目的 探究 miR-140-3p.2靶向程序性死亡受体配体 1（PD-L1）表达对细胞毒性 T细胞（CD8+ T细胞）杀伤肝癌细胞的影响。方法 实时荧光定量 PCR（qRT-PCR）、Western blotting 检测 L-02 和 HepG2、Hep3B、HuH-7 细胞系中 miR-140-3p.2和PD-L1 mRNA和蛋白水平；Targetscan数据库预测miR-140-3p.2和PD-L1的结合位点，并利用双荧光素酶实验进行验证；HepG2 细胞分为过表达 miR-140-3p.2（miR-140-3p.2）组及其对照（miR-NC）组、过表达 PD-L1（PD-L1）组及其对照（NC）组、过表达 miR-140-3p.2 和 PD-L1（miR-140-3p.2＋PD-L1）组、过表达 miR-140-3p.2 和 PD-L1 对照（miR-140-3p.2＋NC）组；qRT-PCR、Western blotting实验分别检测各组细胞中 miR-140-3p.2和 PD-L1 mRNA 和蛋白水平。30只裸鼠分为过表达 miR-140-3p.2（miR-140-3p.2）组及其对照（miR-NC）组，每组各 15 只，采用 sc 过表达 miR-140-3p.2 及其对照（miR-NC）的HepG2细胞制备移植瘤模型；检测肿瘤质量及体积；分离裸鼠脾脏组织CD8+ T细胞，并与各转染HepG2细胞共培养，细胞毒性实验检测细胞裂解率，ELISA法检测细胞培养上清液中肿瘤坏死因子-α（TNF-α）、γ干扰素（IFN-γ）水平；ELISA 法检测移植瘤中 TNF-α、IFN-γ水平，流式细胞术检测移植瘤中 CD8⁺ T 细胞浸润情况。结果 与 L-02细胞相比，HepG2、Hep3B、HuH-7细胞中miR-140-3p.2水平降低，PD-L1 mRNA和蛋白水平升高（P＜0.05）。与miR-NC组相比，miR-140-3p.2组细胞中miR-140-3p.2水平升高，PD-L1 mRNA和蛋白水平降低，细胞裂解率升高，细胞培养上清液中TNF-α、IFN-γ水平升高（P＜0.05）；与 NC组相比，PD-L1组细胞中 miR-140-3p.2水平降低，PD-L1 mRNA 和蛋白水平升高 ，细胞裂解率降低，细胞培养上清液中 TNF- α、IFN- γ 水平降低（P＜0.05）；过表达 PD-L1能部分逆转过表达 miR-140-3p.2 对上述指标的影响（P＜0.05）。裸鼠成瘤 4 周后，与 miR-NC 组相比，miR-140-3p.2 组移植瘤体积和质量明显减小（P＜0.05），移植瘤中 miR-140-3p.2 水平明显升高、PD-L1 mRNA 和蛋白水平明显降低（P＜0.05），CD8⁺ T 细胞浸润水平明显升高（P＜0.05），TNF-α、IFN-γ水平明显升高（P＜0.05）。结论 miR-140-3p.2靶向PD-L1表达增强CD8⁺ T细胞对肝癌细胞的杀伤作用。

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-γ levels 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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