Abstract:

Objective To discuss the effect of circadion rhythm gene TIMELESS （TIM） silencing on immune escape of the ovarian cancer cells， and to clarify its related mechanism. Methods The CD8+T lymphocytes were isolated and identified by flow cytometry to detect the proportion of CD3+/CD8+ cell subsets. The human ovarian cancer SK-OV-3 cells were cultured in vitro and divided into interference plasmid transfected with TIM small interfering （siRNA） （si-TIM）， negative control plasmid （si-NC）， programmed death ligand 1 （PD-L1） over-expression plasmid （oe-PD-L1）， and negative control plasmid （oe-NC） groups. The cells were further divided into blank control group （BC group， non-transfection）， si-NC group （transfected with si-NC）， si-TIM group（transfected with si-TIM）， si-NC+oe-NC group （transfected with si-NC and oe-NC）， and si-TIM+oe-PD-L1 group （transfected with si-TIM and oe-PD-L1）. Real-time fluorescence quantitative PCR（RT-qPCR） and Western blotting methods were used to detect the expression levels of TIM mRNA and protein in the SK-OV-3 cells to verify TIM gene silencing. The transfected SK-OV-3 cells were co-cultured with activated CD8+T lymphocytes and divided into BC group （SK-OV-3 cells cultured alone）， BC/T group， si-NC/T group， si-TIM/T group， si-NC+oe-NC/T group， and si-TIM+oe-PD-L1/T group. CCK-8 method was used to detect the survival rates of the SK-OV-3 cells in various groups； flow cytometry was used to detect the apoptotic rates of the SK-OV-3 cells and positive expression rate of PD-L1 on surface of the cells in various groups； enzyme-linked immunosorbent assay （ELISA） method was used to detect the levels of interferon-γ （IFN-γ） and tumor necrosis factor-α （TNF-α） in the co-culture supernatant； lactate dehydrogenase （LDH） release assay was used to detect the cytotoxicity of the CD8+T lymphocytes in various groups； RT-qPCR method was used to detect the expression levels of TIM and PD-L1 mRNA in the SK-OV-3 cells in various groups； Western blotting method was used to detect the expression levels of TIM and PD-L1 proteins in the SK-OV-3 cells in various groups. Results After scparated with immune magnetic bead method， the proportion of CD8+T lymphocyte （CD3+/CD8+） subsets was （96.56%±0.59%）， indicating high purity of the extracted CD8+T lymphocytes. Compared with BC group， the expression levels of TIM mRNA and protein in the cells in si-TIM group were significantly decreased （P<0.01）， suggesting successful TIM gene silencing in the ovarian cancer SK-OV-3 cells. The CCK-8 results showed that compared with BC group， the survival rate of the SK-OV-3 cells in BC/T group was significantly decreased （P<0.01）； compared with BC/T group， the survival rate of the SK-OV-3 cells in si-TIM/T group was significantly decreased （P<0.01）. The flow cytometry results showed that compared with BC group， the apoptotic rate of the SK-OV-3 cells in BC/T group was significantly increased （P<0.01）； compared with BC/T group， the apoptotic rate of the SK-OV-3 cells in si-TIM/T group was significantly increased （P<0.01）； compared with si-TIM/T group， the apoptotic rate of the SK-OV-3 cells in si-TIM+oe-PD-L1/T group was significantly decreased （P<0.01）. Compared with BC group， the positive expression rate of PD-L1 on surface of the SK-OV-3 cells in si-TIM group was significantly decreased （P<0.01）. The ELISA results showed that compared with BC/T group， the levels of IFN-γ and TNF-α in the culture supernatant in si-TIM/T group were significantly increased （P<0.01）； compared with si-TIM/T group， the levels of IFN-γ and TNF-α in the supernatant in si-TIM+oe-PD-L1/T group were significantly decreased （P<0.01）. The LDH release assay results showed that compared with BC/T group， the cytotoxicity of the CD8+T lymphocytes in si-TIM/T group was significantly increased （P<0.01）； compared with si-TIM/T group， the cytotoxicity of the CD8+T lymphocytes in si-TIM+oe-PD-L1/T group was significantly weakened （P<0.01）. The RT-qPCR and Western blotting results showed that compared with BC group， the expression levels of PD-L1 mRNA and protein in the SK-OV-3 cells in si-TIM group were significantly decreased （P<0.01）； compared with si-TIM group， the expression level of PD-L1 protein in the cells in si-TIM+oe-PD-L1 group was significantly increased （P<0.01）. Conclusion TIM gene silencing enhances the cytotoxic effect of CD8+T lymphocytes on ovarian cancer SK-OV-3 cells and inhibits immune escape， and its mechanism may be related to the regulation of PD-L1 protein expression.

Key words: Ovarian cancer, Immune escape, TIMELESS gene, CD8+T lymphocytes, Programmed death ligand 1