Li Yao, Cai Yi, Duan Guangliang, Du Yulin, Hu Junjie, Han Xue, Tang Xiaolong, Jiang Liangxian, Ren Junqi, Huo Qi
Anhui Provincial Key Laboratory of Immunology in Chronic Diseases, Anhui Provincial Key Laboratory of Infection and Immunology, and Department of Laboratory Medicine, Bengbu Medical University, Anhui, China.
Department of Laboratory Medicine, School of Medicine, Beihua University, Jilin, China.
Sci Prog. 2025 Jul-Sep;108(3):368504251381592. doi: 10.1177/00368504251381592. Epub 2025 Sep 22.
ObjectiveSorafenib is a key targeted therapeutic agent for hepatocellular carcinoma (HCC). However, the emergence of drug resistance greatly limits its clinical efficacy. We found that GPR89A is markedly overexpressed in both sorafenib-resistant HCC cell lines and clinical tumor specimens. Nevertheless, the exact biological role of GPR89A in sorafenib-resistant HCC remains to be elucidated.MethodsTo generate sorafenib-resistant HCC cell lines, cells were cultured in medium with 10 μM sorafenib. GPR89A expression in these cells was examined by quantitative reverse transcription-polymerase chain reaction and Western blot. The impact of GPR89A inhibition on cell proliferation was assessed using cell counting kit-8 and colony formation assays. Bioinformatics predicted miR-199a-5p binding sites in the GPR89A promoter region; luciferase reporter assays were conducted with wild-type or mutant promoter sequences. Glutamate in culture supernatants was measured by enzyme-linked immunosorbent assay, and exogenous glutamate showed dose-dependent inhibition of resistant cell proliferation (MTT assay). Clinical correlation analysis involved 30 pairs of advanced HCC samples. Pearson analysis revealed significant inverse correlations among miR-199a-5p, GPR89A, and metabotropic glutamate receptor 1 (mGluR1) expression.ResultsWe found that the cell surface transmembrane protein GPR89A was substantially overexpressed in both sorafenib-resistant HCC cell lines and clinical tumor specimens. Importantly, higher GPR89A expression levels were positively correlated with an increased degree of sorafenib resistance in clinical samples. In sorafenib-resistant HCC tissues, the expression of both GPR89A and mGluR1 was significantly upregulated, whereas miR-199a-5p levels were markedly reduced. Correlation analysis revealed a negative association between GPR89A and miR-199a-5p expression, and a positive association between GPR89A and mGluR1. Mechanistically, miR-199a-5p directly targets GPR89A mRNA to suppress its expression, thereby downregulating mGluR1 and reducing glutamate levels.ConclusionsThese findings uncover a novel regulatory axis linking GPR89A overexpression, glutamate metabolic reprogramming, and the development of acquired sorafenib-resistant HCC.
目的
索拉非尼是肝细胞癌（HCC）的关键靶向治疗药物。然而，耐药性的出现极大地限制了其临床疗效。我们发现GPR89A在索拉非尼耐药的HCC细胞系和临床肿瘤标本中均显著过表达。然而，GPR89A在索拉非尼耐药HCC中的确切生物学作用仍有待阐明。
方法
为构建索拉非尼耐药的HCC细胞系，将细胞在含10 μM索拉非尼的培养基中培养。通过定量逆转录-聚合酶链反应和蛋白质免疫印迹法检测这些细胞中GPR89A的表达。使用细胞计数试剂盒-8和集落形成试验评估GPR89A抑制对细胞增殖的影响。生物信息学预测了miR-199a-5p在GPR89A启动子区域的结合位点；用野生型或突变型启动子序列进行荧光素酶报告基因试验。通过酶联免疫吸附试验测定培养上清液中的谷氨酸，外源性谷氨酸对耐药细胞增殖有剂量依赖性抑制作用（MTT试验）。临床相关性分析涉及30对晚期HCC样本。Pearson分析显示miR-199a-5p、GPR89A和代谢型谷氨酸受体1（mGluR1）表达之间存在显著负相关。
结果
我们发现细胞表面跨膜蛋白GPR89A在索拉非尼耐药的HCC细胞系和临床肿瘤标本中均大量过表达。重要的是，在临床样本中，较高的GPR89A表达水平与索拉非尼耐药程度增加呈正相关。在索拉非尼耐药的HCC组织中，GPR89A和mGluR1的表达均显著上调，而miR-199a-5p水平明显降低。相关性分析显示GPR89A与miR-199a-5p表达呈负相关，与mGluR1呈正相关。机制上，miR-199a-5p直接靶向GPR89A mRNA以抑制其表达，从而下调mGluR1并降低谷氨酸水平。
结论
这些发现揭示了一个新的调控轴，将GPR89A过表达、谷氨酸代谢重编程与获得性索拉非尼耐药HCC的发生联系起来。
