微小RNA-575靶向黑色素瘤缺乏因子2调控胃癌细胞的生物学特性

MicroRNA-575 regulates the biological characteristics of gastric cancer cells by targeting absent in melanoma 2

目的 通过寻找调控黑色素瘤缺乏因子2(AIM2)的微小RNA(miRNA,miR),分析miRNA/AIM2轴对胃癌(GC)细胞生物学特性的影响.方法 采用R studio软件Limma软件包分析数据集GSE113255和GSE118897差异表达基因,通过韦恩图筛选出与细胞焦亡相关的共同基因,确定目标基因.收集2022年1月至2023年1月在山西医科大学附属运城市中心医院胃肠外科就诊的56例GC患者癌组织及癌旁组织,检测目标基因表达.在人GC细胞MKN45中过表达AIM2,采用碘化丙锭(PI)染色法、细胞计数试剂盒(CCK-8)法和Matrigel侵袭实验检测细胞凋亡、增殖及侵袭.利用Targetscan网站预测调控AIM2的miRNA,并采用荧光素酶报告基因检测AIM2启动子区与其结合情况.在MKN45细胞中转染miR-575类似物(miR-575 mimics)/阴性对照(NC mimics)或miR-575 抑制剂(miR-575 inhibitor)/阴性对照(NC inhibitor),采用 PI 染色法、CCK-8 法和 Matrigel侵袭实验检测细胞凋亡、增殖及侵袭能力.在MKN45细胞给予Ad-GFP/Ad-AIM2以及NC mimics/miR-575 mimics处理,采用蛋白质免疫印迹法、酶联免疫吸附试验(ELISA)检测miR-575/AIM2轴对细胞焦亡和增殖的调控作用.组间差异采用t检验或方差分析(ANOVA)进行统计.结果 经分析发现AIM2蛋白差异最为明显,癌组织中AIM2蛋白及RNA水平均显著低于癌旁组织(蛋白:0.52±0.14 比 0.99±0.17,t=7.023,P<0.01;RNA:0.58±0.11 比 1.01±0.18,t=6.379,P<0.01).PI染色法、CCK-8法和Matrigel侵袭实验结果显示,Ad-AIM2组细胞凋亡数目显著高于Ad-GFP组[(43.52±3.08)%比(17.72±2.21)%,t=6.114,P<0.01],细胞增殖和侵袭能力显著低于Ad-GFP组(48h:1.17±0.18 比 1.45±0.22,t=5.027,P<0.05;72 h:1.58±0.36 比 2.31±0.42,t=4.822,P<0.05).利用Targetscan网站预测,并通过荧光素酶报告基因实验验证,发现miR-575与AIM2有互补的核苷酸序列,miR-575 mimics与野生型AIM2共转染后,miR-575 mimics组细胞荧光素酶活性显著低于 NC mimics 组(0.52±0.06 比 0.98±0.14,t=5.323,P<0.05);而 miR-575 mimics与突变型AIM2共转染后,miR-575 mimics组细胞荧光素酶活性较NC mimics组无显著变化(0.93±0.08 比 1.00±0.12,t=0.972,P>0.05).免疫印迹法结果显示,Ad-AIM2+miR-575 mimics组细胞凋亡相关斑点样蛋白(ASC)、裂解的半胱氨酰天冬氨酸特异性蛋白酶1(cleaved Caspase-1)/Caspase-1以及高迁移率族蛋白B1(HMGB1)的表达水平显著高于Ad-GFP+miR-575 mimics 组(ASC:1.62±0.31 比 0.73±0.19,t=4.302,P<0.05;cleaved Caspase-1/Caspase-1:1.44±0.21 比 0.78±0.16,t=3.198,P<0.05;HMGB1:1.32±0.14 比 0.81±0.11,t=5.029,P<0.05).结论 AIM2在GC中表达降低,miR-575通过靶向抑制AIM2的表达,进而抑制细胞焦亡,促进MKN45细胞的增殖侵袭.

Objective To investigate the role of absent in melanoma 2(AIM2),an important mol-ecule in the pyroptosis signaling pathway,in gastric cancer(GC),and to further analyze the effect of mi-croRNA(miRNA,miR)/AIM2 axis on the biological characteristics of GC cells by searching for micrornas that regulate AIM2.Methods The limma package of R studio software was used to analyze the differenti-ally expressed genes in GSE113255 and GSE118897 datasets,and the common genes related to pyroptosis were screened by Venn diagram to determine the target genes.Cancer tissues and adjacent tissues of 56 GC patients who were treated in the Gastroenterology Department of Yuncheng Central Hospital Affiliated to Shanxi Medical University from January 2022 to January 2023 were collected to detect the expression of tar-get genes.AIM2 was overexpressed in human GC cell line MKN45,and cell death and proliferation were detected by propidium iodide(PI)staining,cell counting kit-8(CCK-8)assay and Matrigel invasion as-say.Targetscan website was used to predict the mirnas regulating AIM2,and luciferase reporter gene was used to detect the binding between AIM2 promoter region and AIM2.MKN45 cells were transfected with miR-575 mimic(miR-575 mimics)/negative control(NC mimics)or miR-575 inhibitor(miR-575 inhibitor)/negative control(NC inhibitor).PI staining,CCK-8 assay and Matrigel invasion assay were used to detect cell death and proliferation.MKN45 cells were treated with Ad-GFP/Ad-AIM2 and NC mimics/miR-575 mimics.Lactate dehydrogenase release assay,CCK-8 assay and Matrigel invasion assay were used to detect cell death and proliferation.The differences between groups were analyzed by t-test or analysis of variance(ANOVA).Results PI staining,CCK-8 assay and Matrigel invasion assay showed that the number of cell death in Ad-AIM2 group was significantly greater than that in Ad-GFP group[(43.52±3.08)%vs.(17.72±2.21)%,t=6.114,P<0.01].Cell proliferation and invasion ability were significantly lower than Ad-GFP group(48 h:1.17±0.18 vs.1.45±0.22,t=5.027,P<0.05;72 h:1.58±0.36 vs.2.31±0.42,t=4.822,P<0.05).Using Targetscan website prediction and verified by luciferase reporter gene experiment,it was found that miR-575 and AIM2 had complementary nucleotide sequences.After co-transfection of miR-575 mimics and wild type AIM2,the luciferase activity of miR-575 mimics group was significantly lower than that of NC mimics group(0.52±0.06 vs.0.98±0.14,t=5.323,P<0.05).However,after co-transfection of miR-575 mimics and mutant AIM2,the luciferase activity of cells in miR-575 mimics group was not significantly different from that in NC mimics group(0.93±0.08 vs.1.00±0.12,t=0.972,P>0.05).Western blotting results showed that The expression levels of apopto-sis-associated speck-like protein(ASC),cleaved cysteinyl aspartate-specific protease(Caspase)-1/Caspase-1 and high mobility group box 1 protein(HMGB1)in Ad-AIM2+miR-575 mimics group were sig-nifiicantly higher than those in Ad-GFP+group miR-575 mimics group(ASC:1.62±0.31 vs.0.73±0.19,t=4.302,P<0.05;cleaved Caspase-1/Caspase-1:1.44±0.21 vs.0.78±0.16,t=3.198,P<0.05;HMGB1:1.32±0.14 vs.0.81±0.11,t=5.029,P<0.05).Conclusion The expression of AIM2 is decreased in GC.miR-575 inhibits pyroptosis and promotes the proliferation and invasion of MKN45 cells by targeting AIM2 expression.