Cholecystokinin-B receptor epigenetic regulation and targeted therapy in hepatocellular carcinoma

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.