PO.TB10.01 · 肿瘤生物学
SLC12A2通过抑制脂解作用驱动肝细胞癌中促肿瘤微环境和不良预后
SLC12A2 drives a tumor-promoting microenvironment and poor prognosis in hepatocellular carcinoma by suppressing lipolysis
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
肝细胞癌（HCC）常发生于脂肪变性和纤维化的肝脏，然而将代谢失调与促肿瘤肝微环境联系起来的分子因素仍未完全明确。我们旨在鉴定与HCC风险相关的转运体基因，并阐明其在脂肪性肝炎相关肝癌发生中的机制作用。使用GSE10143队列，我们基于总生存期的Cox比例风险模型，对从转运体分类数据库（Transporter Classification Database）整理的617个转运体基因进行预后相关性筛选。候选基因通过对2016至2020年间获取的94例代谢功能障碍相关脂肪性肝病（MASLD）患者（其中12例合并HCC）肝活检标本的RNA测序进行验证，研究已获机构伦理批准。功能研究在C57BL/6小鼠中进行，通过AAV8在肝脏过表达溶质载体家族12成员2（SLC12A2）——一种Na⁺-K⁺-Cl⁻协同转运体——随后喂养胆碱缺乏、L-氨基酸限定的高脂饮食（CDAHFD）6周以诱导饮食相关脂肪性肝炎。体外实验使用正常肝细胞系和稳定过表达SLC12A2或经shRNA介导敲低的Hep3B细胞进行。从转运体基因筛选中，SLC12A2对不良生存显示出最高的风险比，并且还与肝切除术后晚期（>2年）复发增加相关，提示背景肝组织的致癌潜能增强。在MASLD队列中，HCC病例的肝脏SLC12A2表达显著更高，并与纤维化分期和肝脏硬度呈正相关。在小鼠中，肝脏SLC12A2过表达加剧了CDAHFD诱导的脂肪性肝炎——表现为肝重量、血清ALT水平、肝脏甘油三酯含量、天狼星红阳性面积和纤维化基因表达的增加。切割型caspase-3和Ki-67染色显示肝细胞凋亡和代偿性增殖增加，这是慢性肝损伤的典型特征。原位植入Hepa1-6细胞导致肿瘤显著增大，证明了促肿瘤微环境。体外，SLC12A2过表达增强了棕榈酸诱导的脂滴形成，而shRNA介导的敲低则抑制了脂滴形成。参与脂肪酸合成和氧化的基因表达保持不变，但甘油释放减少，表明脂解抑制是主要机制。我们的发现提示，SLC12A2通过抑制脂解作用促进肝脂肪变性并培育促肿瘤微环境。其过表达可识别出易发生HCC的高风险MASLD肝脏，并代表代谢性肝病相关肝癌发生的潜在治疗靶点。
查看英文原文 English abstract
Hepatocellular carcinoma (HCC) frequently arises from steatotic and fibrotic livers, yet molecular factors linking metabolic dysregulation to tumor-promoting hepatic microenvironments remain incompletely defined. We aimed to identify transporter genes associated with HCC risk and elucidate their mechanistic role in steatohepatitis-related hepatocarcinogenesis. Using the GSE10143 cohort, we screened 617 transporter genes curated from the Transporter Classification Database for prognostic relevance based on Cox proportional hazards modeling of overall survival. Candidate genes were validated by RNA sequencing of liver biopsy specimens from 94 patients with metabolic dysfunction-associated steatotic liver disease (MASLD) (12 with HCC) obtained between 2016 and 2020, following institutional ethical approval. Functional studies were conducted in C57BL/6 mice with hepatic overexpression of solute carrier family 12 member 2 (SLC12A2)-a Na⁺-K⁺-Cl⁻ cotransporter-via AAV8, followed by feeding with a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) for six weeks to induce diet-associated steatohepatitis. In vitro assays were performed using a normal hepatocyte cell line and Hep3B cells with stable SLC12A2 overexpression or shRNA-mediated knockdown. From the transporter gene screening, SLC12A2 showed the highest hazard ratio for poor survival and was also associated with increased late (>2 years) post-hepatectomy recurrence, implying enhanced carcinogenic potential of background liver tissue. In the MASLD cohort, hepatic SLC12A2 expression was significantly higher in HCC cases and positively correlated with fibrosis stage and liver stiffness. In mice, hepatic SLC12A2 overexpression exacerbated CDAHFD-induced steatohepatitis-evidenced by increased liver weight, serum ALT levels, hepatic triglyceride content, Sirius red-positive area, and fibrogenic gene expression. Cleaved caspase-3 and Ki-67 staining indicated increased hepatocellular apoptosis and compensatory proliferation, features typical of chronic liver injury. Orthotopic implantation of Hepa1-6 cells resulted in significantly larger tumors, demonstrating a tumor-promoting microenvironment. In vitro, SLC12A2 overexpression enhanced palmitate-induced lipid droplet formation, whereas shRNA-mediated knockdown suppressed it. Expression of genes involved in fatty acid synthesis and oxidation remained unchanged, but glycerol release was decreased, indicating suppression of lipolysis as the primary mechanism. Our findings suggest that SLC12A2 promotes hepatic steatosis and fosters a tumor-promoting microenvironment by repressing lipolysis. Its overexpression identifies high-risk MASLD livers predisposed to HCC and represents a potential therapeutic target for metabolic liver disease-associated hepatocarcinogenesis.
利益披露 Disclosure
Y. Kudo, None..
R. Tateishi, None..
M. Fujishiro, None.