Effects of specific small interfering RNA-mediated cyclinD1 gene silencing on the growth of human keloid fibroblasts / 中国组织工程研究

ABSTRACT

AIM:Cyclin is a decisive factor of regulating cell cycle,and RNA interference as an effective and specific gene silencing technique,can induce cell express the phenotype of specific gene deficiency. This study is to apply cyclinD1 specific small interfering RNA(siRNA) on inhibiting cyclinD1 gene expression and investigate the effect of cyclinD1 specific siRNA on the cell cycle and multiplication in keloid fibroblasts. METHODS:The experiment was conducted at the Institute of Genetic Engineering(Grade BSL-2) in Southern Medical University from July 2006 to May 2007.①siRNA was designed with siRNA target finder of ambion Company,and synthesized chemically in Shanghai GeneChem,Co.,Ltd. Then double-strand siRNA was obtained following degenerative renaturation. Keloid fibroblasts were sampled from the patients in the Department of Plastic Surgery,Southern Medical University(informed consents were obtained from the patients or their relatives).②The keloid fibroblasts were divided into experimental group and control group. The cyclinD1 specific siRNA was transfected into the keloid fibroblast of the experimental group by the liposome-mediated gene transfection method. The untreated cells were set as controls.③At hours 24,48 and 72 of transfection,light microscope was used to observe cell morphologic change and flow cytometry was used to examine cell cycle. The viable cells were counted by MTT colorimetry and a cell growth curve was drawn. RESULTS:①Abnormal change of cell morphology that became into spherical shape or oval-shape from normal long fusiform after transfection may be the apoptosis or necrosis cells.②The G1 stage of cell cycle extended and the S stage decurtated. At 24,48 and 72 hours after transfection,the radio of G1 stage cell was 60.13%,66.22% and 67.53%,which were all significantly higher than that in the control group(54.53%);the radio of S stage cell(18.25%,17.11% and 11.15%) was also significantly lower than that in the control group(22.31%),indicating that the proportion of the cells blocked in G1 stage and those in S stage decreased in the keloid fibroblast.③siRNA-cyclinD1 inhibited the growth of keloid fibroblasts obviously by using MTT assay,and the cell growth curves indicated that the proliferation of cell transfected with cyclinD1 specific siRNA was inhibited significantly when compared with controls. CONCLUSION:CyclinD1 specific siRNA effectively inhibits the expression of cyclinD1 in keloid fibroblasts thus arresting the cell cycle at G1 stage and enhancing cell apoptosis.