Cyclin D_1基因沉默诱发肾癌ACHN细胞增殖受抑的研究  

Inhibition of Proliferation Induced by Cyclin D_1 Gene Silence in Human ACHN Cells

作　　者：王江[1] 郭风劲[1] 陈安民[1] 袁林[1] 杨彩虹[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院骨科,武汉430030

出　　处：《华中科技大学学报（医学版）》2010年第2期141-146,共6页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家重点基础研究发展计划资助项目(No.2002CB513107)

摘　　要：目的证实Cyclin D1基因作为肿瘤基因治疗新靶点的可行性及发卡环RNA(shRNA)表达载体介导该基因沉默的有效性。方法设计合成Cyclin D1-shRNA模板片段,与Pgenesil-1-U6载体连接,构建shRNA真核表达载体;将Pgenesil-Cyclin D1-shRNA转入ACHN肾癌细胞株。RT-PCR、Western blot分析Cyclin D1基因mRNA及蛋白表达;MTT比色法绘制细胞生长曲线;流式细胞术测细胞凋亡率;Transwell小室体外侵袭实验检测细胞侵袭。结果Pgene-sil-Cyclin D1-shRNA构建成功。Cyclin D1-shRNA实验组Cyclin D1mRNA抑制效果显著(0.10±0.04),明显高于Pgenesil-NC阴性对照组(0.92±0.03)及ACHN空白对照组(0.94±0.04)(均P<0.05)。同样,实验组Cyclin D1蛋白表达明显下调。流式细胞术分析提示,Cyclin D1-shRNA组细胞早期及晚期凋亡细胞均较Pgenesil-NC组和ACHN组显著增高。生长曲线显示Cyclin D1-shRNA组细胞生长明显缓慢(P<0.05)。Transwell实验同样发现该组细胞侵袭能力显著下降(P<0.05)。结论Cyclin D1可作为肾癌ACHN细胞生物学行为的评判标准。经载体介导shRNA干扰的高效性为肾癌基因治疗提供了实验依据。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 construct the recombinant plasmid Pgenesil-Cyclin D1-shRNA.The identified recombinant plasmid was transfected into ACHN cells with lipofactamine.Cyclin D1 mRNA and protein expression was detected by RT-PCR and Western blot.MTT method was used for observing cell proliferation and drawing growth curve.The cell cycle and percentage of apoptotic cells were assessed by flow cytometry.The invasion ability of migrating cells was measured by Transwell chamber invasive models.Results The plasmid was constructed successfully.After interference,the expression rate of Cyclin D1 mRNA was decreased to（0.10±0.04） in Cyclin D1-shRNA（experimental） group and significantly lower than in Pgenesil-NC（negative） group（0.92±0.03） and ACHN（blank control） group（0.94±0.04）（P0.05）.The expression rate of Cyclin D1 protein was decreased evidently in experimental group.The flow cytometry revealed that the apoptotic ratio in experimental group was obviously higher than that in the negative group and blank control group.The cell growth curves indicated that the proliferation of cells in the experimental group was inhibited significantly（P0.05） and Transwell results suggested that the invasion abilities of cells transfected with Pgenesil CyclinD1-shRNA were decreased significantly（P0.05）.Conclusion Cyclin D1 gene can be used as the standard to evaluate the prognosis of ACHN cells.The efficiency of vector-mediated shRNA interference offered the possibility of gene therapy for renal cell carcinoma.

关 键 词：肾ACHN细胞 RNA干扰 细胞周期蛋白D1 增殖 

分 类 号：R349.6[医药卫生—基础医学]