具核梭杆菌介导的上皮细胞间质转化对结直肠癌干细胞恶性潜能的影响

Fusobacterium nucleatum-mediated epithelial-mesenchymal transition promotes malignant potentials of colorectal cancer stem cells

目的通过体外内实验明确具核梭杆菌(F_(n))对结直肠肿瘤干细胞(CSCs)恶性潜能的影响并初步探讨相关分子机制。方法采用流式细胞术检测结直肠癌细胞系HCT-116、HT-29、LOVO、SW480和SW620中CD133^(+)CD44^(+)细胞亚群比例,并选择比例最高的细胞系进行筛选。采用细胞计数试剂盒8(CCK-8)、克隆形成及Transwell实验分别检测F_(n)干预后CD133^(+)CD44^(+)细胞亚群增殖、克隆形成、侵袭及迁移能力的改变。将裸鼠随机分为F_(n)干预组和对照组,构建裸鼠皮下移植瘤模型,明确F_(n)干预后CD133^(+)CD44^(+)细胞亚群体内成瘤能力的改变。采用蛋白质印迹法检测F_(n)干预后CD133^(+)CD44^(+)细胞亚群中上皮标志物、间质标志物及细胞增殖指标的表达水平。结果流式细胞术检测发现HCT-116细胞系中CD133^(+)CD44^(+)细胞亚群所占比例最高,为(87.59±0.37)%,因此选择该细胞系用于后续实验。CCK-8实验结果表明,F_(n)干预(F_(组别)=156.53,P_(组别)<0.001)及生长天数(F_(天数)=410.79,P_(天数)<0.001)的主效应差异有统计学意义,且两者联合有协同交互作用(F_(组别×天数)=19.83,P_(组别×天数)<0.001)。F_(n)干预组的克隆形成数目多于对照组,t=19.11,P<0.001。F_(n)干预组的细胞侵袭和迁移数量分别为(115.67±8.14)和(131.00±6.56)个,均高于对照组,均P<0.05。F_(n)干预组及对照组的皮下移植瘤体积分别为(1.66±0.57)和(0.69±0.23)cm^(3),重量分别为(1.66±0.40)和(0.58±0.24)g,均P<0.05。免疫组化实验结果显示,F_(n)干预组皮下移植瘤中Ki-67染色阳性率〔(80.60±5.86)%〕高于对照组〔(60.40±11.99)%〕,t=3.39,P=0.016。蛋白质印迹实验显示,F_(n)干预组较对照组E-cadherin表达水平下调,而N-cadherin、Snail及增殖细胞核抗原的表达水平上调。结论F_(n)通过调控上皮细胞间质转化促进CD133^(+)CD44^(+)结直肠癌细胞亚群的体内外恶性潜能,因此,靶向F_(n)有望成为抑制结直肠CSCs进而预防肿瘤复发转移的新策略。

To clarify the impact of Fusobacterium nucleatum(F_(n))on the malignant potentials of colorectal cancer stem cells(CSCs)and related molecular mechanisms in colorectal cancer.Methods Flow cytometry was used to detect the proportion of CD133^(+)CD44^(+)cell subsets in colorectal cancer cell lines HCT-116,HT-29,LOVO,SW480and SW620,and the cell line with the highest proportion was selected for screening.Cell counting kit 8(CCK-8),clone formation and transwell assays were used to detect the changes of proliferation,clone formation,invasion and migration of CD133^(+)CD44^(+)cell subsets after F_(n)intervention,respectively.The nude mice were randomly divided into the F_(n)intervention group and the control group,and the subcutaneous xenograft tumor model of nude mice was constructed to clarify the changes in the tumorigenic ability of CD133^(+)CD44^(+)cell subsets after F_(n)intervention.Western blotting was used to detect the expression levels of epithelial markers,mesenchymal markers and cell proliferation indexes in CD133^(+)CD44^(+)cell subsets after F_(n)intervention.Results Flow cytometry showed that the CD133^(+)CD44^(+)cell subset accounted for the highest proportion in HCT-116cell line,which was(87.59±0.37)%,so this cell line was selected for subsequent experiments.The results of the CCK-8experiment showed that the main effects of F_(n)intervention(F_(group)=156.53,P_(group)<0.001)and growth days(F_(days)=410.79,P_(days)<0.001)had statistically significant differences,and the combination of the two had significantlly synergistic interaction(F_(group×days)=19.83,P_(group×days)<0.001).The number of clones formed in the F_(n)intervention group was more than that in the control group,t=19.11,P<0.001.The numbers of cell invasion and migration in the F_(n)intervention group were(115.67±8.14)and(131.00±6.56)cells,respectively,which were higher than those in the control group,all P<0.05.The volume of the subcutaneously transplanted tumor in the F_(n)intervention group and the control group were(1.66±0.57)and(0.69±0.23)cm^(3),respectively,and the weight were(1.66±0.40)and(0.58±0.24)g,respectively,all P<0.05.The results of immunohistochemical experiments showed that the positive rate of Ki-67staining in subcutaneous transplanted tumors in F_(n)intervention group[(80.60±5.86)%]was higher than that in control group[(60.40±11.99)%],t=3.39,P=0.016.Western blotting experiments showed that the expression level of E-cadherin in the F_(n)intervention group was down-regulated compared with the control group,while the expression levels of N-cadherin,Snail and proliferating cell nuclear antigen were up-regulated.Conclusions F_(n)promotes the malignant potential of CD133^(+)CD44^(+)colorectal cancer cell subsets invitroand in vivo by regulating epithelial-mesenchymal transition.Therefore,targeting F_(n)is expected to be a new strategy to inhibit colorectal CSCs and prevent tumor recurrence and metastasis.