miR-193a-3p/E2F6调控细胞焦亡在鼻咽癌中的机制研究

Mechanistic study of miR-193a-3p/E2F6 regulation of cell pyroptosis in nasopharyngeal carcinoma

目的探讨miR-193a-3p/E2F6在鼻咽癌(NPC)中对细胞焦亡的调控作用及其潜在的分子机制。方法双荧光素酶基因报告实验评估了miR-193a-3p对E2F6的靶向调控作用。HNE-2细胞分为不同处理组:对照组、空白质粒组、沉默E2F6质粒组、空白质粒(miRNA)组、过表达miR-193a-3p组、过表达miRNA+空白(E2F6)质粒组以及过表达miRNA+过表达(E2F6)质粒组。通过qPCR检测miR-193a-3p的表达水平,Western blot检测E2F6蛋白的表达以及NLRP3/Caspase-1/GSDMD/GSDMD-N信号通路的激活情况。同时,使用扫描电子显微镜(scanning electron microscope,SEM)观察细胞形态,CCK-8法评估细胞增殖情况,进行集落形成实验和Transwell实验来检测细胞集落形成数量和迁移数量。结果与对照组(0.90±0.02)和空白质粒组(0.88±0.02)相比,沉默E2F6质粒组中E2F6蛋白(0.26±0.02)的表达水平显著降低,NLRP3/Caspase-1/GSDMD/GSDMD-N信号通路明显被激活,SEM显示细胞发生肿胀。CCK-8实验、集落形成实验和Transwell实验显示细胞活力、集落形成数量和迁移数量均下降(P均<0.05)。双荧光素酶报告基因实验证实E2F6是miR-193a-3p的靶基因。同对照组(1.01±0.03,1.02±0.02)和空白质粒(miRNA)组(1.02±0.04,0.21±0.03)相比,过表达miR-193a-3p组(2.02±0.32,0.23±0.02)中的miR-193a-3p的表达水平升高,E2F6的表达水平显著降低。而NLRP3/Caspase-1/GSDMD/GSDMD-N信号通路明显被激活,SEM显示细胞发生肿胀。CCK-8实验、集落形成实验和Transwell实验显示细胞活力、集落形成数量和迁移数量均下降(P均<0.05)。同过表达miR-193a-3p组和过表达miRNA+空白(E2F6)质粒组相比,发现过表达miR-193a-3p结合过表达E2F6组中E2F6蛋白的含量上升,NLRP3/Caspase-1/GSDMD/GSDMD-N蛋白的含量下降,SEM显示细胞肿胀减轻,CCK-8实验、集落形成实验和Transwell实验显示细胞活力、集落形成数量和迁移数量均上升(P均<0.05)。结论miR-193a-3p通过靶向抑制E2F6激活NLRP3/Caspase-1/GSDMD/GSDMD-N通路,诱导NPC细胞发生细胞焦亡,并抑制其增殖、克隆和侵袭。

OBJECTIVETo investigate the role of miR-193a-3p/E2F6 in the regulation of cell pyroptosis in nasopharyngeal carcinoma(NPC)and its potential molecular mechanisms.METHODS The dual-luciferase reporter assay was used to evaluate the targeted regulation of E2F6 by miR-193a-3p.HNE-2 cells were divided into different treatment groups:control group,empty vector group,E2F6-silenced vector group,empty vector(miRNA)group,miR-193a-3p overexpression group,miRNA overexpression+empty(E2F6)vector group,and miRNA overexpression+E2F6 overexpression vector group.The expression level of miR-193a-3p was detected by qPCR,the protein expression of E2F6 and the activation of the NLRP3/Caspase-1/GSDMD/GSDMD-N signaling pathway were detected by Western bltting.In addition,scanning electron microscopy(SEM)was used to observe cell morphology,and CCK-8 assay,colony formation assay,and Transwell assay were performed to evaluate cell proliferation,colony formation,and migration,respectively.RESULTS Compared to the control group(0.90±0.02)and empty vector group(0.88±0.02),the E2F6 protein expression level was significantly reduced in the E2F6-silenced vector group(0.26+0.02).The NLRP3/Caspase-1/GSDMD/GSDMD-N signaling pathway was significantly activated,and SEM showed cellswelling.CCK-8 assay,colony formation assay,and Transwell assay demonstrated decreased cell viability,colony formation,and migration in the E2F6-silenced vector group(all P<0.05).The dual-luciferase reporter gene assay confirmed that E2F6 was a target gene of miR-193a-3p.Compared to the control group(1.01±0.03,1.02±0.02)and the empty vector(miRNA)group(1.02±0.04,0.21±0.03),the expression level of miR-193a-3p in the overexpressed miR-193a-3p group(2.02±0.32,0.23+0.02)was elevated,while the expression level of E2F6 was decreased.In the miR-193a-3p overexpression group,the expression level of miR-193a-3p was increased,while the expression level of E2F6 protein was significantly reduced.The NLRP3/Caspase-1/GSDMD/GSDMD-N signaling pathway was significantly activated,and SEM showed cell swelling.CCK-8 assay,colony formation assay,and Transwell assay demonstrated decreased cell viability,colony formation,and migration in the miR-193a-3p overexpression group(all P<0.05).Compared to the miRNA overexpression group and miRNA overexpression+empty(E2F6)vector group,the miR-193a-3p overexpression+E2F6 overexpression vector group showed increased protein content of E2F6,decreased protein content of NLRP3/Caspase-1/GSDMD/GSDMD-N,reduced cell swelling observed by SEM,and increased cell viability,colony formation,and migration observed by CCK-8 assay,colony formation assay,and Transwell assay,respectively(all P<0.05).CONCLUSION miR-193a-3p activates the NLRP3/Caspase-1/GSDMD/GSDMD-N signaling pathway by targeting E2F6,induces cell pyroptosis in NPC,and inhibits cell proliferation,colony formation,and invasion.