背景:肝细胞癌(HCC)是全球最常见的恶性肿瘤之一。全反式维甲酸(ATRA)与FOLFOX化疗的组合已显示出增强HCC患者预后的希望。ATRA,作为化学增敏剂,为治疗应用提供了新的可能性。然而,HCC细胞对ATRA的反应性各不相同。表观遗传修饰剂GSK-126目前正在作为一种潜在的抗肿瘤药物进行研究。我们的目的是探索肝癌患者对ATRA不同敏感性的分子机制。并提出了一种新的联合方案。这项研究旨在为肝癌患者的个性化用药奠定基础。
方法:低表达维甲酸受体Alfa(RARA)的细胞模型,视黄酸受体(RARB),通过siRNA干扰建立视黄酸受体γ(RARG)。RARA表达降低的影响,RARB,使用3-(4,5-二甲基-2-噻唑基)-2,5-二苯基四唑溴化物(MTT)细胞毒性测定,评估RARG对ATRA在Hep3B细胞中的半数最大抑制浓度(IC50)的影响。流式细胞术显示RARG是影响组合敏感性的关键受体。通过相关网站对来自HCC细胞的基因组DNA进行ChIP-qPCR分析表明,RARG启动子上游的组蛋白H3(H3K27me3)上赖氨酸27的三甲基化修饰富集。ChIP-PCR检测证实GSK-126可以降低RARG启动子上的H3K27me3水平,随后升高RARG表达。通过MTT法验证GSK-126与ATRA的协同作用,流式细胞术细胞凋亡测定,细胞周期测定,和细胞划痕分析。
结果:我们的研究揭示了HCC细胞对ATRA的不敏感性可能与RARG的低表达有关。ChIP-qPCR分析说明GSK-126通过减少RARG启动子区域中的H3K27me3富集来激活RARG表达。因此,同时给药的ATRA和GSK-126肝癌细胞表现出协同作用,抑制细胞增殖,诱导细胞凋亡,减少S期细胞的比例。
结论:我们的发现强调GSK-126和ATRA的协同作用通过上调RARG的表达来增强HCC细胞的敏感性。这为个性化HCC治疗提供了潜在的基础。
BACKGROUND: Hepatocellular carcinoma (HCC) stands out as one of the most prevalent malignant tumors globally. The combination of all-trans-retinoic acid (
ATRA) with FOLFOX chemotherapy has shown promise in enhancing the prognosis of HCC patients.
ATRA, serving as a chemosensitizing agent, presents novel possibilities for therapeutic applications. Nevertheless, the responsiveness of HCC cells to
ATRA varies. The epigenetic modifier-GSK-126 is currently under investigation as a potential antitumor drug. Our aim is to explore the molecular mechanisms underlying the diverse sensitivity of HCC patients to
ATRA, and to propose a new combination regimen. This research aims to lay the groundwork for personalized medication approaches for individuals with HCC.
METHODS: A cell model with low expression of retinoic acid receptor Alfa (RARA), retinoic acid receptor belta (RARB), and retinoic acid receptor gamma (RARG) was established through siRNA interference. The impact of reduced expression of RARA, RARB, and RARG on the half maximal inhibitory concentration (IC50) of ATRA in Hep3B cells was assessed using the 3-(4,5-Dimethyl-2-Thiazolyl)-2,5-Diphenyl Tetrazolium Bromide (MTT) cytotoxicity assay. Flow cytometry revealed that RARG emerged as the key receptor influencing the combination\'s sensitivity. Conducting ChIP-qPCR analysis on genomic DNA from HCC cells through relevant websites demonstrated enrichment of the trimethylation modification of lysine 27 on histone H3 (H3K27me3) upstream of the RARG promoter. ChIP-PCR assay confirmed that GSK-126 could diminish H3K27me3 levels on the RARG promoter, subsequently elevating RARG expression. The synergistic efficacy of GSK-126 and ATRA was validated through MTT assay, flow cytometry apoptosis assay, cell cycle assay, and cell scratch assay.
RESULTS: Our study unveiled that the insensitivity of HCC cells to
ATRA could be linked to the low expression of RARG. ChIP-qPCR analysis illuminated that GSK-126 activated RARG expression by diminishing H3K27me3 enrichment in the RARG promoter region. Consequently, the concurrent administration of ATRA and GSK-126 to hepatoma cells exhibited a synergistic effect, inhibiting cell proliferation, inducing cell apoptosis, and reducing the proportion of cells in the S-phase.
CONCLUSIONS: Our findings emphasize that the synergistic action of GSK-126 and ATRA enhances the sensitivity of HCC cells by upregulating the expression of RARG. This presents a potential foundation for personalized HCC treatment.