天马颗粒对结肠直癌细胞中FLI-1启动子甲基化影响

Effects of Tianma Granule on methylation of FLI-1 promoter in colorectal cancer cells

目的研究天马颗粒对结直肠癌细胞中Friend白血病整合素1转录因子(Friend leukemia integration 1,FLI-1)启动子甲基化的作用机制。方法以结直肠癌细胞HCT116、HT29、Caco-2为研究对象,每种细胞分为3组:Blank组、NC组、TMKL组。Blank组为空白对照,NC组予以空白血清干预,TMKL组予以天马颗粒血清干预。采用CCK8筛选天马颗粒血清最佳干预浓度,Western blot检测各组细胞中FLI-1蛋白表达,qPCR法检测各组细胞FLI-1 mRNA表达,ELISA法检测DNA甲基转移酶1(DNA methyltransferase 1,DNMT1)、DNA甲基转移酶3a(DNA methyltransferase 3a,DNMT3a)蛋白表达,甲基化特异性PCR(methylation-Specific PCR,MSP)检测各组细胞中FLI-1启动子甲基化。结果天马颗粒血清对HCT116、HT29和Caco-2细胞增殖有抑制作用,其中20%的天马颗粒血清浓度抑制效果最佳。在HCT116细胞分组和Caco-2细胞分组中,同Blank组、NC组相比,TMKL组FLI-1蛋白及mRNA表达升高(P<0.05),Blank组同NC组相比差异无统计学意义(P>0.05);在HT29细胞分组中,Blank组、NC组和TMKL组FLI-1蛋白及mRNA表达差异无统计学意义(P>0.05)。在HCT116、HT29和Caco-2细胞中,同Blank组、NC组相比,TMKL组DNMT1、DNMT3a的表达量下降(P<0.05),Blank组同NC组相比差异无统计学意义(P>0.05);在HCT116细胞中,同Blank组、NC相比,TMKL组细胞FLI-1启动子产生了部分去甲基化,甲基化减弱,非甲基化增多,NC组同Blank相比无变化;在HT29细胞中,Blank组、NC组和TMKL组细胞FLI-1启动子无甲基化;在Caco-2细胞中,同Blank组、NC相比,TMKL组细胞FLI-1启动子完全去甲基化,NC组同Blank相比无变化。结论天马颗粒可能通过抑制DNMT1、DNMT3a蛋白表达,促使结直肠癌细胞中FLI-1启动子去甲基化,进而上调其表达。

Objective To study the mechanism of Tianma Granule on methylation of friend leukemia integrin 1(FLI-1)transcription factor promoter in colorectal cancer cells.Methods Colorectal cancer cells HCT116,HT29 and Caco-2 were divided into three groups: Blank group, NC group and TMKL group. Blank group was blank control. NC group was intervened with blank serum. TMKL group was intervened with Tianma Granule serum. Cell counting kit-8 (CCK-8) was used to screen the optimal intervention concentration of Tianma Granule serum. Western blot was taken to detect the expression of FLI-1 protein in each group. The expression of FLI-1 mRNA in each group was detected by qPCR. ELISA was used to detect the expression of DNA methyltransferase 1 (DNMT1) and DNA methyltransferase 3a (DNMT3a) protein. Methylation-specific PCR (MSP) was used to detect the methylation of FLI-1 promoter in each group. Results Tianma Granule serum inhibited the proliferation of HCT116, HT29 and Caco-2 cells. And 20% serum concentration of Tianma Granule had the best inhibitory effect. Compared with Blank group and NC group, in HCT116 and Caco-2 cell groups, the expression of FLI-1 protein and mRNA in TMKL group increased (P<0.05), while there was no difference between Blank group and NC group (P>0.05). In HT29 cell group, there was no difference in the expression of FLI-1 protein and mRNA among Blank group, NC group and TMKL group (P>0.05). Compared with Blank group and NC group, in HCT116, HT29 and Caco-2 cells, the expression of DNMT1 and DNMT3a in TMKL group decreased (P<0.05), while there was no difference between Blank group and NC group (P>0.05). Compared with blank group and NC group, in HCT116 cells, FLI-1 promoter in TMKL group produced partial demethylation, decreased methylation and increased non-methylation. And there was no change in NC group compared with blank group. In HT29 cells, there was no methylation of FLI-1 promoter in blank group, NC group and TMKL group. Compared with Blank group and NC group, in Caco-2 cells, the FLI-1 promoter in TMKL group was completely demethylated, and there was no change in NC group compared with Blank group. Conclusion Tianma Granule may promote the demethylation of FLI-1 promoter in colorectal cancer cells by inhibiting the expression of DNMT1 and DNMT3a proteins, which resulted in up-regulating the expression.