PO.TB03.03 · 肿瘤生物学
胆囊收缩素B受体在肝细胞癌中的表观遗传调控与靶向治疗
Cholecystokinin-B receptor epigenetic regulation and targeted therapy in hepatocellular carcinoma
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：代谢功能障碍和膳食饱和脂肪导致肝细胞癌（HCC）发病率上升。胆囊收缩素B受体（CCK-BR）在HCC中表达上调，然而驱动其表达的上游机制仍未明确。新兴证据提示，microRNA失调和DNA甲基化可能促进致癌性受体信号传导。本研究探讨饱和脂肪是否通过对miR-148a的表观遗传抑制诱导CCK-BR表达，并评估CCK-BR拮抗是否可在体内抑制HCC进展。
方法：
表观遗传调控：将HCC细胞用棕榈酸处理8、24和48小时。采用qRT-PCR定量miR-148a和CCK-BR mRNA的表达。为验证miRNA依赖性调控，使用Lipofectamine 2000将miR-148a模拟物或阴性对照瞬时转染Hepa1-6细胞。为评估饮食诱导的DNA甲基化，将高脂饮食（HFD）小鼠和对照小鼠肝脏的基因组DNA进行亚硫酸氢盐转化，并在miR-148启动子的CpG富集区进行扩增。PCR产物克隆入pCR2.1-TOPO载体，使用PyroMark MD仪器和亚硫酸氢盐焦磷酸测序进行测序。为检验可逆性，将HCC细胞用5-氮杂胞苷处理，并重新评估miR-148a和CCK-BR的表达。
体内治疗靶向：将表达荧光素酶的RIL-175 HCC细胞原位注射至C57BL/6小鼠肝脏。恢复后，小鼠（每组N=10）分别饮用含丙谷胺处理或未处理的水。每周通过IVIS成像监测肿瘤生长和转移。第5周时，通过ELISA评估血清甲胎蛋白（AFP），并分析肝脏的肿瘤负荷和转移扩散。
结果：预期棕榈酸抑制miR-148a并增加CCK-BR表达，而miR-148a模拟物转染逆转此效应。预测HFD肝脏显示miR-148启动子高甲基化，并与CCK-BR升高相关。5-氮杂胞苷应可恢复miR-148a并下调CCK-BR。在体内，预期丙谷胺治疗可降低肿瘤通量、AFP水平、肿瘤大小和转移。
结论：这些研究界定了一种将饱和脂肪暴露与HCC中CCK-BR表达上调相联系的表观遗传机制，并提供了支持将CCK-BR拮抗作为代谢驱动性肝癌治疗策略的临床前证据。
查看英文原文 English abstract
Background: Metabolic dysfunction and dietary saturated fat contribute to rising hepatocellular carcinoma (HCC) rates. The cholecystokinin-B receptor (CCK-BR) is upregulated in HCC, yet the upstream mechanisms driving its expression remain undefined. Emerging evidence suggests that microRNA dysregulation and DNA methylation may promote oncogenic receptor signaling. This study investigates whether saturated fat induces CCK-BR expression through epigenetic repression of miR-148a and evaluates whether CCK-BR antagonism suppresses HCC progression in vivo.
Methods:
Epigenetic Regulation: HCC cells were treated with palmitic acid for 8, 24, and 48 hours. qRT-PCR quantified miR-148a and CCK-BR mRNA expression. To validate miRNA-dependent regulation, Hepa1-6 cells were transiently transfected with miR-148a mimics or negative controls using Lipofectamine 2000. To assess diet-induced DNA methylation, genomic DNA from livers of high-fat diet (HFD) and control mice was bisulfite-converted and amplified across CpG-rich regions of the miR-148 promoter. PCR products were cloned into pCR2.1-TOPO vectors and sequenced using PyroMark MD instrument and bisulfite pyrosequencing. To test reversibility, HCC cells are treated with 5-azacytidine, and expression of miR-148a and CCK-BR reassessed.
In Vivo Therapeutic Targeting: Luciferase-expressing RIL-175 HCC cells are orthotopically injected into C57BL/6 mouse livers. After recovery, mice (N=10/group) received either proglumide-treated or untreated water. Tumor growth and metastasis were monitored weekly via IVIS imaging. At week 5, serum alpha-fetoprotein (AFP) was assessed by ELISA, and livers were analyzed for tumor burden and metastatic spread.
Results: Palmitic acid is expected to suppress miR-148a and increase CCK-BR expression, with miR-148a mimic transfection reversing this effect. HFD livers are predicted to show hypermethylation of the miR-148 promoter correlating with elevated CCK-BR. 5-azacytidine should restore miR-148a and downregulate CCK-BR. In vivo, proglumide therapy is anticipated to reduce tumor flux, AFP levels, tumor size, and metastasis.
Conclusions: These studies define an epigenetic mechanism linking saturated fat exposure to CCK-BR upregulation in HCC and provide preclinical evidence supporting CCK-BR antagonism as a therapeutic strategy for metabolically driven liver cancer.
利益披露 Disclosure
M. Gay, None..
H. Cao, None..
W. Chen, None..
J. P. Smith, None.