MiR-140-3p.2 enhances killing effect of CD8+T cells on liver cancer cells by targeting PD-L1 expression
Objective To explore the effect of miR-140-3p.2 on cytotoxic T cells(CD8+T cells)killing liver cancer cells by targeting programmed death-ligand 1(PD-L1)expression.Methods QRT-PCR and Western blotting were used to detect miR-140-3p.2,PD-L1 mRNA and protein levels in L-02,HepG2,Hep3B,and HuH-7 cell lines.Targetscan database was used to predict the binding sites of miR-140-3p.2 and PD-L1,and validated using dual luciferase assay.HepG2 cells were divided into overexpressing of miR-140-3p.2(miR-140-3p.2)group and its control(miR-NC)group,overexpressing of PD-L1(PD-L1)group and its control(NC)group,overexpressing of miR-140-3p.2+PD-L1(miR-140-3p.2+PD-L1)group and its control(miR-140-3p.2+NC)group.QRT-PCR and Western blotting were used to detect miR-140-3p.2,PD-L1 mRNA and protein levels in cells.Thirty nude mice were divided into overexpression of miR-140-3p.2(miR-140-3p.2)group and control(miR-NC)group,with 15 mice in each group.Subcutaneous injection of HepG2 cells was used to prepare transplanted tumor model.CD8+T cells from nude mouse spleen tissue was isolated and cocultured with HepG2 cells.Cytotoxicity experiment was used to detect cell lysis rate;ELISA was used to detect TNF-α,IFN-γlevels in cell culture supernatant and transplanted tumors;Flow cytometry was used to detect CD8+T cell infiltration in transplanted tumors.Results Compared with L-02 cells,miR-140-3p.2 levels in HepG2,Hep3B,and HuH-7 cells were decreased,while PD-L1 mRNA and protein levels were increased(P＜0.05);Compared with miR-NC group,miR-140-3p.2 level in miR-140-3p.2 group was increased,PD-L1 mRNA and protein levels were decreased,Cell lysis rate was increased,TNF-α,IFN-γ levels in cell culture supernatant were increased(P＜0.05);Compared with NC group,miR-140-3p.2 level in PD-L1 group was decreased,PD-L1 mRNA and protein levels were inicreased,cell lysis rate was decreased,TNF-α,IFN-γ levels in cell culture supernatant were decreased(P＜0.05);Overexpression of PD-L1 could partially reverse the effect of overexpression of miR-140-3p.2 on the above indicators(P＜0.05).Overexpression of miR-140-3p.2 could enhance the killing ability of CD8+T cells against liver cancer cells in vivo(P＜0.05).After four weeks of tumor formation in nude mice,the tumor volume and mass in miR-140-3p.2 group were significantly decreased compared with that in miR-NC group(P＜0.05),the level of miR-140-3p.2 in transplanted tumors was significantly increased,and the mRNA and protein levels of PD-L1 were significantly decreased(P＜0.05).CD8+T cell infiltration level was significantly increased(P＜0.05),TNF-α,IFN-γ levels were significantly increased(P＜0.05).Conclusion MiR-140-3p.2 enhances the killing effect of CD8+T cells on liver cancer cells by targeting PD-L1 expression.
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