华蟾素通过支链氨基酸氨基转移酶1调控直肠癌细胞增殖的机制研究

Study on the mechanism of controlling the proliferation of rectal cancer cells by cinobufacini on the branched-chain amino acid transferase-1

目的探究华蟾素通过支链氨基酸氨基转移酶1 (branched-chain amino acid transferase-1,BCAT1)调控直肠癌细胞增殖的机制。方法 SW480细胞分为四组:正常对照组、低浓度华蟾素组(22.5μg/L)、中浓度华蟾素组(45μg/L)、高浓度华蟾素组(90μg/L),分别处理12 h、24 h、48 h。QPCR、蛋白质免疫印迹法(Western blot)检测SW480细胞中BCAT1表达水平。采用siRNA沉默BCAT1后,观察SW480细胞的生长状态和凋亡率。各组细胞处理后,CCK-8检测华蟾素对细胞的增殖影响;Q-PCR检测各组细胞BCAT1 mRNA表达水平;Western blot检测各组细胞BCAT1蛋白表达水平。JC-1检测各组细胞线粒体膜电位。结果 Western blot和Q-PCR结果显示SW480细胞中BCAT1高表达。不同浓度华蟾素作用直肠癌细胞后,低浓度华蟾素处理后就可以抑制SW480细胞的增殖,且在不同时间点,华蟾素能够以浓度依赖的方式显著抑制SW480细胞的活力,差异有统计学意义(P<0.05)。分别采用不同浓度华蟾素处理SW480细胞48 h后,检测各组细胞线粒体膜电位,结果显示,低浓度的华蟾素处理后,SW480细胞的线粒体膜电位降低,且随着华蟾素浓度的增高,线粒体膜电位下降显著,差异有统计学意义(P<0.05)。Q-PCR检测各组细胞BCAT1 mRNA的表达量,Western blot检测其蛋白表达量,结果显示,与正常对照组细胞相比,低浓度的华蟾素处理48 h后,BCAT1mRNA、蛋白的表达量出现明显下降,且随着华蟾素浓度的升高,BCAT1 mRNA。蛋白的表达量下降更为明显,呈浓度依赖,差异有统计学意义(P<0.05)。沉默BCAT1后,细胞较正常的SW480细胞增殖率降低,且差异有统计学意义(P<0.05)。结论华蟾素可以通过抑制BCAT1的活性来抑制直肠癌细胞的增殖。

Objective To explore cinobufacini through branched-chain amino acid transferase 1 regulation mechanism of cell proliferation of colon cancer.Methods SW480 cells were divided into four groups:normal control group,low concentration group(22.5 μg/L),medium concentration group(45 μg/L) and high concentration group(90 μg/L).The expression of BCAT1 in SW480 cells was detected by Q-PCR and Western blot.After silencing BCAT1 with siRNA,the growth status and apoptosis rate of SW480 cells were observed.After cell treatment in each group,CCK-8 was used to detect the effect of sinopadin on cell proliferation.The expression level of BCAT1 mRNA in each group was detected by Q-PCR.BCAT1 protein expression was detected by Western blot.The mitochondrial membrane potential was determined by JC-1.Results Western blot and Q-PCR results showed that BCAT1 was highly expressed in SW480 cells.After the action of different concentrations of cinobufacini on rectal cancer cells,low concentration of cinobufacini can inhibit the proliferation of SW480 cells,and at different time points,cinobufacini can significantly inhibit the activity of SW480 cells in a concentrate-dependent manner,the difference is statistically significant(P<0.05).Cinobufacini to deal with different concentrations were used respectively to SW480 cells after 48 h,testing each cell mitochondrial membrane potential,the results show that the low concentration of cinobufacini after processing,the mitochondrial membrane potential is reduced,and with higher cinobufacini concentration,mitochondrial membrane potential decreased more significantly,the difference was statistically significant(P<0.05).Q-PCR was used to detect the expression level of BCAT1 mRNA in each group,and Western blot was used to detect the protein expression level.Compared with the normal control group,the expression levels of BCAT1 mRNA and protein decreased significantly after 48 h of treatment with low concentration of cinobufacini,and BCAT1 mRNA increased with the concentration of cinobufacini.The decrease of protein expression was more obvious,which was concentration-dependent,and the difference was statistically significant(P<0.05).After the silencing of BCAT1,the cell proliferation rate was reduced compared with that of normal SW480 cells,and the difference was statistically significant(P<0.05).Conclusion Cinobufacini can inhibit the proliferation of rectal cancer cells by inhibiting the activity of branched-chain amino acid transferase 1.