RNA干扰神经纤毛蛋白-2基因对结肠癌细胞HCT-8增殖和凋亡的影响  

Effect of RNA interference targeting neuropilin-2 gene on proliferation and apoptosis of colon cancer cell line HCT-8

作　　者：丁冠一 徐卫国 郭尚 詹琪琪 杨鑫 贾纯亮[2] Ding Guanyi;Xu Weiguo;Guo shang;Zhan Qiqi;Yang Xin;Jia Chunliang(Department of Surgical Oncology,Affiliated Hospital of North China University of Science and Technology,Tangshan 063000,China;Department of Gastrointestinal surgery,Tangshan People′s Hospital,Tangshan 063000,China)

机构地区：[1]华北理工大学附属医院肿瘤外科,唐山063000 [2]唐山市人民医院胃肠外科,唐山063000

出　　处：《中华医学杂志》2020年第48期3879-3883,共5页National Medical Journal of China

摘　　要：目的探讨应用RNAi技术降低神经纤毛蛋白-2(NRP-2)表达后对结肠癌细胞HCT-8增殖和凋亡的影响。方法通过脂质体lip2000分别将NRP2-siRNA和NControl-siRNA转染入HCT-8中作为转染组和阴性对照组,加入磷酸盐缓冲液作为空白对照组,通过实时荧光定量PCR(RT-qPCR)和Western印迹法验证转染效果,采用细胞计数试剂盒(CCK)法、平板克隆实验和Ki-67蛋白染色实验检测3组细胞的增殖情况,吖啶橙/碘化丙啶(AO/PI)荧光染色实验检测3组细胞的凋亡情况。结果RT-qPCR和Western印迹法结果表明转染组细胞NRP-2 mRNA相对表达量和NRP-2蛋白含量降低(0.46±0.05比0.99±0.05和1.00±0.06,1.04±0.06比1.73±0.09和1.65±0.11)(均P<0.05),CCK法表明,转染组各时段的增殖能力较阴性对照组和空白对照组显著降低(24 h为0.53±0.04比0.82±0.07和0.87±0.07,48 h为0.54±0.05比1.00±0.09和1.17±0.05,72 h为0.75±0.05比1.31±0.13和1.50±0.03,96 h为1.05±0.04比1.46±0.09和1.86±0.06)(均P<0.05);平板克隆实验显示转染组细胞较其他两组克隆形成能力显著下降(134.67±8.74比245.33±19.14和300.33±14.01,P<0.05);Ki-67蛋白检测显示与对照组相比,转染组的细胞Ki-67含量显著下降(5.93±0.22比8.36±0.09和8.70±0.21,P<0.05);AO/PI实验显示转染组较对照组相比凋亡细胞/活细胞比例显著上升(0.43±0.07比0.14±0.04和0.11±0.04,P<0.05)。结论降低NRP-2表达可使结肠癌细胞HCT-8的增殖能力下降,凋亡能力上升。Objective To investigate the effects of down-regulation of expression of neuropilin-2(NRP-2)by RNA interference(RNAi)technique on proliferation and apoptosis of HCT-8 colon cancer cells.Methods NRP2-siRNA and negative control(NControl)-siRNA were transferred into HCT-8 colon cancer cells by liposomes(lip2000)as transfection group and negative control group,and phosphate buffered solution(PBS)was added as blank control group.Quantitative reverse transcription PCR(RT-qPCR)and Western blot were used to detect the transfection effect.The proliferation of cells in the three groups was examined by cell counting kit(CCK)assay,colony-forming unit assay and Ki-67 protein staining assay,respectively.Moreover,the apoptosis of cells in the three groups was determined by acridine orange/propranidine iodide(AO/PI)staining method.Results The results of RT-qPCR and Western blot showed that the relative expression of NRP-2 mRNA and the content of NRP-2 protein in the transfer group decreased(0.46±0.05 vs 0.99±0.05 and 1.00±0.06;1.04±0.06 vs 1.73±0.09 and 1.65±0.11)(all P<0.05).The results of CCK-8 demonstrated that the optical density of transfection group was significantly lower than that of the negative control group and the blank control group(24 h:0.53±0.04 vs 0.82±0.07 and 0.87±0.07;48 h:0.54±0.05 vs 1.00±0.09 and 1.17±0.05;72 h:0.75±0.05 vs 1.31±0.13 and 1.50±0.03;96 h:1.05±0.04 vs 1.46±0.09 and 1.86±0.06)(all P<0.05).The results of colony-forming unit assay indicated that the proliferation ability of the cells in the transfer group was significantly lower than that in the other two groups(134.67±8.74 vs 245.33±19.14 and 300.33±14.01,P<0.05).The results of Ki-67 protein staining assay showed that compared with the negative control group and blank control group,the expression of Ki-67 protein was significantly decreased in the transfection group(5.93±0.22 vs 8.36±0.09 and 8.70±0.21,P<0.05).The results of AO/PI assay revealed that the ratio of apoptotic cells to living cells in the transfer group was s

关 键 词：结肠肿瘤 Neuropilin-2 细胞增殖 细胞凋亡 

分 类 号：R735.35[医药卫生—肿瘤]