miR-199a-5p/GPR89A axis modulates sorafenib resistance in hepatocellular carcinoma through glutamate metabolic reprogramming.

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.