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Zheng S.,Wuhan General Hospital of Guangzhou Millitary Command | Wang F.,Air force Radar Academy | Ding Y.,Wuhan General Hospital of Guangzhou Millitary Command | Zhang L.,Wuhan General Hospital of Guangzhou Millitary Command | And 2 more authors.
Chinese-German Journal of Clinical Oncology | Year: 2010

Objective: The aim of the study was to construct the eukaryotic expression vector of human angiopoietin-like protein 4 (ANGPTL4) and observe the effect of ANGPTL4 overexpression on the growth of esophageal carcinoma EC9706 cells. Methods: Total RNA was extracted from normal hepatic tissue, and ANGPTL4 cDNA was amplified by RT-PCR. The PCR product was doubly digested by XbaI and SalI, and then recombined into eukaryotic expression vector. Then, pIRES-GFP-ANGPTL4 was obtained by G418 selection, then pIRES-GFP-ANGPTL4 and pIRES-GFP were transfected into EC9706 cells with lipidosome-packaged method. Meanwhile, the transfected cells were selected by G418, and then stable transfected cell lines were obtained. ANGPTL4 mRNA levels, the cell cycles and growth curves of EC9706 cells in experiment group (transfected with pIRES-GFP-ANGPTL4), empty vector group (transfected with pIRES-GFP) and blank control group (EC9706 cells without transfection) were detected with RT-PCR, flow cytometry and MTT methods, respectively. Results: Eukaryotic ANGPTL4 expression vector pIRES-GFP-ANGPTL4 was successfully constructed. The ANGPTL4 mRNA level (0.21 ± 0.03) in experiment group was significantly higher than that of the empty vector group (0.04 ± 0.008) and the blank control group (0.05 ± 0.007), with significant differences (P < 0.01). The proportion of cells in S phase in experiment group was significantly different with those of the other two groups (P < 0.05). The cell growth of EC9706 cells in experiment group was slower than those of the other two groups. From the third day, the differences began to be significant. Conclusion: ANGPTL4 overexpression in esophageal carcinoma EC9706 cells could inhibit the growth of EC9706 cells. © Springer-Verlag Berlin Heidelberg 2010. Source


Zheng S.,Wuhan General Hospital of Guangzhou Millitary Command | Jing Q.,Health Care Agency of Air Force Early Institute | Zheng Y.,Wuhan General Hospital of Guangzhou Millitary Command | Ding Y.,Wuhan General Hospital of Guangzhou Millitary Command | And 2 more authors.
Chinese-German Journal of Clinical Oncology | Year: 2012

Objective: The aim of our study was to investigate the effects of down-regulation Bmi-1 by RNA interference (RNAi) in T Lymphocytic leukemia Jurkat cells. Methods: Two complementary oligonucleotide strands were synthesized based on the siRNA sequence targeting Bmi-1 gene. After annealing, siRNA strands were recombined into the pRNATU6.2 vector, and then DNA sequencing was carried out following transformation and amplification. The recombinant was transfected into Jurkat cells with liposomes. Positive colonies were obtained through G418 selection. The mRNA and protein expressions of Bmi-1 were detected by RT-PCR and Western-blot, respectively. Effects of Bmi-1 silence on cell proliferation, cell cycle and cell aging of Jurkat cells were detected by MTT assay, flow cytometry, colony formation assay and SA-β-Gal staining, respectively. Results: The siRNA recombinant targeting Bmi-1 gene was successfully constructed. All three siRNA recombinants could significantly inhibit the expression of Bmi-1. The siRNA targeting 825nt-843nt (GACCAGACCACTACT GAAT) has the strongest inhibitory effect on Bmi-1 expression, with almost complete inhibition on Bmi-1 mRNA and protein expressions. Compared with the non-transfection group and the empty vector group, growth velocity and colony formation ability were significantly decreased, while the proportion of cells in G1 phase and the percentage of senile cells were significantly increased in highly transfected group (P < 0.05). Conclusion: Down-regulation Bmi-1 by RNA interference (RNAi) could significantly inhibit the growth of Jurkat cells in vitro. © 2012 Springer-Verlag Berlin Heidelberg. Source

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