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

doi:10.13481/j.1671-587X.20250310

摘要/Abstract

摘要：

目的探讨沉默生物节律基因TIMELESS（TIM）对卵巢癌细胞免疫逃逸的影响，并阐明其相关作用机制。方法分离CD8+T淋巴细胞，并采用流式细胞术进行鉴定，检测细胞中CD3+/CD8+细胞亚群比例。体外培养人卵巢癌SK-OV-3细胞，分别转染TIM小干扰RNA（siRNA）干扰质粒（si-TIM）、阴性对照质粒（si-NC）、程序性死亡配体1（PD-L1）过表达质粒（oe-PD-L1）及其阴性对照质粒（oe-NC），分为空白对照组（BC组，不进行转染）、si-NC组（转染si-NC）、si-TIM组（转染si-TIM）、si-NC+oe-NC组（转染si-NC和oe-NC）和si-TIM+oe-PD-L1（转染si-TIM和oe-PD-L1）组。采用实时荧光定量PCR（RT-qPCR）法和Western blotting法检测SK-OV-3细胞中TIM mRNA及蛋白表达水平，验证TIM基因沉默情况。将转染后的SK-OV-3细胞与激活的CD8+T淋巴细胞共培养，分为BC组（单独培养SK-OV-3细胞）、BC/T组、si-NC/T组、si-TIM/T组、si-NC+oe-NC/T组和si-TIM+oe-PD-L1/T组，采用CCK-8法检测各组SK-OV-3细胞存活率，流式细胞术检测各组SK-OV-3细胞凋亡率和细胞表面PD-L1阳性表达率，酶联免疫吸附试验（ELISA）法检测共培养上清液中干扰素γ（IFN-γ）和肿瘤坏死因子α（TNF-α）水平，乳酸脱氢酶（LDH）释放法检测各组CD8+T淋巴细胞杀伤力，RT-qPCR法检测各组SK-OV-3细胞中TIM和PD-L1 mRNA表达水平，Western blotting法检测各组SK-OV-3细胞中TIM和PD-L1蛋白表达水平。结果免疫磁珠法分离后，CD8+T淋巴细胞（CD3+/CD8+）亚群比例为96.56%±0.59%，提示所提取CD8+T淋巴细胞纯度较高。与BC组比较，si-TIM组细胞中TIM mRNA和蛋白表达水平均明显降低（P<0.01），提示本研究成功获得TIM基因沉默的卵巢癌SK-OV-3细胞。CCK-8法，与BC组比较，BC/T组SK-OV-3细胞存活率明显降低（P<0.01）；与BC/T组比较，si-TIM/T组SK-OV-3细胞存活率明显降低（P<0.01）。流式细胞术，与BC组比较，BC/T组SK-OV-3细胞凋亡率明显升高（P<0.01）；与BC/T组比较，si-TIM/T组SK-OV-3细胞凋亡率明显升高（P<0.01）；与si-TIM/T组比较，si-TIM+oe-PD-L1/T组SK-OV-3细胞凋亡率明显降低（P<0.01）。与BC组比较，si-TIM组SK-OV-3细胞表面PD-L1阳性表达率明显降低（P<0.01）。ELISA法，与BC/T组比较，si-TIM/T组细胞培养上清液中IFN-γ和TNF-α水平均明显升高（P<0.01）；与si-TIM/T组比较，si-TIM+oe-PD-L1/T组细胞培养上清液中IFN-γ和TNF-α水平均明显降低（P<0.01）。LDH释放法，与BC/T组比较，si-TIM/T组CD8+T淋巴细胞杀伤力明显增强（P<0.01）；与si-TIM/T组比较，si-TIM+oe-PD-L1/T组CD8+T淋巴细胞杀伤力明显减弱（P<0.01）。RT-qPCR法和Western blotting法，与BC组比较，si-TIM组SK-OV-3细胞中PD-L1 mRNA和蛋白表达水平均明显降低（P<0.01）；与si-TIM组比较，si-TIM+oe-PD-L1组细胞中PD-L1蛋白表达水平明显升高（P<0.01）。结论 TIM基因沉默可增强CD8+T淋巴细胞对卵巢癌SK-OV-3细胞的杀伤作用，抑制其免疫逃逸，其作用机制可能与调控PD-L1蛋白表达有关。

关键词: 卵巢癌, 免疫逃逸, TIMELESS基因, CD8+T淋巴细胞, 程序性死亡配体1

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

中图分类号:

R737.31

顾雨玲,郑翠,汤云仙. 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[J]. 吉林大学学报(医学版), 2025, 51(3): 653-662.

Yuling GU,Cui ZHENG,Yunxian TANG. Inhibitory effect of silencing of circadian rhythm gene TIMELESS on immune escape of ovarian cancer SK-OV-3 cells and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2025, 51(3): 653-662.

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Group	IFN-γ	TNF-α
BC/T	72.63±6.16	103.79±9.71
Si-NC/T	68.71±9.57	108.86±9.29
Si-TIM/T	133.32±9.91^*	202.29±14.00^*

Group	IFN-γ	TNF-α
BC/T	72.89±4.55	106.78±6.01
Si-NC+oe-NC/T	74.03±6.13	109.44±10.03
Si-TIM/T	134.83±11.28^*	209.53±9.59^*
Si-TIM+oe-PD-L1/T	92.57±4.25^△	153.55±8.23^△