Inhibitory effect of silencing of circadian rhythm gene TIMELESS on immune escape of ovarian cancer SK-OV-3 cells and its mechanism
10.13481/j.1671-587X.20250310
- VernacularTitle:沉默生物节律基因TIMELESS对卵巢癌SK-OV-3细胞免疫逃逸的抑制作用及其机制
- Author:
Yuling GU
1
;
Cui ZHENG
;
Yunxian TANG
Author Information
1. 南京医科大学附属苏州医院 苏州市立医院妇产科,江苏 南京 215000
- Keywords:
Ovarian cancer;
Immune escape;
TIMELESS gene;
CD8+T lymphocytes;
Programmed death ligand 1
- From:
Journal of Jilin University(Medicine Edition)
2025;51(3):653-662
- CountryChina
- Language:Chinese
-
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.
