二烯丙基二硫在RORα抑制人胃癌细胞上皮间质转化中的作用

Up-regulation of RORα inhibits EMT in human gastric MGC803 cells induced by diallyl disulfide

摘要:

目的探讨二烯丙基二硫（DADS）是否上调维甲酸相关孤核受体α（RORα）抑制人胃癌细胞株MGC803 细胞上皮- 间质转化（EMT）。方法相差显微镜观察MGC803 细胞形态的改变。逆转录PCR（RTPCR）、蛋白免疫印迹法（Western blot）、免疫荧光与免疫组织化学检测EMT 的相关分子表达。裸鼠实验检测DADS 与沉默RORα对移植瘤生长的影响。结果相差显微镜显示，沉默RORα细胞较MGC803 细胞大小与形状差异更明显。DADS作用后，细胞大小较一致，呈圆形或椭圆形，梭形细胞减少，异型性下降。RT-PCR与Western blot显示，DADS 处理后较处理前各组RORα mRNA 与蛋白表达上调（p <0.05）。DADS 作用对照组与空载体组较沉默组效果更为明显（p <0.05）。RORα沉默较对照组和空载体组细胞锌指转录因子（Snail）和波形蛋白（Vimentin）mRNA与蛋白表达上调，而E- 钙黏蛋白（E-cadherin）mRNA 与蛋白下调（p <0.05）。DADS处理后，各组Snail蛋白下调、Vimentin mRNA与蛋白下调和E-cadherin mRNA与蛋白上调（p <0.05）。免疫荧光显示，沉默组细胞Snail与Vimentin阳性表达较对照组增强，而E-cadherin阳性表达减弱。DADS作用后，结果相反。裸鼠移植瘤实验显示，RORα沉默组移植瘤较对照组生长加快和体重增加（p <0.05），增殖细胞核抗原（Ki-67）、Snail、CD34及Vimentin 阳性表达较对照组增加，而E-cadherin阳性降低。DADS 处理各组移植瘤生长与体积减慢与减小，Ki-67、Snail、CD34与Vimentin阳性表达较对照组减弱，而E-cadherin阳性表达增强。结论DADS 通过上调RORα 可体内外抑制人胃癌MGC803 细胞EMT。

Abstract:

Objective To investigate whether diallyl disulfide (DADS) inhibits epithelial mesenchymal trasition (EMT) in human gastric cancer MGC803 cells by up-regulation of retinoid acid receptor related orphan receptor α(RORα). Methods The morphological effect of MGC803 cells was observed by phase contrast microscope. The expressions of molecules correlated to EMT were detected by RT -PCR, Western blot, immunofluorescence and immunohistochemistry. The influence of DADS and silencing RORαon the growth of xenograft tumor in athymic mice was observed. Results Phase contrast microscopy showed that MGC803 cells AGAAATCCTGCTCTCCTCGCCTof RORα silence were discordancy in size and shape and the number of spindle cells increased. Besides, the MGC803 cells treated by DADS were found with characteristics such as size unification, round or ellipse shape, decreased spindle cells and less atypia. RT-PCR and Western blot revealed up-regulation of RORα mRNA and protein in each group treated by DADS (p < 0.05). Moreover, the effect in control group and the vector group increased compared to the RORα silence group treated by DADS (p < 0.05). RT-PCR and Western blot showed the up-regulation of Snail protein and Vimentin mRNA and protein, and down-regulation of E-cadherin mRNA and protein in the RORα silence cells (p < 0.05). However, in the MGC803 cells treated by DADS Snail and Vimentin were down-regulated and E-cadherin was up-regulated (p < 0.05).Immunofluorescence showed that the positive expressions of Snail and Vimentin in the RORα silence cells were strengthened, but E-cadherin was attenuated. Nevertheless, the positive expressions of Snail and Vimentin were attenuated, and E-cadherin was strengthened in the MGC803 cells treated by DADS. The growth of the xenograft tumor was accelerated, and the weight of the transplantation tumor increased in the RORα silence group compred to the control group (p < 0.05). The positive expressions of Ki-67, Snail, CD34 and Vimentin were obviously increased, while the positive rate of E-cadherin also increased in the RORα silence group. Inversely, the rate of the xenograft tumor growth slowed down and the volume of tumor was significantly diminished. The expressions of Ki-67, Snail, CD34 and Vimentin decreased and the positive expression of Ecadherin increased in the MGC803 cells treated by DADS. Conclusions DADS can inhibit EMT in MGC803 cell in vivo and in vitro through up-regulation of RORα.