Inhibition of Proliferation Induced by Cyclin D1 Gene Silence in Human Renal Carcinoma ACHN Cells
Abstract
Objective: To confirm feasibility of Cyclin D1 gene as a new target for cancer gene therapy and to verify the effectiveness of shRNA expression vector-mediated gene silencing.
Methods: A RNA interference DNA template targeting Cyclin D1 gene was designed and synthesized. By ligation, the fragment was inserted into Pgenesil-1-U6 to constract the recombinant plasmid Pgenesil-shRNA- Cyclin D1. The identified recombinant plasmid was transfected into ACHN cells with lipofactamine. Cyclin D1 mRNA and protein expression was analyzed by RT-PCR and western-blotting. MTT method was used for observing cell proliferation and drawing growth curve. The cell cycle and ratios of apoptotic cell were assessed by flow cytometric detection. The ability of invasion of cell migration was detected by Transwell chamber invasive models.
Results: The plasmid was constructed successfully. After interference, The expression rate of Cyclin D1 mRNA decreased to 0.10±0.04 in Cyclin D1-shRNA(experimental) group and were significantly lower than Pgenesil-NC (negative) group (0.92±0.03) and ACHN (blank control) group(0.94±0.04)(P<0.05). As well, the expression rate of Cyclin D1 protein was decreased evidently in experimental group. The results of flow cytometric detection showed that, including early and late apoptotic cells, the apoptotic ratio of experimental group increased to (37.26±0.60)% significantly, while, the negative group and blank control group were only (4.62±0.40)% and (5.95±1.20)%, respectively. The cell growth curves indicated that the proliferation of experimental group cells was inhibited significantly(P<0.05) and Transwell results suggested that the abilities of invasion cells transfected with Pgenesil–CyclinD1-shRNA decreased conspicuously(P<0.05).
Conclusion: The shRNA can inhibit Cyclin D1 expression, specifically and persistently. The down-regulation of Cyclin D1 expression can inhibit the proliferation and induce the apoptosis of renal cell adenocarcinoma cell line ACHN.
Methods: A RNA interference DNA template targeting Cyclin D1 gene was designed and synthesized. By ligation, the fragment was inserted into Pgenesil-1-U6 to constract the recombinant plasmid Pgenesil-shRNA- Cyclin D1. The identified recombinant plasmid was transfected into ACHN cells with lipofactamine. Cyclin D1 mRNA and protein expression was analyzed by RT-PCR and western-blotting. MTT method was used for observing cell proliferation and drawing growth curve. The cell cycle and ratios of apoptotic cell were assessed by flow cytometric detection. The ability of invasion of cell migration was detected by Transwell chamber invasive models.
Results: The plasmid was constructed successfully. After interference, The expression rate of Cyclin D1 mRNA decreased to 0.10±0.04 in Cyclin D1-shRNA(experimental) group and were significantly lower than Pgenesil-NC (negative) group (0.92±0.03) and ACHN (blank control) group(0.94±0.04)(P<0.05). As well, the expression rate of Cyclin D1 protein was decreased evidently in experimental group. The results of flow cytometric detection showed that, including early and late apoptotic cells, the apoptotic ratio of experimental group increased to (37.26±0.60)% significantly, while, the negative group and blank control group were only (4.62±0.40)% and (5.95±1.20)%, respectively. The cell growth curves indicated that the proliferation of experimental group cells was inhibited significantly(P<0.05) and Transwell results suggested that the abilities of invasion cells transfected with Pgenesil–CyclinD1-shRNA decreased conspicuously(P<0.05).
Conclusion: The shRNA can inhibit Cyclin D1 expression, specifically and persistently. The down-regulation of Cyclin D1 expression can inhibit the proliferation and induce the apoptosis of renal cell adenocarcinoma cell line ACHN.