核仁素表达下调对宫颈癌细胞Hela生物学行为的影响  

Effect of nucleolin dow n-regulation on biological behaviors of HeLa cells in human cervical carcinoma

作　　者：刘洪丽[1] 张友忠[1] 吕昌帅[1] 丁佰娟[1] 王颉[1] 

机构地区：[1]山东大学齐鲁医院妇产科,济南250012

出　　处：《山东大学学报（医学版）》2013年第11期46-50,共5页Journal of Shandong University:Health Sciences

基　　金：国家自然科学基金(81072122)

摘　　要：目的探讨核仁素表达下调对宫颈癌Hela细胞增殖、凋亡、侵袭等生物学行为的影响。方法实验分4组:3个靶向人核仁素基因的实验组(siN1组、siN2组、siN3组)及1个阴性对照组(NC组)。体外化学合成各组siRNA并转染HeLa细胞;利用荧光实时定量RT-PCR法和Western blotting法分别从mRNA和蛋白水平检测核仁素基因的干扰效率,并筛选出干扰效率最高的片段;利用细胞增殖及细胞毒性检测试剂盒(CCK-8)法检测核仁素下调对HeLa细胞增殖活性的影响;利用流式细胞学Annexin V-FITC检测转染后HeLa细胞凋亡率的改变;Transwell实验检测转染后细胞侵袭能力的改变。结果与NC组相比,各实验组核仁素mRNA表达水平均下降(P<0.05);蛋白表达水平也相应下调(P<0.05)。成功筛选出最佳干扰片段siN2,用于后续实验。siN2组HeLa细胞生长速率明显受到抑制(P<0.05);流式细胞学Annexin V-FITC检测siN2组HeLa细胞凋亡率明显高于NC组(P<0.05);Transwell实验显示siN2组HeLa细胞侵袭能力减弱(P<0.05)。结论核仁素表达下调明显抑制宫颈癌Hela细胞增殖与侵袭能力,促进细胞凋亡,为宫颈癌的基因治疗提供了理论依据。Objective To study the effect of nucleolin down-regulation on the biological behaviors of HeLa cells in human cervical carcinoma, including proliferation, apoptosis and invasion. Methods Four groups were enrolled in the experiment, including the siN1, siN2, and siN3 groups, which targeted human nucleolin gene, and siC group, which served as negative control. The chemically synthetic siRNA was transfected to cervical neoplasm HeLa cell lines in vitro by RNAi Max transfection reagent. The expression of nucleolin mRNA was detected by real time PCR （RT-PCR）, and nucleolin protein was evaluated by Western blotting, in order to select the siRNA with the highest transfection efficiency. The proliferative activity of HeLa cells was assessed by Cell Counting Kit-8 （CCK-8） assay; the apoptosis was measured by flow cytometry （FCM） with Annexin V/PI staining; the ability of cellular invasion was tested by Transwell experiment. Results Sequence-specific siRNAs of nucleolin down-regulated the mRNA levels of nucleolin compared with its negative control siRNA （P〈0.05）. The protein expression was down-regulated after transfection （P〈0.05）. The siRNA with the highest transfection efficiency was selected. Compared with the negative control, the proliferative cellular invasion abilities of HeLa cells in the siN2 group were inhibited significantly （P〈0.05）, while its apoptosis was effectively enhanced （P〈0.05）. Conclusion The down-regulated expression of nucleolin mRNA and its protein is able to inhibit cell proliferation and invasion, and promote apoptosis, which indicates gene therapy may be feasible in the treatment of cervical neoplasms.

关 键 词：宫颈肿瘤 HELA细胞 siRNA 小分子干扰 核仁素 

分 类 号：R711.7[医药卫生—妇产科学]