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目的通过基于DLEC1基因表观遗传学调控探讨重楼皂苷Ⅰ抗卵巢癌作用及其分子机制。方法 MTT法检测不同浓度重楼皂苷Ⅰ(5、10、15与20μmol/L)对卵巢癌SK-OV-3及OVCAR-3细胞增殖的影响;实时荧光定量PCR及Western blot法检测重楼皂苷Ⅰ对SK-OV-3与OVCAR-3细胞DLEC1表达的影响;RNAi技术及MTT法检测DLEC1基因对重楼皂苷Ⅰ抑制SK-OV-3及OVCAR-3细胞增殖作用的影响;甲基化特异性PCR及甲基化DNA免疫沉淀-qPCR技术检测重楼皂苷Ⅰ对SK-OV-3及OVCAR-3细胞中DLEC1基因启动子甲基化的影响。结果重楼皂苷Ⅰ可显著抑制SK-OV-3及OVCAR-3细胞增殖(P<0.05),且具有一定的浓度依赖性;可显著上调SK-OV-3及OVCAR-3细胞中DLEC1基因m RNA转录及蛋白的表达水平(P<0.05);可显著增强SK-OV-3及OVCAR-3细胞中DLEC1基因启动子的去甲基化作用(P<0.05)。同时,下调DLEC1表达可明显减弱重楼皂苷1对SK-OV-3及OVCAR-3细胞增殖抑制作用(P<0.05)。结论重楼皂苷Ⅰ可通过表观遗传学调控对DLEC1基因启动子发挥去甲基化作用,进而诱导DLEC1的表达而发挥抗卵巢癌作用。
OBJECTIVE: To investigate the anti-ovarian cancer effect and its molecular mechanism by polysaccharide Ⅰ based on the regulation of epigenetic regulation of DLEC1 gene. Methods The proliferation of SK-OV-3 ovarian cancer cells and OVCAR-3 ovarian cancer cells were detected by MTT assay. The effects of polysaccharide saponins Ⅰ (5, 10, 15 and 20 μmol / L) Ⅰ on the expression of DLEC1 in SK-OV-3 and OVCAR-3 cells. The effect of DLEC1 gene on the proliferation of SK-OV-3 and OVCAR-3 cells was detected by RNAi and MTT assay. The methylation-specific PCR and methylation-specific DNA immunoprecipitation (qPCR) were used to detect the methylation of DLEC1 promoter in SK-OV-3 and OVCAR-3 cells. Results Saponin Ⅰ significantly inhibited the proliferation of SK-OV-3 and OVCAR-3 cells (P <0.05) in a concentration-dependent manner and significantly up-regulated the expression of DLEC1 gene in SK-OV-3 and OVCAR- RNA transcription and protein expression (P <0.05). The demethylation of DLEC1 gene promoter in SK-OV-3 and OVCAR-3 cells was significantly enhanced (P <0.05). Meanwhile, down-regulation of DLEC1 expression significantly attenuated the inhibitory effect of polysaccharide Saponin 1 on the proliferation of SK-OV-3 and OVCAR-3 cells (P <0.05). Conclusions Saponin Ⅰ can demethylate the promoter of DLEC1 gene through epigenetic regulation, and then induce the expression of DLEC1 to exert its anti-ovarian cancer effect.