背景:SGI-1027是公认的DNA甲基转移酶1(DNMT1)抑制剂,早期的研究表明,胃癌(GC)中DNMT1表达失调与视网膜母细胞瘤1(RB1)基因表达呈负相关。尽管有这些知识,SGI-1027在GC细胞中作用的确切机制仍未被充分理解。本研究的主要目的是阐明SGI-1027对GC细胞行为的影响,包括生长和转移潜力等方面,通过干预DNMT1,从而影响RB1基因表达的调控。
方法:获取正常胃粘膜细胞系GES-1和人胃癌细胞系MKN45后,采用Westernblot(WB)和定量逆转录-聚合酶链反应(qRT-PCR)技术评价RB1和DNMT1在这两种细胞系中的表达水平。随后,MKN45细胞系在含有不同浓度SGI-1027的培养基中培养,并使用WB和qRT-PCR技术重新评估SGI-1027对GC细胞中RB1和DNMT1调节的影响。为了仔细检查SGI-1027对GC细胞的影响,我们利用3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2H四唑溴化物(MTT)测定来确定细胞增殖,并进行Transwell实验来评估细胞迁移和侵袭能力.在整个过程中,我们还使用WB评估细胞周期相关蛋白(CyclinD1,CyclinE1和CyclinB1)和细胞凋亡相关蛋白(BCL-2相关蛋白X凋亡调节因子(BAX)和B细胞淋巴瘤2凋亡调节因子(BCL-2))的水平.此外,我们将以最佳浓度的SGI-1027培养的MKN45细胞系和MKN45细胞系分别注射入小鼠皮下和通过尾静脉注射5天和10天,将它们划分为模型组,型号+SGI-10275d组,和Model+SGI-102710D组。我们监测了小鼠肿瘤大小和体积的变化,收集肿瘤组织和肺组织进行苏木精和曙红(HE)染色。最后,使用WB和免疫组织化学(IHC)技术检测GC组织中的DNMT1表达水平。此外,使用WB评估GC组织中的RB1表达水平。
结果:与GES-1细胞相比,MKN45细胞表现出独特的特征谱,其特征在于DNMT1表达增加和RB1表达减少(p<0.05)。然而,在引入SGI-1027后,观察到GC细胞内DNMT1水平显着降低,伴随着RB1基因表达的升高,以25μmol/LSGI-1027为最佳浓度(p<0.05)。功能测定表明,SGI-1027处理的GC细胞表现出明显的抑制增殖特征,迁移,当与未处理的MKN45细胞相比时(p<0.05)。此外,在SGI-1027处理的GC细胞中,CyclinD1,CyclinE1,CyclinB1和BCL-2的水平显着降低,而BAX的表达水平升高(p<0.05)。值得注意的是,在25μmol/LSGI-1027时观察到最明显的影响,进一步强调了其对肿瘤细胞行为的调节作用(p<0.05)。在动物实验中,模型组肿瘤体积显著增加,HE染色结果提示大部分胃组织广泛坏死和明显的肺转移征象,伴随着DNMT1表达增加和RB1基因表达减少。相比之下,SGI-1027组显示胃肿瘤体积减少,坏死减少,肺肿瘤转移减少(p<0.05)。此外,DNMT1的表达在SGI-1027处理的GC细胞中显著降低,而RB1表达增加(p<0.05),进一步证实了SGI-1027对肿瘤生长和转移的抑制作用。
结论:SGI-1027通过下调DNMT1和促进RB1的表达,有效阻碍GC细胞的增殖和播散。
BACKGROUND: SGI-1027 is a recognized inhibitor of DNA methyltransferase 1 (DNMT1), and earlier investigations have indicated an inverse correlation between dysregulated DNMT1 expression in gastric cancer (GC) and retinoblastoma 1 (RB1) gene expression. Despite this knowledge, the precise mechanisms underlying the action of SGI-1027 in GC cells remain inadequately comprehended. The primary objective of this study is to elucidate the impact of SGI-1027 on the behavior of GC cells, encompassing aspects such as growth and metastatic potential, by intervening in DNMT1, thereby influencing the regulation of RB1 gene expression.
METHODS: The acquisition of the normal gastric mucosal cell line GES-1 and the human gastric cancer cell line MKN45 was followed by employing Western blot (WB) and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) techniques to evaluate the expression levels of RB1 and DNMT1 in these two cell lines. Subsequently, the MKN45 cell line was cultured in medium containing varying concentrations of SGI-1027, and the impact of SGI-1027 on the regulation of RB1 and DNMT1 in GC cells was reassessed using WB and qRT-PCR techniques. To scrutinize the effect of SGI-1027 on GC cells, we utilized the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay to determine cell proliferation and performed Transwell experiments to assess cell migration and invasion capabilities. Throughout this process, we also employed WB to assess the levels of cell cycle-associated proteins (Cyclin D1, Cyclin E1, and Cyclin B1) and proteins related to apoptosis (BCL-2 associated protein X apoptosis regulator (BAX) and B-cell lymphoma 2 apoptosis regulator (BCL-2)). Furthermore, we injected the MKN45 cell line and MKN45 cell line cultured with the optimal concentration of SGI-1027 for 5 days and 10 days into mice subcutaneously and through the tail vein, dividing them into the Model group, Model+SGI-1027 5d group, and Model+SGI-1027 10d group. We monitored changes in tumor size and volume in mice, and tumor tissues as well as lung tissues were collected for hematoxylin and eosin (HE) staining. Finally, DNMT1 expression levels in GC tissues were detected using both WB and immunohistochemistry (IHC) techniques. Additionally, RB1 expression levels in GC tissues were assessed using WB.
RESULTS: In contrast to GES-1 cells, MKN45 cells displayed a distinctive profile characterized by increased DNMT1 expression and decreased RB1 expression (p < 0.05). However, upon the introduction of SGI-1027, a notable decrease in DNMT1 levels within GC cells was observed, concomitant with an elevation in RB1 gene expression, with 25 μmol/L SGI-1027 identified as the optimal concentration (p < 0.05). Functional assays demonstrated that SGI-1027-treated GC cells exhibited pronounced features of inhibited proliferation, migration, and invasion when compared to untreated MKN45 cells (p < 0.05). Moreover, in SGI-1027-treated GC cells, the levels of Cyclin D1, Cyclin E1, Cyclin B1, and BCL-2 were significantly reduced, while the expression level of BAX increased (p < 0.05). Notably, the most pronounced impact was observed at 25 μmol/L SGI-1027, further underscoring its regulatory effects on tumor cell behavior (p < 0.05). In animal experiments, the Model group exhibited a substantial increase in tumor volume, with HE staining results indicating extensive necrosis in most gastric tissues and noticeable signs of lung metastasis, accompanied by increased DNMT1 expression and decreased RB1 gene expression. In contrast, the SGI-1027 group displayed a reduction in gastric tumor volume, decreased necrosis, and reduced lung tumor metastasis (p < 0.05). Additionally, the expression of DNMT1 was significantly reduced in SGI-1027-treated GC cells, while RB1 expression increased (p < 0.05), further confirming the inhibitory effects of SGI-1027 on tumor growth and metastasis.
CONCLUSIONS: SGI-1027 effectively hinders the proliferation and dissemination of GC cells by downregulating DNMT1 and promoting the expression of RB1.