摘要目的 探讨shRNA干扰富含亮氨酸重复序列G-蛋白偶联受体5(Lgr5)的表达对结直肠癌肿瘤干细胞(CSCs)恶性行为的影响及其作用机制.方法 采用实验研究方法.流式细胞术分选Lgr5+结直肠癌CSCs,转染shLgr5慢病毒载体设为实验组;转染相应对照病毒,设为对照组.流式细胞术检测Lgr5+细胞比例,荧光定量PCR(qRT-PCR)检测Lgr5 mRNA的相对表达量;成球实验检测细胞自我更新能力变化;平板克隆形成实验及裸鼠皮下成瘤实验分别检测体内外成瘤能力变化;qRT-PCR检测细胞中干细胞基因(Oct4、Sox2、Nanog、KLF4)、CSCs基因(CD133、CD44、ALDH)及Wnt/β-catenin信号通路关键基因(Axin2、Wnt5a、Wnt3a、Fzd3、c-myc、VEGF、Ascl2、claudin-1) mRNA表达变化.正态分布的计量资料以-x±s表示,组间比较采用t检验.结果 (1) shRNA慢病毒介导转染效率,流式细胞术检测结果显示:实验组Lgr5+细胞为6.8％±1.0％,对照组为92.7％±3.3％,两组比较,差异有统计学意义(t=43.148,P＜0.05).qRT-PCR检测两组CSCs中Lgr5 mRNA相对表达量:实验组和对照组分别为0.168±0.057和1.148±0.004,两组比较,差异有统计学意义(t=28.778,P＜0.05).(2) Lgr5下调对结直肠癌CSCs成球和成瘤能力的影响,成球实验结果显示:实验组和对照组成球数量分别为(29±6)个和(410±10)个,两组比较,差异有统计学意义(t=41.070,P＜0.05).平板克隆成瘤实验结果显示:实验组和对照组克隆成瘤数量分别为(72±4)个和(412±19)个,两组比较,差异有统计学意义(t=31.433,P＜0.05).裸鼠皮下成瘤实验结果显示:实验组和对照组裸鼠肿瘤体积分别为(81±15)mm3和(328±24) mm3,两组比较,差异有统计学意义(t=11.304,P＜0.05).(3) Lgr5下调对相关基因的影响,qRT-PCR检测结果显示:①实验组结直肠癌CSCs中干细胞基因Oct4、Sox2、Nanog、KLF4 mRNA的相对表达量分别为0.377±0.093、0.662±0.104、3.591±0.300、0.425±0.091;对照组结直肠癌CSCs中上述基因mRNA的相对表达量分别为1.957±0.026、2.137±0.015、5.831±0.165、1.536±0.014;两组上述指标比较,差异均有统计学意义(t=23.079,22.261,8.446,19.186,P＜0.05).②实验组结直肠癌CSCs中CSCs基因CD133、CD44、ALDH mRNA相对表达量分别为1.490±0.155、5.535±0.487、1.640±0.039;对照组结直肠癌CSCs中上述基因mRNA的相对表达量分别为2.488±0.061、9.908±0.332、5.718±0.292;两组上述指标比较,差异均有统计学意义(t=8.170,9.667,27.849,P＜0.05).③实验组结直肠癌CSCs中Wnt/β-catenin通路相关基因Axin2、Wnt5a、Wnt3a、Fzd3、c-myc、VEGF、Ascl2、claudin-1 mRNA相对表达量分别为1.592±0.267、0.528±0.138、2.153±0.078、1.480±0.064、0.248±0.128、1.492±0.025、0.658±0.095、1.647±0.087;对照组结直肠癌CSCs中上述基因mRNA的相对表达量分别为3.651±0.224、2.570±0.093、2.301±0.157、1.636±0.058、1.415±0.080、2.610±0.159、2.480±0.123、3.432±0.273;两组结直肠癌CSCs中Axin2、Wnt5a、c-myc、VEGF、Ascl2、claudin-1比较,差异均有统计学意义(t=7.316,15.332,12.649,12.320,14.831,9.063,P＜0.05).两组结直肠癌CSCs中Wnt3a、Fzd3比较,差异均无统计学意义(t=2.887,2.242,P＞0.05).结论 shRNA干扰结直肠癌CSCs中Lgr5表达后,细胞的恶性行为受到抑制,这种作用可能是通过抑制Wnt/β-catenin通路活性发挥生物学效应.

abstractsObjective To investigate the effects and mechanisms of shRNA interfered with expression of leucine-rich repeat containing G-protein coupled receptor 5 (Lgr5) on the malignant behaviors of colorectal cancer stem cells (CSCs).Methods The experimental study was conducted.The CSCs expressing Lgr5+ were sorted by fluorescence activated cell sorting.Lgr5+ cells that were transfected with Lgr5-shRNA lentiviral vector and nontarget shRNA lentiviral vector were respectively allocated into the experimental group and control group.The percentage of Lgr5+ cells was analyzed by flow cytometery.The relative expression of Lgr5 mRNA was detected by fluorescence quantitative real-time polymerase chain reaction (qRT-PCR).The capacity of self-renewal was detected by sphere forming assay.The tumorigenesis in vitro and in vivo were respectively measured by colony formation assay and xenografting experiment.The mRNA expressions of stem cells related genes (Oct4,Sox2,Nanog,KLF4),CSCs genes (CD133,CD44,ALDH) and Wnt/β-catenin pathway key genes (Axin2,Wnt5a,Wnt3a,Fzd3,c-myc,VEGF,Ascl2,claudin-1) were detected by qRT-PCR.Measurement data with normal distribution were represented as-x±s.Comparison between groups was analyzed using the t test.Results (1)Transfection efficiency of shRNA lentiviral vector induced Lgr5 by flow cytometery was respectively 6.8％± 1.0％ in the experimental group and 92.7％±3.3％ in the control group,with a statistically significant difference (t =43.148,P＜0.05).The relative expression of Lgr5 mRNA measured by qPT-PCR was respectively 0.168±0.057 in the experimental group and 1.148±0.004 in the control group,with a statistically significant difference (t=28.778,P＜0.05).(2) The capacity of self-renewal was detected by sphere forming assay.The results of sphere forming assay:the number of spheres was 29±6 in the experimental group and 410± 10 in the control group,with a statistically significant difference (t =41.070,P＜0.05).The results of colony formation assay:the numbers of colonies in the experimental group and control group were respectively 72±4 and 412± 19,showing a statistically significant difference (t =31.433,P＜ 0.05).The results of tumorigenesis:the volumes of tumors in the experimental group and control group were respectively (81± 15)mm3 and (328±24)mm3,with a statistically significant difference (t=11.304,P＜0.05).(3) The effects of Lgr5 down-regulation on related genes,results of qRT-PCR detection:① The mRNA relative expressions of Oct4,Sox2,Nanog and KLF4 (stem cells related genes) were 0.377±0.093,0.662±0.104,3.591±0.300,0.425±0.091 in the experimental group and 1.957± 0.026,2.137±0.015,5.831±0.165,1.536±0.014 in the control group,with statistically significant differences (t=23.079,22.261,8.446,19.186,P＜0.05).② The mRNA relative expressions of CD133,CD44 and ALDH (CSCs genes) were 1.490±0.155,5.535±0.487,1.640±0.039 in the experimental group and 2.488± 0.061,9.908±0.332,5.718±0.292 in the control group,with statistically significant differences (t =8.170,9.667,27.849,P＜0.05).③The mRNA relative expressions of Axin2,Wnt5a,Wnt3a,Fzd3,c-myc,VEGF,Ascl2 and claudin-1 genes in the Wnt/β-catenin pathway were respectively 1.592±0.267,0.528±0.138,2.153±0.078,1.480±0.064,0.248±0.128,1.492±0.025,0.658±0.095,1.647±0.087 in the experimental group and 3.651±0.224,2.570±0.093,2.301±0.157,1.636±0.058,1.415±0.080,2.610±0.159,2.480±0.123,3.432±0.273 in the control group.There were statistically significant differences in the mRNA relative expressions of Axin2,Wnt5a,c-myc,VEGF,Ascl2 and claudin-1 genes between the 2 groups (t =7.316,15.332,12.649,12.320,14.831,9.063,P＜0.05),and no statistically significant difference in the mRNA relative expressions of Wnt3a and Fzd3 between the 2 groups (t =2.887,2.242,P＞0.05).Conclusion The malignant behaviors of colorectal CSCs are suppressed after shRNA lentivirus interfered with expression of Lrg5,and its mechanism is related to inhibiting activity of Wnt/β-catenin pathway.