富含亮氨酸重复序列G-蛋白耦联受体5基因沉默对结肠癌干细胞生物学行为的影响  

Effect of leucine - rich repeat - containing G - protein coupled receptor 5 gene silencing on biological behaviors of colon cancer stem cells

作　　者：陈翔[1] 曾兵[1] 魏波[2] 黄勇[2] 郑宗珩[2] 卫洪波[2] 

机构地区：[1]广州医科大学附属第六医院清远市人民医院胃肠外科,清远511518 [2]中山大学附属第三医院胃肠外科,广州510630

出　　处：《中华实验外科杂志》2017年第10期1699-1701,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金（81000960）;清远市科技计划项目（2016857）

摘　　要：目的观察富含亮氨酸重复序列G-蛋白耦联受体5（LGR5）基因沉默对具有＂干性＂结肠癌球囊细胞生物学行为影响。方法采用RNA干扰技术沉默LGR5基因表达。利用脂质体Lipofectamine RNAi MAX将LGR5小干扰RNA （LGR5-siRNA）转染至HT29结肠癌球囊细胞，而转染对照siRNA细胞和未转染细胞分别作为阴性对照组和空白对照组。实时定量聚合酶链反应（Real-time PCR）和Western blot法检测转染48 h后球囊细胞LGR5 mRNA和蛋白表达；细胞计数试剂盒（CCK-8）法转染1、2、3、4 d球囊细胞增殖能力，球囊形成实验、Transwell小室实验、流式细胞术检测转染48 h后细胞自我更新、侵袭能力和细胞周期。结果转染48 h后，实验组LGR5 mRNA表达量（0.32±0.02）较空白对照组（1.00±0.16）和阴性对照组（0.99±0.12）明显下降（P＝0.000），蛋白表达亦显著下调（P＝0.000）；CCK-8实验结果显示，实验组细胞增殖能力明显减弱，4 d时空白对照组、阴性对照组和实验组吸光度（A）值分别为0.540±0.010、0.528±0.016、0.390±0.022（P＝0.000）；球囊形成实验结果显示实验组形成子代球囊数目[（21.33±2.08）个]明显少于空白对照组[（72.33±3.06）个]和阴性对照组[（70.67±2.52）个，P＝0.000]；流式细胞术检测结果显示实验组细胞明显阻滞于G0/G1期（P＝0.000）；Transwell小室实验结果显示，实验组穿膜细胞数[（35.60±6.74）个]较空白对照组[（86.47±7.85）个]和阴性对照组[（83.53±8.58）个]明显减少（P＝0.000）。结论LGR5基因沉默可抑制结肠癌球囊细胞增殖、自我更新及侵袭，LGR5对结肠癌干细胞生物学行为有重要调控作用。ObjectiveTo investigate the effect of leucine-rich repeat-containing G-protein coupled receptor 5 gene （LGR5） silencing on biological behaviors of colon cancer spheroid cells with stemness.MethodsRNA interfering methods were used to knock down the LGR5 expression. Small interfering RNA （siRNA） targeting LGR5 and control siRNA were transfected into HT29 colon cancer spheroid cells by Lipofectamine RNAi MAX. Cells transfected with control siRNA or untransfected were used as negative control and blank control respectively. The expression of LGR5 mRNA and protein was detected by real-time quantitative polymerase chain reaction （Real-time PCR） and Western blotting. The proliferation of spheroid cells was detected by cell counting kit-8 （CCK-8） assay at 1st, 2nd, 3rd, and 4th day after transfection respectively. The self-renewal, invasion and cell cycle of spheroid cells were determined by sphere formation assay, Transwell assay and flow cytometry respectively at 48th h after transfection. ResultsAt 48 h after transfection, the expression of LGR5 mRNA in experimental group （0.32±0.02） was remarkably lower than that in blank control group （1.00±0.16） and negative control group （0.99±0.12） （P=0.000）. The expression of LGR5 protein in experimental group was also significantly down-regulated （P=0.000）. CCK-8 assay showed that the absorbance （A） value in blank control group, negative control group and experimental group at 4th day was 0.540±0.010, 0.528±0.016 and 0.390±0.022, respectively （P=0.000）. In addition, the experimental group （21.33±2.08） formed fewer secondary spheres than blank control group （72.33±3.06） and negative control group （70.67±2.52, P=0.000）. Flow cytometry revealed that the cells in the experimental group were significantly arrested in G0/G1 phase （P=0.000）. Furthermore, the number of cells passing through the Transwell membrane in experimental group （35.60±6.74） was remarkably decreased as compared with that in the blank contr

关 键 词：结肠癌 肿瘤干细胞 富含亮氨酸重复序列G-蛋白耦联受体5 增殖 侵袭 

分 类 号：R735.35[医药卫生—肿瘤]