RNA干扰抑制KATO-Ⅲ CD133阳性胃癌细胞CD133基因表达及对其生物学特性的影响

Effect of RNA interference inhibition to expression of CD133 on tumor cell biological characteristics in KATO-Ⅲ CD133＋ cells of human gastric cancer

目的 探讨CD133基因表达被抑制后对胃癌细胞增殖、侵袭、克隆球形成及化疗药物敏感性的影响.方法 通过免疫磁珠分选KATO-Ⅲ胃癌细胞中的CD133阳性细胞,将合成的CD133小干扰核糖核酸分子（siRNA）转染至KATO-ⅢCD133阳性胃癌细胞内,使用荧光标记的siRNA （FAM-siRNA）检测转染效率,通过RT-PCR、Western-blot方法检测CD133基因表达的沉默效果及上皮-间质转化（EMT）相关因子（E-cadherin、Snail和N-cadherin）的蛋白表达,采用CCK-8、Transwell侵袭实验、单克隆球形成实验和CCK- 8等方法分别检测细胞增殖、侵袭、克隆球形成能力及对化疗药物5-氟尿嘧啶（5-FU）的敏感性.结果 转染24 h后,转染效率可达到（87.7±8.1）％.干扰组CD133 mRNA及蛋白的表达显著低于阴性对照组（P＜0.01）.转染24、48和72 h后,与阴性对照组比较,干扰组细胞的增殖活性均得到显著抑制,差异均有统计学意义（P＜0.01）；转染72 h,干扰组细胞的增殖活性较阴性对照组降低了（52.1±8.0）％.与阴性对照组比较,干扰组的细胞侵袭数减少[（41.7±6.0）比 （130.3±11.0）,P＜0.05],克隆球形成率降低[（24.3±4.3）％比（45.1±6.4）％,P＜0.01],Snail和N- cadherin蛋白表达降低（P＜0.01）,而E-cadherin蛋白表达增高（P＜0.01）.干扰组细胞对化疗药物5-FU的敏感性显著增强,5-FU对干扰组细胞的抑制率为（62.4±3.3）％,较阴性对照组的（21.5±2.2）％增加（P＜0.01）.结论 CD133基因在胃癌细胞的增殖、侵袭、克隆球形成和化疗抵抗性等方面具有重要作用,可能是胃癌干细胞新型标志物,有望成为胃癌生物治疗的新靶点.

Objective To investigate the changes in proliferation,invasiveness,clone sphere formation and chemosensitivity of human gastric cancer cell lines of KATO-Ⅲ CD133＋ cells transfected with small interfering RNA （siRNA） against CD133 gene.Methods CD133＋ cells of KATO-Ⅲ cell lines were isolated by magnetic activated cell sorting （MACS）.CD133 siRNA was designed and synthesized,and then transfected into KATO-Ⅲ CD133 ＋ cells.Cell fluorescence counting under confocal laser scanning microscope was used to determine the transfection efficiency after transfection with the CD133 FITC-siRNA.The knock-down effect of the CD133 gene and expression of epithelialmesenchymal transition （EMT）-related factors were detected by RT-PCR and Western blotting.Cell counting kit-8 assay （CCK-8）,transwell chamber and colony sphere forming assay were performed to measure the variation of cell proliferative,invasive,colony formation viability and chemosensitivity to 5FU after the above-mentioned treatment.Results The transfection efficiency was （87.7±8.1）%.The CD133 mRNA and protein expression levels in the interference group were lower than those in negative control group.Twenty-four,48 and 72 hours after transfection,cells proliferation activity was significantly inhibited in the interference group compared with negative control group,（all P〈0.01）.Seventy-two hours after transfection,compared with negative control group,cells proliferation activity was reduced by （52.1±8.0）%.The invasive cell number reduced（41.7±6.0 vs.130.3±11.0,P〈0.05） and clone formation rate decreased significantly[（24.3±4.3）% vs.（45.1±6.4）%,P〈0.01] in the interference group.EMT-related gene E-cadherin protein expression increased,while the Snail and N-cadherin protein expression reduced in the interference group （all P〈0.01）.The cells sensitivity to 5-FU was significantly enhanced in the interference group,and the cell inhibition rate of 5-Fu was （62.4±3.3）%,higher than that in negative control group [（21.5±2.2）%,P〈0.01].Conclusions The expression of CD133 gene plays an important role in cell proliferation,invasiveness,colony formation and resistance to chemotherapy of KATO-Ⅲ CD133＋ gastric cancer cells.It suggests that CD133 can be used as one of surface markers for detection of gastric cancer stem cells.Inhibination of CD133 expression may be a promising way for gastric cancer biotherapy.