CAJ | 학술논문

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.
목적 탐토CD133기인표체피억제후대위암세포증식、침습、극륭구형성급화료약물민감성적영향.방법 통과면역자주분선KATO-Ⅲ위암세포중적CD133양성세포,장합성적CD133소간우핵당핵산분자(siRNA)전염지KATO-ⅢCD133양성위암세포내,사용형광표기적siRNA (FAM-siRNA)검측전염효솔,통과RT-PCR、Western-blot방법검측CD133기인표체적침묵효과급상피-간질전화(EMT)상관인자(E-cadherin、Snail화N-cadherin)적단백표체,채용CCK-8、Transwell침습실험、단극륭구형성실험화CCK-8등방법분별검측세포증식、침습、극륭구형성능력급대화료약물5-불뇨밀정(5-FU)적민감성.결과 전염24 h후,전염효솔가체도(87.7±8.1)％.간우조CD133 mRNA급단백적표체현저저우음성대조조(P＜0.01).전염24、48화72 h후,여음성대조조비교,간우조세포적증식활성균득도현저억제,차이균유통계학의의(P＜0.01)；전염72 h,간우조세포적증식활성교음성대조조강저료(52.1±8.0)％.여음성대조조비교,간우조적세포침습수감소[(41.7±6.0)비(130.3±11.0),P＜0.05],극륭구형성솔강저[(24.3±4.3)％비(45.1±6.4)％,P＜0.01],Snail화N-cadherin단백표체강저(P＜0.01),이E-cadherin단백표체증고(P＜0.01).간우조세포대화료약물5-FU적민감성현저증강,5-FU대간우조세포적억제솔위(62.4±3.3)％,교음성대조조적(21.5±2.2)％증가(P＜0.01).결론 CD133기인재위암세포적증식、침습、극륭구형성화화료저항성등방면구유중요작용,가능시위암간세포신형표지물,유망성위위암생물치료적신파점.
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.