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Objective To construct recombinant lentiviral vector of microRNA-34a and observe the cell viability,cell cycle and apoptosis of hepatocellular carcinoma cells transfected with the vector system and treated with Doxorubicin.Methods Recombinant lentiviral vector containing microRNA-34a gene was constructed and transfected into 3 hepatocellular carcinoma cell lines,and cells were treated with Doxorubicin.The expression of microRNA-34a gene was detected by real-time PCR.The effect of microRNA-34a overexpression on hepatocellular carcinoma cells proliferation were quantified via MTT assay,cell cycle and apoptosis was evaluated by flow cytometry.Western blotting was used to evaluate the expression of cell cycle and apoptosis related protein.Results The successful construction of microRNA-34a recombinant lentiviral vector was confirmed by plasmid enzyme digestion and DNA sequencing.Compared with the control group,relative expression of microRNA-34a gene in hepatocellular carcinoma cells significantly increased ((HepG2:t=15.36,P＜0.01;Hep3B:t=36.75,P＜0.01;Bel-7402:t=24.17,P＜0.01)).Cells viability decreased (HepG2:t =7.12,P ＜ 0.01;Hep3B:t =8.89,P ＜ 0.01;Bel-7402:t =13.62,P ＜0.01),G1 phase cells increased significantly(HepG2:F =137.65,P ＜ 0.01;Hep3B:F =143.39,P ＜0.01;Bel-7402:F =1 306.47,P ＜ 0.01) and cell apoptosis increased(HepG2:F =386.14,P ＜ 0.01;Hep3B:F =881.94,P ＜ 0.01;Bel-7402:F =885.89,P ＜ 0.01).Conclusions MicroRNA-34a recombinant lentiviral vector (LV-hsa-mir-34a) transfected hepatocellular carcinoma cells overexpress microRNA-34a,reduce the malignant biological behavior.MicroRNA-34a recombinant lentiviral vector (LV-hsa-mir-34a) enhance the in vitro inhibitory effects of Doxorubicin on hepatocellular carcinoma cell lines.
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Objective To observe the inhibitory effect and mechanism of 9-nitrocamptothecin liposomes on HepG2 liver carcinoma cells. Methods HepG2 cells were incubated with 9-nitrocampto-thecin(9NC) or with 9-nitrocamptothecin liposomes(9NC-LP) for 24 h, 48 h and 72 h. Cell viability was then measured by the MTT assay. Cell cycle and apoptosis were evaluated by flow cytometry.Western Blot was used to determine the expression of cell cycle and apoptosis related proteins. HepG2tumor-bearing mouse models were then established. The HepG2 tumor-bearing mice were randomly divided into control group, free liposomes group, DMSO group, 9NC low dose group, 9NC high dose group, 9NC-LP low dose group and 9NC-LP high dose group. There were 10 mice in each group.Drugs were administered by tail vein and tumor volume and body weight were observed 28 days after administration. Then animals were sacrificed and the expression of proteins from tumor homogenates was analyzed by Western blotting. Results In vitro, HepG2 cell viability was apparently inhibited by 9NC and 9NC-LP, and the inhibitory effect increased in a time-dependent and dose-dependent manner.Both S and G2/M phase arrests were observed after incubation with drugs. HepG2 cells were completely arrested in S phase with 9NC concentration over than 0.1 μmol/L after incubation for 24 h,while more than 95% of cells arrested in G2/M phase when 9NC concentration was 0.1 μmol/L after incubation for 72 h. In vivo, compared with the control group, the average tumor volume was reduced in both the 9NC and 9NC-LP group (P50% of animals died 14 days after drug administration. Conclusion 9NC and 9NC-LP can inhibit HepG2 cell growth via cell cycle arrest and apoptosis induction. 9NC-LP has a more potent anti-tumor effect and fewer side effects in vivo,which means 9NC-LP is a promising compound for cancer therapy via intravenous administration.
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Objective To investigate the modulating effects and explore their mechanism of 9-nitrocamptothecin and its liposomes to induce apoptosis and inhibit cell cycle in HepG2 and L02 cell lines. Methods Cells were incubated with 9-nitrocamptothecin(9NC) or with 9-nitrocamptothecin liposomes for 24 h, 48 h and 72 h, then, the cell viability was measured via MTT assay; cell cycle and apoptosis was evaluated by flow cytometry after stained by PI and Annexin V-PE/7AAD. Additional, Western Blot was used to evaluate the expression of cell cycle and apoptosis related protein. Results Both cells viability were apparently inhibited by the 9-nitrocamptothecin and 9-nitrocamptothecin liposomes, the inhibitory effect showed a time-dependent and dose-dependent manner. Both S and G2/M phases arrest were observed after incubated with drugs. HepG2 cell was completely arrested in S phase when 9NC concentration over than 0. 1 μmol/L after incubation for 24 h, while more than 95% cells arrested in G2/M phase when 9NC concentration is 0. 1 μmol/L after incubation for 72 h. Apoptosis induction effect also showed a time-dependent and dose-dependent manner. Western Blot results showed the expression of Bax and Caspase-3 were upregulated while Cyclin A, Cdk2, Cyclin E and Bcl-2 were downregulated. More importantly, the compounds were more cytotoxic to the cancer cell lines than to the normal liver cell. Conclusions 9-nitrocamptothecin and 9-nitrocamptothecin liposomes can potently inhibit cell growth via regulation of cell cycle and induction of apoptosis, and this effect was preferentially in cancer cell. Inhibitory of 9-nitrocamptothecin liposomes was slightly better than the 9-nitrocamptothecin.
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Objective The article introduces the present status of the application of comparative proteomics in study of tumor marker. Methods This essay review the present status and advances of the application of comparative proteomics in study of tumor marker through refer considerable literatures about proteome, proteomics and tumor marker. Results Follow the study of human genome deepening; the paradox between the finiteness of genes’ number and stability of genes’ structure and the variety of the life phenomena is more conspicuous. Then, the study of proteomics was pushed to the advancing front of life science research. The application of comparative proteomics to tumor research becomes a hot spot nowadays. Conclusion Screening tumor marker via comparative proteomics is an extremely promising research.