MicroRNA-363-mediated Downregulation Of S1PR1Suppresses The Proliferation Of Hepatocellular Carcinoma Cells

The sphingosine-1-phosphate receptor (S1PR1), a direct signaling molecule engagedwith sphingosine1-phosphate (S1P), elicits various downstream responses involved incell proliferation, migration, anti-apoptosis and angiogenesis. S1PR1, a mediator ofsphingosine1-phosphate (S1P), belongs to a family of five G-protein-coupled receptors(S1PR1~5) and exists in various kinds of cells. It is known that S1PR1is an importantsignaling molecular that regulate various signaling pathways.Previous studies have suggested S1PR1plays a crucial role in the occurrence anddevelopment of hepatocellular carcinoma (HCC) acting as an oncogene affecting severalsignaling pathway. At first, HCC cell lines cell express high level of S1PR1compared thenormal hepatic cell L02. Second, S1PR1regulating some genes expression throughinterfering with some classic signaling pathway, include ERK and STAT3. Third, ERKand STAT3signaling pathways are involved in the occurrence of many tumors,promoting tumors development.In summary, S1PR1is an important signaling molecular, over expressed in varioussubtypes of HCC. Therefore, we tried to find an effective approach to down regulate theexpression of S1PR1and deeply understand the mechanisms of the occurrence anddevelopment of HCC.We focused on the microRNA regulatory mechanism of S1PR1gene expression andgot these achievements:1. Bioinformatic analysis predicted a putative binding site of miR-363within the3’-UTR of S1PR1mRNA. And we have identified that the binding site of mir-363onS1PR13’-UTR located at nt1256–1262with the Luciferase reporter assay.2. We have checked the expression levels of endogenous mir-363and S1PR1epxression on HCC cell lines and normal hepatic cell line. We found that HCC cellsexpress high level of S1PR1and low level of miR-363with qPCR and Western Blot.3. With qPCR and Western Blot, we demonstrated that S1PR1could be down regulated by mir-363at both mRNA and protein levels.4. We have miR-363inhibits the proliferation of HCC cells with CCK-8.5. After transfection of mir-363mimics, we indentified that downregulation ofS1PR1by miR-363blocks the activation of ERK and STAT3with Western Blot.6. Employing siRNA targeting S1PR1, we have got similar results to that oftransfection of miR-363into HCC cells.Taken together, the present study demonstrated that miR-363was a negativeregulator of S1PR1expression in HCC cells and inhibited cell proliferation, suggestingthat miR-363/S1PR1pathway might be a novel target for the treatment of HCC.