华蟾素毒基通过JAK2/STAT3途径对人结肠癌细胞增殖、迁移及侵袭的影响

Effects of cinbufagin on proliferation,migration and invasion of human colon cancer cells via JAK2/STAT3 pathway

目的 研究华蟾素毒基(CB)对人结肠细胞HCT116增殖、迁移和侵袭能力以及上皮-间质转化(EMT)的影响。方法 取对数生长期的HCT116细胞,随机分为空白组以及低、中、高剂量实验组,空白组不做任何处理(0 nmol·L^(-1)),低、中、高剂量实验组分别用含17.5、35、70 nmol·L^(-1)华蟾素毒基的1640培养基培养48 h。用细胞计数-8(CCK-8)法检测华蟾素毒基对HCT116细胞存活率的影响;用克隆实验检测华蟾素毒基对HCT116细胞增殖的影响;用细胞划痕实验和Transwell实验检测华蟾素毒基对HCT116细胞迁移和侵袭能力的影响;用蛋白质印迹法检测HCT116细胞中酪氨酸蛋白激酶2(JAK2)/信号转导和转录激活因子3(STAT)途径以及EMT相关蛋白表达水平。结果 空白组和低、中、高剂量实验组的克隆形成数分别为(122.67±24.42)、(73.67±15.82)、(44.33±4.51)和(21.67±1.53)个,划痕迁移率分别为(44.64±9.15)%、(26.91±2.94)%、(19.28±1.52)%和(6.33±2.30)%,侵袭细胞数分别为(120.33±1.15)、(58.33±9.07)、(33.33±1.53)和(18.33±3.21)个,磷酸化JAK-2(p-JAK-2)/JAK-2的蛋白相对表达水平分别为1.02±0.06、0.94±0.05、0.75±0.22和0.49±0.22,磷酸化STAT3(p-STAT3)/STAT3的蛋白相对表达水平分别为0.89±0.10、0.72±0.04、0.65±0.06和0.52±0.18,E-cadherin蛋白相对表达水平分别为0.30±0.14、0.41±0.13、0.49±0.14和0.69±0.17,N-cadherin的蛋白相对表达水平分别为0.96±0.11、0.78±0.04、0.69±0.12和0.40±0.15,Snail蛋白相对表达水平分别为0.89±0.08、0.62±0.15、0.44±0.15和0.27±0.09,Vimentin蛋白相对表达水平分别为0.92±0.09、0.76±0.13、0.63±0.01和0.43±0.09,高剂量实验组的上述指标与空白组比较,在统计学上差异均有统计学意义(均P<0.05)。结论 华蟾素毒基可以通过JAK2/STAT3通路抑制上皮-间质转化从而抑制人结肠癌细胞HCT116的侵袭与转移。

Objective To investigate the effects of cinbufagin(CB) on the proliferation,migration and invasion ability as well as epithelialmesenchymal transition(EMT) of human colon cells HCT116.Methods Logarithmically grown HCT116 cells were randomly divided into blank group and experimental-L,-M,-H groups;the blank group did not receive any treatment(0 nmol·L^(-1)),and experimental-L,-M,-H groups were cultured in 1 640 medium containing 17.5,35 and70 nmol·L^(-1) cinbufagin for 48 h.Cell counting kit-8(CCK-8) was used to detect the effect of cinbufagin on the survival rate of HCT116 cells;cloning assay was used to detect the effect of cinbufagin on the proliferation of HCT116 cells;cell scratch assay and Transwell assay were used to detect the effect of cinbufagin on the migration and invasive ability of HCT116 cells;Western blot was used to detect the expression levels of janus kinase 2(JAK2)/signal transducers and activators of transcription 3(STAT3) pathway and EMTrelated proteins of HCT116 cells.Results The number of clone formation in blank group and experimental-L,-M,-H groups were 122.67±24.42,73.67±15.82,44.33±4.51 and 21.67±1.53;the rates of migration of scratches were(44.64±9.15) %,(26.91±2.94) %,(19.28±1.52) % and(6.33±2.30) %;the number of invaded cells were 120.33±1.15,58.33±9.07,33.33±1.53 and 18.33±3.21;the relative protein expression of phosphorylated JAK-2(p-JAK-2)/JAK-2 were 1.02±0.06,0.94±0.05,0.75±0.22 and 0.49±0.22;relative protein expression of phosphorylated STAT3(p-STAT3)/STAT3 were 0.89±0.10,0.72±0.04,0.65±0.06 and0.52±0.18;relative protein expression of E-cadherin were 0.30±0.14,0.41±0.13,0.49±0.14 and0.69±0.17;relative protein expression of N-cadherin were 0.96±0.11,0.78±0.04,0.69±0.12 and0.40±0.15;Snail relative protein expression were 0.89±0,08,0.62±0.15,0.44±0.15 and 0.27±0.09;Vimentin relative protein expression were 0.92±0.09,0.76±0.13,0.63±0.01 and 0.43±0.09,respectively.The above indexes in experimental-H group showed statistically significant differences compared to blank group(all P <0.05).Conclusion HCT116 can inhibit the invasion and metastasis of human colorectal cancer cells HCT116 by inhibiting epithelial-mesenchymal transition through JAK2/STAT3 pathway.