目的 探究 miR-140-3p.2靶向程序性死亡受體配體 1（PD-L1）表達(dá)對(duì)細(xì)胞毒性 T細(xì)胞（CD8+ T細(xì)胞）殺傷肝癌細(xì)胞的影響。方法 實(shí)時(shí)熒光定量 PCR（qRT-PCR）、Western blotting 檢測(cè) L-02 和 HepG2、Hep3B、HuH-7 細(xì)胞系中 miR-140-3p.2和PD-L1 mRNA和蛋白水平；Targetscan數(shù)據(jù)庫(kù)預(yù)測(cè)miR-140-3p.2和PD-L1的結(jié)合位點(diǎn)，并利用雙熒光素酶實(shí)驗(yàn)進(jìn)行驗(yàn)證；HepG2 細(xì)胞分為過(guò)表達(dá) miR-140-3p.2（miR-140-3p.2）組及其對(duì)照（miR-NC）組、過(guò)表達(dá) PD-L1（PD-L1）組及其對(duì)照（NC）組、過(guò)表達(dá) miR-140-3p.2 和 PD-L1（miR-140-3p.2＋PD-L1）組、過(guò)表達(dá) miR-140-3p.2 和 PD-L1 對(duì)照（miR-140-3p.2＋NC）組；qRT-PCR、Western blotting實(shí)驗(yàn)分別檢測(cè)各組細(xì)胞中 miR-140-3p.2和 PD-L1 mRNA 和蛋白水平。30只裸鼠分為過(guò)表達(dá) miR-140-3p.2（miR-140-3p.2）組及其對(duì)照（miR-NC）組，每組各 15 只，采用 sc 過(guò)表達(dá) miR-140-3p.2 及其對(duì)照（miR-NC）的HepG2細(xì)胞制備移植瘤模型；檢測(cè)腫瘤質(zhì)量及體積；分離裸鼠脾臟組織CD8+ T細(xì)胞，并與各轉(zhuǎn)染HepG2細(xì)胞共培養(yǎng)，細(xì)胞毒性實(shí)驗(yàn)檢測(cè)細(xì)胞裂解率，ELISA法檢測(cè)細(xì)胞培養(yǎng)上清液中腫瘤壞死因子-α（TNF-α）、γ干擾素（IFN-γ）水平；ELISA 法檢測(cè)移植瘤中 TNF-α、IFN-γ水平，流式細(xì)胞術(shù)檢測(cè)移植瘤中 CD8⁺ T 細(xì)胞浸潤(rùn)情況。結(jié)果 與 L-02細(xì)胞相比，HepG2、Hep3B、HuH-7細(xì)胞中miR-140-3p.2水平降低，PD-L1 mRNA和蛋白水平升高（P＜0.05）。與miR-NC組相比，miR-140-3p.2組細(xì)胞中miR-140-3p.2水平升高，PD-L1 mRNA和蛋白水平降低，細(xì)胞裂解率升高，細(xì)胞培養(yǎng)上清液中TNF-α、IFN-γ水平升高（P＜0.05）；與 NC組相比，PD-L1組細(xì)胞中 miR-140-3p.2水平降低，PD-L1 mRNA 和蛋白水平升高 ，細(xì)胞裂解率降低，細(xì)胞培養(yǎng)上清液中 TNF- α、IFN- γ 水平降低（P＜0.05）；過(guò)表達(dá) PD-L1能部分逆轉(zhuǎn)過(guò)表達(dá) miR-140-3p.2 對(duì)上述指標(biāo)的影響（P＜0.05）。裸鼠成瘤 4 周后，與 miR-NC 組相比，miR-140-3p.2 組移植瘤體積和質(zhì)量明顯減?。≒＜0.05），移植瘤中 miR-140-3p.2 水平明顯升高、PD-L1 mRNA 和蛋白水平明顯降低（P＜0.05），CD8⁺ T 細(xì)胞浸潤(rùn)水平明顯升高（P＜0.05），TNF-α、IFN-γ水平明顯升高（P＜0.05）。結(jié)論 miR-140-3p.2靶向PD-L1表達(dá)增強(qiáng)CD8⁺ T細(xì)胞對(duì)肝癌細(xì)胞的殺傷作用。

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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