S-腺苷甲硫氨酸通过hepaCAM抑制膀胱癌细胞增殖迁移

S-adenosylmethionine inhibits proliferation and migration of bladder cancer cells via hepaCAM

摘要:

目的：探究S-腺苷甲硫氨酸（S-adenosylmethionine，SAM）通过hepaCAM对膀胱癌（bladder cancer，BCa）细胞增殖和迁移的影响及其可能的分子机制。方法：不同浓度SAM作用于体外培养的T24和BIU细胞，四甲基偶氮唑盐（3-（4，5-dimethylth－iazol-2-yl）-2，5-diphenyltetrazolium bromide，MTT）法检测SAM对细胞增殖的影响。用不同浓度的SAM（0、0.8、1.6 mmol/L）处理T24和BIU细胞72 h后，Real-time PCR和Western blot法检测甲基结合蛋白-2（methyl DNA-binding domain protein，MBD2）、组蛋白去乙酰化酶-1（histone deacetylases，HDAC1）以及肝细胞黏附分子（hepatocyte cell adhesion molecule，hepaCAM）的表达；划痕及Transwell实验检测各组细胞的迁移能力；克隆形成实验检测各组细胞的克隆形成能力。结果：SAM抑制T24和BIU细胞生长且与药物浓度和作用时间呈正相关。与0 mmol/L组相比，0.8 mmol/L的SAM处理T24和BIU细胞72 h后MBD2 mRNA表达明显降低（P=0.048；P=0.038），蛋白表达明显降低（P=0.001；P=0.000）；0.8 mmol/L的SAM处理T24细胞72 h后HDAC1 mRNA和蛋白水平明显降低（P=0.000；P=0.001）；0.8 mmol/L的SAM处理BIU细胞72 h后HDAC1 mRNA水平无统计学差异（P=0.140），蛋白表达明显降低（P=0.004）；0.8 mmol/L的SAM处理T24和BIU细胞72 h后hepaCAM mRNA水平明显增加（P=0.003；P=0.008），蛋白表达无统计学差异（P=0.770；P=0.381）；1.6 mmol/L的SAM处理T24和BIU细胞72 h后MBD2 mRNA水平明显降低（P=0.003；P=0.000），蛋白表达明显降低（P=0.001；P=0.000）；HDAC1 mRNA水平明显降低（均P=0.000），蛋白表达明显降低（P=0.001；P=0.000）；hepaCAM mRNA和蛋白表达均明显增加（均P=0.000）。划痕及Transwell实验结果显示SAM处理T24和BIU细胞72 h后迁移能力受到明显抑制（P<0.05）。克隆形成实验结果显示SAM处理T24和BIU细胞72 h后细胞克隆形成能力明显降低（P<0.05）。结论：SAM可通过MBD2/HDAC1逆转hepaCAM的表达，抑制BCa细胞的增殖及迁移能力。

Abstract:

Objective：To study the effect of S-adenosylmethionine（SAM） on the proliferation and migration of bladder cancer（BCa） cells via hepatocyte cell adhesion molecule（hepaCAM） and its potential molecular mechanism. Methods：T24 and BIU cells cultured in vitro were treated with different concentrations of SAM，and the effect of SAM on cell proliferation was evaluated by MTT assay. After treatment of T24 and BIU cells with different concentrations（0，0.8，1.6 mmol/L） of SAM for 72 h，real-time PCR and Western blot were used to measure the expression of methyl-CpG-binding domain protein 2（MBD2），histone deacetylase 1（HDAC1），and hepaCAM；wound healing assay and Transwell assay were used to measure the migration of cells in each group；colony formation assay was used to measure the colony formation ability of cells in each group. Results：SAM showed an inhibitory effect on the growth of T24 and BIU cells in a manner positively correlated with the con－centration and duration of treatment. After treatment of T24 and BIU cells with 0.8 mmol/L SAM for 72 h，the mRNA and pro－tein expression levels of MBD2 decreased significantly com－pared with those in the 0 mmol/L group（mRNA：P=0.048 and 0.038，respectively；protein：P=0.001 and 0.000，respectively）；the mRNA expression level of HepaCAM increased significantly（P=0.003 and 0.008，respectively），but there were no significant differences in the protein expression level of HepaCAM（P=0.770 and 0.381，respectively）. After treatment of T24 cells with 0.8 mmol/L SAM for 72 h，the mRNA and protein expression levels of HDAC1 decreased significantly（P=0.000 and 0.001，respectively）；after treatment of BIU cells with 0.8 mmol/L SAM for 72 h，the protein expression level of HDAC1 decreased significantly（P=0.004），but there was no sig－nificant difference in the mRNA expression level of HDAC1（P=0.14）. After the treatment of T24 and BIU cells with 1.6 mmol/L SAM for 72 h，there were significant decreases in the mRNA and protein expression levels of MBD2（mRNA：P=0.003 and 0.000，re－spectively；protein：P=0.001 and 0.000，respectively） and HDAC1（mRNA：P=0.000 and 0.000，respectively；protein：P=0.001 and 0.000，respectively），but there were significant increases in the mRNA and protein expression levels of hepaCAM（all P=0.000）. After treat－ment of T24 and BIU cells with SAM for 72 h，cell migration was significantly inhibited as demonstrated by the wound healing assay and Transwell assay（P<0.05），and cell colony formation ability was significantly reduced as demonstrated by the colony formation assay（P<0.05）. Conclusion：SAM can inhibit the proliferation and migration of BCa cells by reversing the expression of hepaCAM via MBD2/HDAC1.