HIF-1α通过调控miR-544对A549肺癌细胞免疫逃逸及NCR1/NKP46通路的影响

Effect of HIF-1α on immune escape and NCR1/NKP46 pathway of A549 lung cancer cells by regulating miR-544

目的探究缺氧诱导因子-1α(HIF-1α)在A549肺癌细胞免疫逃逸中的作用,并初步探究可能作用机制。方法体外培养A549肺癌细胞,转染si-HIF-1α质粒分为Control组、si-NC组、si-HIF-1α组;转染miR-544 mimics质粒分为Control组、NC组、miR-544 mimics组。免疫印迹法检测转染后细胞中HIF-1α蛋白表达;实时荧光定量PCR(qRT-PCR)法miR-544表达;将转染后三组细胞与NK-92MI细胞以靶效比1:5共培养,Elisa法检测培养液中干扰素-γ(IFN-γ)、白介素-2(IL-2)、肿瘤坏死因子-ɑ(TNF-ɑ)水平;CCK8法检测NK-92MI细胞免疫杀伤率;荧光素酶活性检测HIF-1α、miR-544(miR-544、NCR1)调控关系;流式细胞术检测细胞NKP46水平。结果与Control、si-NC组相比,si-HIF-1α组A549细胞中HIF-1α表达降低,共培养组细胞IFN-γ、IL-2、TNF-ɑ水平升高,NK-92MI细胞对A549细胞免疫杀伤率升高,miR-544表达升高(P<0.05)。在HIF-1α3’UTR-Wt细胞中,与si-NC组相比,miR-544 mimics组荧光素酶活性降低(P<0.05)。与Control、NC组相比,miR-544 mimics组IFN-γ、IL-2、TNF-ɑ水平降低,NK-92MI细胞对A549肺癌细胞免疫杀伤率降低,NCR1表达、NKP46比例降低(P<0.05)。在NCR1 3’UTR-Wt细胞中,与NC组相比,miR-544 mimics组荧光素酶活性降低(P<0.05)。结论体外抑制A549肺癌细胞中HIF-1α表达后能够促进NK细胞释放免疫因子,增强NK细胞免疫杀伤性,进而抑制A549细胞免疫逃逸,其作用可能与调控miR-544靶向抑制NCR1/NKP46通路实现的。

Objective To investigate the role of hypoxia inducible factor-1α(HIF-1α) in immune escape of A549 lung cancer cells, and to explore the possible mechanism. Methods A549 lung cancer cells were cultured in vitro. The transfected si-HIF-1α plasmid was divided into the control group, the si-NC group and the si-HIF-1α group. The transfected miR-544 MICs plasmid was divided into the control group, the NC group and the miR-544 MICs group. The expression of HIF-1α protein in transfected cells was detected by Western blot, and real-time fluorescence quantitative PCR(qRT-PCR) was used to detect the expression of miR-544. After transfection, cells in the three groups were co-cultured with NK-92 MI cells at a target-to-effect ratio of 1:5, the levels of IFN-γ, IL-2 and TNF-α in the culture medium were detected by Elisa method, the killing rate of NK-92 MI cells was detected by CCK8 assay, luciferase activity was used to detect the regulation of HIF-1α, miR-544(miR-544, NCR1), and the NKP46 level was detected by flow cytometry. Results Compared with the control and si-NC groups, the expression of HIF-1α in A549 cells in the si-HIF-1α group decreased, the levels of IFN-γ, IL-2 and TNF-α in the co-culture group increased, the immune killing rate of NK-92 MI cells increased, and the expression of miR-544 increased(P<0.05). In HIF-1α 3’UTR-Wt cells, compared with the si-NC group, the luciferase activity in the miR-544 mimimics group decreased(P<0.05). Compared with the control group and the NC group, the levels of IFN-γ, IL-2 and TNF-α in the miR-544 mimics group decreased,NK-92 MI cells reduced the immune killing rate of A549 lung cancer cells,the expression of NCR1 and the proportion of NKP46 decreased( P < 0. 05). In NCR1 3’UTR-Wt cells,compared with the NC group,the luciferase activity in the miR-544 mimics group decreased( P < 0. 05). Conclusion Inhibiting the expression of HIF-1α in A549 lung cancer cells in vitro can promote NK cells to release immune factors,enhance NK cells immune killing ability,and then inhibit A549 cells’ immune escape,which may be achieved by targeting the inhibition of NCR1/NKP46 pathway by miR-544.