干扰技术靶向P13K/AKT信号通路抵制胃腺癌SGC7901细胞侵袭转移的研究

Objective To observe the inhibitory effects on the invasion of gastric adenocarcinoma SGC7901 cells by small hairpin RNA targeting PIK3R1 and AKT1 in vitro. Methods The recombinant adenovirus vector plasmid expression vector which contained PIK3R1 and AKT1 shRNA was transfected into SGC7901 cells. Real time PCR and Western blot were used to detect the expression of P1K3R1 and AKT1. ELESA was used to measure the change in the MMP-2 and MMP-9 expression. The invasion ability of the tumor ceils was examined by Scarification, Transwell and 3-dimensional matrigel matrix tests. Re-sults rAdS-A-P mediated shRNA targeting PIK3R1 ,and AKT1 dramatically down-regulated their expres-sion in SGC7901 cells. MMP-2 and MMP-9 were downregulated,and TIMP-2 was upregulated. The extra-cellular levels of MMP-2 and MMP-9 were decreased. Scarification test indicated that the invision ability was decreased obviously. Transwell showed that the number of cells invading through the matrigel in con-trol. nonsense sequence, and rAd5-A-P transfection groups was 105.0±4.0,102.5±6.4, and 67.0±3.9 respectively. In the rAdS-A-P transfection group, cells formed only small aggregates as compared with the control and nonsense sequence groups in 3-dimensional matrigel matrix growth test. Conclusion ShRNA targeting PIK3R1 ,and AKTI downregulated significantly their expression in a sequence-specific manner, and inhibited the invasion of gastric adenocarcinoma SGC7901 cells in vitro.     

RNA干扰技术靶向P13K/AKT信号通路抵制胃腺癌SGC7901细胞侵袭转移的研究

Objective To observe the inhibitory effects on the invasion of gastric adenocarcinoma SGC7901 cells by small hairpin RNA targeting PIK3R1 and AKT1 in vitro. Methods The recombinant adenovirus vector plasmid expression vector which contained PIK3R1 and AKT1 shRNA was transfected into SGC7901 cells. Real time PCR and Western blot were used to detect the expression of P1K3R1 and AKT1. ELESA was used to measure the change in the MMP-2 and MMP-9 expression. The invasion ability of the tumor ceils was examined by Scarification, Transwell and 3-dimensional matrigel matrix tests. Re-sults rAdS-A-P mediated shRNA targeting PIK3R1 ,and AKT1 dramatically down-regulated their expres-sion in SGC7901 cells. MMP-2 and MMP-9 were downregulated,and TIMP-2 was upregulated. The extra-cellular levels of MMP-2 and MMP-9 were decreased. Scarification test indicated that the invision ability was decreased obviously. Transwell showed that the number of cells invading through the matrigel in con-trol. nonsense sequence, and rAd5-A-P transfection groups was 105.0±4.0,102.5±6.4, and 67.0±3.9 respectively. In the rAdS-A-P transfection group, cells formed only small aggregates as compared with the control and nonsense sequence groups in 3-dimensional matrigel matrix growth test. Conclusion ShRNA targeting PIK3R1 ,and AKTI downregulated significantly their expression in a sequence-specific manner, and inhibited the invasion of gastric adenocarcinoma SGC7901 cells in vitro.     

RNA干扰技术靶向P13K/AKT信号通路抵制胃腺癌SGC7901细胞侵袭转移的研究

Objective To observe the inhibitory effects on the invasion of gastric adenocarcinoma SGC7901 cells by small hairpin RNA targeting PIK3R1 and AKT1 in vitro. Methods The recombinant adenovirus vector plasmid expression vector which contained PIK3R1 and AKT1 shRNA was transfected into SGC7901 cells. Real time PCR and Western blot were used to detect the expression of P1K3R1 and AKT1. ELESA was used to measure the change in the MMP-2 and MMP-9 expression. The invasion ability of the tumor ceils was examined by Scarification, Transwell and 3-dimensional matrigel matrix tests. Re-sults rAdS-A-P mediated shRNA targeting PIK3R1 ,and AKT1 dramatically down-regulated their expres-sion in SGC7901 cells. MMP-2 and MMP-9 were downregulated,and TIMP-2 was upregulated. The extra-cellular levels of MMP-2 and MMP-9 were decreased. Scarification test indicated that the invision ability was decreased obviously. Transwell showed that the number of cells invading through the matrigel in con-trol. nonsense sequence, and rAd5-A-P transfection groups was 105.0±4.0,102.5±6.4, and 67.0±3.9 respectively. In the rAdS-A-P transfection group, cells formed only small aggregates as compared with the control and nonsense sequence groups in 3-dimensional matrigel matrix growth test. Conclusion ShRNA targeting PIK3R1 ,and AKTI downregulated significantly their expression in a sequence-specific manner, and inhibited the invasion of gastric adenocarcinoma SGC7901 cells in vitro.     

RhoA基因沉默对肝癌HepG2细胞增殖和迁移能力的影响

Objective To construct a RhoA-siRNA expression vector and determine its role on the malig-nant behavior of HepG2 cells.Methods A RhoA-siRNA DNA fragment was synthesized and cloned into the expression vector of pGenesil-1.The constructed Rhon-siRNA DNA plasmid was stably transfected into HerG2 cells by lipofectamine,and then HepG2 cells were divided into the HepG2/RhoA-siRNA group (HepG2 cells were transfected with pGenesil-1-RhoA-siRNA),HepG2/control group(HepG2 cells were transfected with control plasmid) and HepG2 group (without plasmid transfection).The inbibitory effect of RhoA-siRNA on RhoA protein expression was shown by Western blot.The proliferation,migration,growth potentiality and cell cycle of transfected HepG2 cells were evaluated by MTT assay,wounded healing,the plate cloning formation test and flow cytometry,respectively.All data were analyzed by one-way analysis of variance (ANOVA) and chi-square test.Results The expression of RhoA protein in the HepG2/RhoA-siRNA group was,significantly decreased compared with that in the other two groups (F=178.19,P<0.05).Scratched cells were healed within 48 hours in the HepG2/control group and HepG2 group,but not in the HepG2/RhoA-siRNA group.The clone formation rates in the HepG2/RhoA-siRNA group,HepG2 group and HepG2/control group were 39%±3%,67%±5%and 70%±6%,respectively,with a significant difference among the three groups(χ2=33.34,38.69,P<0.05).Flow cytometry showed that the number of cells transfected with RhoA-siRNA was highest in the G0/G1 phase and lowest in the S phase(F=70.46,76.57.P<0.05).Conclusion The RhoA-siRNA expression vector can effectively suppress the proliferation and migration of HepG2 cells,which may provide a novel gene therapy for hepatocellular carcinoma.     

RhoA基因沉默对肝癌HepG2细胞增殖和迁移能力的影响

Objective To construct a RhoA-siRNA expression vector and determine its role on the malig-nant behavior of HepG2 cells.Methods A RhoA-siRNA DNA fragment was synthesized and cloned into the expression vector of pGenesil-1.The constructed Rhon-siRNA DNA plasmid was stably transfected into HerG2 cells by lipofectamine,and then HepG2 cells were divided into the HepG2/RhoA-siRNA group (HepG2 cells were transfected with pGenesil-1-RhoA-siRNA),HepG2/control group(HepG2 cells were transfected with control plasmid) and HepG2 group (without plasmid transfection).The inbibitory effect of RhoA-siRNA on RhoA protein expression was shown by Western blot.The proliferation,migration,growth potentiality and cell cycle of transfected HepG2 cells were evaluated by MTT assay,wounded healing,the plate cloning formation test and flow cytometry,respectively.All data were analyzed by one-way analysis of variance (ANOVA) and chi-square test.Results The expression of RhoA protein in the HepG2/RhoA-siRNA group was,significantly decreased compared with that in the other two groups (F=178.19,P<0.05).Scratched cells were healed within 48 hours in the HepG2/control group and HepG2 group,but not in the HepG2/RhoA-siRNA group.The clone formation rates in the HepG2/RhoA-siRNA group,HepG2 group and HepG2/control group were 39%±3%,67%±5%and 70%±6%,respectively,with a significant difference among the three groups(χ2=33.34,38.69,P<0.05).Flow cytometry showed that the number of cells transfected with RhoA-siRNA was highest in the G0/G1 phase and lowest in the S phase(F=70.46,76.57.P<0.05).Conclusion The RhoA-siRNA expression vector can effectively suppress the proliferation and migration of HepG2 cells,which may provide a novel gene therapy for hepatocellular carcinoma.     

HCCR-2反义核酸对肝癌HepG2细胞的作用

Objective To investigate the effects of antisense recombinant euraryotic expression vector of HCCR-2 on the proliferation and apoptosis of HepG2. Methods The antisense recombinant eukaryotic expression vector of HCCR-2 was constructed. The vector was stably transfected to the HepG2 cells, and positive clones were selected by G418 (antiseuse vector group), pIRES2-EGFP vector was transfected into the HepG2 cells in the same way (pIRES2-EGFP group). The conditions of the nontransfected HepG2 cells were used as control (HepG2 group). Changes in cell growth curve, cell cycle, cell apoptosis and morphology of HepG2 cells after the transfec-tion were detected by MTT method, flow cytometry and transmission electron microscopy, respectively. All the data were analyzed by one-way ANOVA and chi-square test. Results The expression level of HCCR-2 mRNA was down-regulated to 0.39±0.04 in antisense vector group, and the expression level of HCCR-2 mRNA in pIRES2-EGFP group and HepG2 group were 0.62±0.06 and 0.72±0.03, respectively, with significant difference among the 3 groups (F=43.701, P<0.05). The apoptotic rate of HepG2 cells in antisense vector group, pIRES2-EGFP grop and HepG2 group were 13.30%, 2.51% and 2.07%, respectively, with significant difference among the 3 group (χ2=6.793, 8.721, P<0.05). The growth of HepG2 cells in antisense vector group was retarded, and was blocked in G0/G1 stage. Conclusions The HCCR-2 antisense recombinant eukaryotic expression vector can inhibit the mRNA expression of HCCR-2 and promote the apoptosis of cells. HCCR-2 may be involved in cell regulation and the proliferation of hepatocellular carcinoma cells.     

HCCR-2反义核酸对肝癌HepG2细胞的作用

Objective To investigate the effects of antisense recombinant euraryotic expression vector of HCCR-2 on the proliferation and apoptosis of HepG2. Methods The antisense recombinant eukaryotic expression vector of HCCR-2 was constructed. The vector was stably transfected to the HepG2 cells, and positive clones were selected by G418 (antiseuse vector group), pIRES2-EGFP vector was transfected into the HepG2 cells in the same way (pIRES2-EGFP group). The conditions of the nontransfected HepG2 cells were used as control (HepG2 group). Changes in cell growth curve, cell cycle, cell apoptosis and morphology of HepG2 cells after the transfec-tion were detected by MTT method, flow cytometry and transmission electron microscopy, respectively. All the data were analyzed by one-way ANOVA and chi-square test. Results The expression level of HCCR-2 mRNA was down-regulated to 0.39±0.04 in antisense vector group, and the expression level of HCCR-2 mRNA in pIRES2-EGFP group and HepG2 group were 0.62±0.06 and 0.72±0.03, respectively, with significant difference among the 3 groups (F=43.701, P<0.05). The apoptotic rate of HepG2 cells in antisense vector group, pIRES2-EGFP grop and HepG2 group were 13.30%, 2.51% and 2.07%, respectively, with significant difference among the 3 group (χ2=6.793, 8.721, P<0.05). The growth of HepG2 cells in antisense vector group was retarded, and was blocked in G0/G1 stage. Conclusions The HCCR-2 antisense recombinant eukaryotic expression vector can inhibit the mRNA expression of HCCR-2 and promote the apoptosis of cells. HCCR-2 may be involved in cell regulation and the proliferation of hepatocellular carcinoma cells.     

HCCR-2反义核酸对肝癌HepG2细胞的作用

Objective To investigate the effects of antisense recombinant euraryotic expression vector of HCCR-2 on the proliferation and apoptosis of HepG2. Methods The antisense recombinant eukaryotic expression vector of HCCR-2 was constructed. The vector was stably transfected to the HepG2 cells, and positive clones were selected by G418 (antiseuse vector group), pIRES2-EGFP vector was transfected into the HepG2 cells in the same way (pIRES2-EGFP group). The conditions of the nontransfected HepG2 cells were used as control (HepG2 group). Changes in cell growth curve, cell cycle, cell apoptosis and morphology of HepG2 cells after the transfec-tion were detected by MTT method, flow cytometry and transmission electron microscopy, respectively. All the data were analyzed by one-way ANOVA and chi-square test. Results The expression level of HCCR-2 mRNA was down-regulated to 0.39±0.04 in antisense vector group, and the expression level of HCCR-2 mRNA in pIRES2-EGFP group and HepG2 group were 0.62±0.06 and 0.72±0.03, respectively, with significant difference among the 3 groups (F=43.701, P<0.05). The apoptotic rate of HepG2 cells in antisense vector group, pIRES2-EGFP grop and HepG2 group were 13.30%, 2.51% and 2.07%, respectively, with significant difference among the 3 group (χ2=6.793, 8.721, P<0.05). The growth of HepG2 cells in antisense vector group was retarded, and was blocked in G0/G1 stage. Conclusions The HCCR-2 antisense recombinant eukaryotic expression vector can inhibit the mRNA expression of HCCR-2 and promote the apoptosis of cells. HCCR-2 may be involved in cell regulation and the proliferation of hepatocellular carcinoma cells.     

HCCR-2反义核酸对肝癌HepG2细胞的作用

Objective To investigate the effects of antisense recombinant euraryotic expression vector of HCCR-2 on the proliferation and apoptosis of HepG2. Methods The antisense recombinant eukaryotic expression vector of HCCR-2 was constructed. The vector was stably transfected to the HepG2 cells, and positive clones were selected by G418 (antiseuse vector group), pIRES2-EGFP vector was transfected into the HepG2 cells in the same way (pIRES2-EGFP group). The conditions of the nontransfected HepG2 cells were used as control (HepG2 group). Changes in cell growth curve, cell cycle, cell apoptosis and morphology of HepG2 cells after the transfec-tion were detected by MTT method, flow cytometry and transmission electron microscopy, respectively. All the data were analyzed by one-way ANOVA and chi-square test. Results The expression level of HCCR-2 mRNA was down-regulated to 0.39±0.04 in antisense vector group, and the expression level of HCCR-2 mRNA in pIRES2-EGFP group and HepG2 group were 0.62±0.06 and 0.72±0.03, respectively, with significant difference among the 3 groups (F=43.701, P<0.05). The apoptotic rate of HepG2 cells in antisense vector group, pIRES2-EGFP grop and HepG2 group were 13.30%, 2.51% and 2.07%, respectively, with significant difference among the 3 group (χ2=6.793, 8.721, P<0.05). The growth of HepG2 cells in antisense vector group was retarded, and was blocked in G0/G1 stage. Conclusions The HCCR-2 antisense recombinant eukaryotic expression vector can inhibit the mRNA expression of HCCR-2 and promote the apoptosis of cells. HCCR-2 may be involved in cell regulation and the proliferation of hepatocellular carcinoma cells.