Background Sphingosine-1-phosphate receptor 3 (S1PR3) plays an important role in the development and progression of various tumors; however, its specific function in hepatocellular carcinoma (HCC) remains unclear.

Objective To investigate the impact of S1PR3 on the malignant biological behavior of hepatocellular carcinoma (HCC) and elucidate its underlying mechanisms.

Methods The human hepatocellular carcinoma cell lines Huh7 and HepG2 were randomly devided into two groups, and treated with the SIPR3-specific inhibitor TY-52156 and dimethyl sulfoxide (DMSO) separately. Cell proliferation was assessed by CCK-8 assay; migration and invasion abilities were evaluated using Transwell assays; differentially expressed genes and potential signaling pathways were identified by transcriptome sequencing; key molecular expression levels were verified by Western blot; additionally, a hepatocellular carcinoma organoid model was established, and cell viability after inhibitor treatment was measured by CellTiter-Glo assay.

Results Compared with the control group, CCK-8 and Transwell assays showed that TY-52156 significantly inhibited the proliferation, migration, and invasion of Huh7 and HepG2 cells (P < 0.05). Transcriptome sequencing and subsequent analyses suggested that argininosuccinate synthetase 1 (ASS1) night act as a downstream effector of S1PR3 in this regulatory process. Western blot results demonstrated that S1PR3 inhibition upregulated ASS1 expression (P < 0.05). In the organoid model, CellTiter-Glo assay revealed that TY-52156 treatment induced significant death of tumor organoids (P < 0.001).

Conclusion Inhibition of S1PR3 reduces the proliferation, migration, and invasion capacities of HCC cell lines. As a downstream differentially expressed molecule of S1PR3, ASS1 is negatively regulated by S1PR3. The S1PR3 inhibitor effectively kills HCC organoids.