摘要
目的:通过观察环状RNA(circRNA)BIRC6(circBIRC6)在非小细胞肺癌(NSCLC)中的表达探讨其在肿瘤细胞免疫逃逸中的作用及分子机制。方法:体外培养人NSCLC细胞系(H1975、HCC827、H1650、H1299和A549)和人正常肺上皮细胞系(BEAS-2B),qRT-PCR检测检测circBIRC6、miR-944和分化簇44(CD44)、程序性细胞死亡配体1(PD-L1)的表达。取A549细胞,分为正常对照(NC)组、si-NC组、si-circBIRC6组、si-circBIRC6+anti-NC组、si-circBIRC6+anti-miR-944组。转染48h后,采用qRT-PCR和Western blot检测细胞中circBIRC6、miR-944和CD44、PD-L1的mRNA和蛋白表达水平;MTT法检测细胞增殖活性,Transwell小室检测细胞迁移、侵袭能力;将A549细胞与CD8^(+)T细胞共培养,台盼蓝染色测定共培养系统中CD8^(+)T细胞活力,流式细胞术检测共培养系统中CD8^(+)T细胞凋亡,ELISA法检测共培养细胞培养上清液中可溶性PD-L1(sPD-L1)、干扰素-γ(INF-γ)、肿瘤坏死因子α(TNF-α)、颗粒酶B和穿孔素水平,验证敲低circBIRC6在NSCLC免疫逃逸中的作用。通过双荧光素酶报告(DLRA)、RNA免疫沉淀(RIP)和RNA pull-down实验验证A549细胞中circBIRC6、miR-944和CD44的调控机制。结果:circBIRC6和CD44、PD-L1在NSCLC中呈高表达,miR-944呈低表达。敲低circBIRC6(0.36±0.05比1.01±0.12)可上调miR-944(3.45±0.38比0.99±0.10),抑制CD44 mRNA(0.39±0.05比1.00±0.10)、CD44蛋白(0.25±0.05比0.64±0.07)和PD-L1 mRNA(0.31±0.04比1.00±0.13)、PD-L1蛋白(0.17±0.03比0.52±0.06)表达,降低A549细胞增殖活力和迁移、侵袭能力(均P<0.05)。在共培养系统中,敲低circBIRC6的A549细胞可激活CD8^(+)T细胞[(68.35±7.20)%比(35.82±4.14)%],抑制CD8^(+)T细胞凋亡[(23.85±3.76)%比(56.01±6.22)%],降低sPD-L1水平,增加IFN-γ、TNF-α、颗粒酶B和穿孔素水平(均P<0.05)。下调miR-944可减弱circBIRC6敲低对A549细胞增殖、迁移和侵袭及CD8^(+)T细胞活化的影响。DLRA、RIP和RNA pull-down实验证实circBIRC6可以靶向并结合A549细胞中的miR-944,且CD44是miR-944的靶标。结论:敲低A549细胞中的circBIRC6可通过下调PD-L1的表达和分泌,激活肿瘤微环境中的CD8^(+)T细胞,进而抑制NSCLC的免疫逃逸;其分子机制可能与miR-944/CD44轴对PD-L1的调控作用有关。
Objective:To observe the expression of circular RNA(circRNA)BIRC6(circBIRC6)in non-small cell lung cancer(NSCLC)and to explore its role and molecular mechanism in tumor cell immune escape.Methods:Human NSCLC cell lines(H1975,HCC827,H1650,H1299,and A549)and human normal lung epithelial cell lines(BEAS-2B)were cultured in vitro.The expressions of circBIRC6,miR-944,cluster of differentiation 44(CD44)and programmed cell death ligand 1(PD-L1)were detected by qRT-PCR.A549 cells were collected and separated into normal control(NC)group,si-NC group,si-circBIRC6 group,si-circBIRC6+anti-NC group,and si-circBIRC6+anti-miR-944 group.48h after transfection,qRT-PCR and Western blot were applied to detect the mRNA and protein expression levels of circBIRC6,miR-944,CD44 and PD-L1 in cells;MTT assay was applied to detect cell proliferation activity;Transwell chambers were used to detect cell migration and invasion ability;A549 cells were co-cultured with CD8^(+)T cells;Trypan blue staining was applied to determine CD8^(+)T cell viability in co-culture systems;flow cytometry was applied to detect CD8^(+)T cell apoptosis in co-culture systems;ELISA was applied to detect the levels of soluble PD-L1(sPD-L1),interferon-γ(INF-γ),tumor necrosis factor alpha(TNF-α),granzyme B,and perforin in culture supernatants of co-cultured cells;the role of knockdown of circBIRC6 in NSCLC immune escape was validated.Dual-luciferase reporter(DLRA),RNA immunoprecipitation(RIP)and RNA pull-down experiments were applied to validate the regulatory mechanisms of circBIRC6,miR-944 and CD44 in A549 cells.Results:CircBIRC6,CD44 and PD-L1 were highly expressed in NSCLC,while miR-944 was lowly expressed in NSCLC.Knockdown of circBIRC6(0.36±0.05 vs 1.01±0.12)could up-regulate miR-944(3.45±0.38 vs 0.99±0.10),inhibit the expression of CD44 mRNA(0.39±0.05 vs 1.00±0.10),CD44 protein(0.25±0.05 vs 0.64±0.07)and PD-L1 mRNA(0.31±0.04 vs 1.00±0.13),PD-L1 protein(0.17±0.03 vs 0.52±0.06),and greatly reduce the proliferation,migration and invasion abilities of A549 cells(all P<0.05).In the co-culture system,knockdown of circBIRC6 in A549 cells could activate CD8^(+)T cells[(68.35±7.20)%vs(35.82±4.14)%],inhibit the apoptosis of CD8^(+)T cells[(23.85±3.76)%vs(56.01±6.22)%],reduce the level of sPD-L1 in the supernatant,increase the levels of IFN-γ,TNF-α,granzyme B and perforin(all P<0.05).Down-regulation of miR-944 increased the expressions of CD44 and PD-L1,and attenuated the effects of circBIRC6 knockdown on the proliferation,migration and invasion of A549 cells and activation of CD8^(+)T cells(all P<0.05).DLRA,RIP and RNA pull-down experiments confirmed that circBIRC6 could target and bind to miR-944 in A549 cells,and CD44 was the target of miR-944.Conclusion:Knockdown of circBIRC6 in A549 cells can activate CD8^(+)T cells in the tumor microenvironment by down-regulating the expression and secretion of PD-L1,thereby inhibiting the immune escape of NSCLC.The molecular mechanism may be related to the regulatory effect of miR-944/CD44 axis on PD-L1.
作者
张田
任丹
李杰
ZHANG Tian;REN Dan(Xianyang First People's Hospital,Shaanxi Xianyang 712000,China)
出处
《河北医学》
CAS
2022年第12期1969-1977,共9页
Hebei Medicine
基金
湖北省卫生健康委科研项目,(编号:WJ2019M238)。
