摘要
目的探讨miR-223通过调控花生四烯酸12-脂加氧酶(ALOX12)的表达对胃癌细胞铁死亡的影响.方法收集2016-09-13-2018-11-25北京市朝阳区桓兴肿瘤医院胃肠内科23例胃癌患者癌和癌旁组织(距癌周≥5 cm).人胃癌细胞系MKN45、MGC803和NCI-N87,以及人正常乳腺上皮细胞系MCF10A和胃黏膜细胞系GES-1均购自上海中科院细胞库.利用定量逆转录聚合酶链反应(RT-qPCR)和蛋白质印迹法检测ALOX12的表达水平和miR-223的相对表达量.分别用miR-223-agomir、miR-223-antagomir+miR-223-agomir和miR-223-NC转染MKN45细胞,空白培养细胞做对照组.MTT法测定细胞活力.双荧光素酶实验验证ALOX12与miR-223的互作关系.20μmol/L铁死亡诱导剂Erastin给药胃癌MKN45细胞检测miR-223对细胞铁死亡的影响.结果ALOX12在胃癌组织中的表达量低于癌旁正常组织,t=-8.205,P=0.014;miR-233在胃癌组织中的表达量高于癌旁正常组织,t=5.699,P=0.018.同样,ALOX12在胃癌细胞MKN45、MGC803和NCI-N87中的表达量相对于人正常乳腺上皮细胞系MCF10A和胃黏膜细胞系GES-1下调,F=41.720,P<0.001,且ALOX12的蛋白水平检测结果与转录水平一致.miR-233在MKN45、MGC803和NCI-N87中的表达量与GES-1和MCF10A相比上调,F=74.639,P<0.001.双荧光素酶实验显示,ALOX12是miR-223的靶基因,且通过对癌组织中miR-233和ALOX12的表达量进行分析,发现其具有负相关性,r=-0.613,P=0.002.在转染miR-223-agomir后MKN45细胞中的ALOX12蛋白表达下调,而miR-223-agomir与miR-223-antagomir的共转染则部分回补了ALOX12的表达;miR-223过表达可增强MKN45细胞的增殖能力,F=7.010,P=0.027;铁死亡诱导剂Erastin处理实验发现miR-223减缓MKN45细胞中p53介导的铁死亡过程.结论miR-223通过抑制ALOX12表达促进胃癌的发生与发展,而miR-223与ALOX12在铁死亡过程中的具体机制还需要进一步的研究.
Objective To explore the effect of miR-223on ferroptosis in gastric cancer cells by regulating the expression of Arachidonate-12-Lipoxygenase(ALOX12).Methods Totally 23patients with gastric cancer and adjacent normal tissues(≥5cm)were collected from the Department of Gastroenterology,Huanxing Cancer Hospital,Chaoyang District,Beijing,from September 2018to November 25,2018.Human gastric cancer cell lines MKN45,MGC803and NCI-N87,human normal breast epithelial cell line MCF10Aand gastric cell line GES-1were purchased from the cell bank of Shanghai Chinese Academy of Sciences.The expression of ALOX12and miR-223 were detected by quantitative reverse transcriptase polymerase chain reaction(reverse transcription quantitative PCR,RT-qPCR)and Western blotting(Western blot).miR-223-agomir,miR-223-antagomir+miR-223-agomir and miR-223-NC were transfected into MKN45cells,respectively,and blank cultured cells were used as control group.The cell viability was determined by MTT method.The interaction between ALOX12and miR-223was verified by double luciferase experiment.Twentyμmol/L ferroptosis inducer Erastin was used to detect the effect of miR-223on ferroptosis in gastric cancer MKN45cells.The mean values of measurement data samples with normal distribution were compared by single factor analysis of variance and pairwise multiple comparison by LSD-t test,while the samples that did not accord with normal distribution were tested by nonparametric analysis Kruskal-Wallis method,and the test level wasα=0.05(bilateral).Results Experiments showed that the relative expression level of ALOX12in gastric cancer tissue was significantly lower than that in normal tissues(t=-8.205,P=0.014),besides,the protein level of ALOX12were similar with the results of qRT-PCR.The expression of miR-233in gastric cancer tissue was extremely higher than it in normal tissues(t=5.699,P=0.018).The expression of ALOX12in gastric cancer cells MKN45,MGC803and NCI-N87was also down-regulated compared to the expression in normal cells,GES-1and MCF10A(F=41.720,P<0.001).The protein level of ALOX12were similar with the results of RT-qPCR.The expression levels of miR-223in gastric cancer cells MKN45,MGC803and NCI-N87were also higher than the expression in normal cells,GES-1and MCF10A(F=74.639,P<0.001).The dual-luciferase experiment showed that ALOX12is the target gene of miR-223,and by analyzing the expression of miR-233and ALOX12in cancer tissues,it was found to have a negative correlation with r=-0.613and P=0.002.After transfection of miR-223-agomir,the expression of ALOX12protein in MKN45cells was down-regulated,while co-transcription of miR-223-agomir and miR-223-antagomir partially complemented the expression of ALOX12.miR-223overexpression enhanced the proliferation ability of MKN45cells(F=7.013,P=0.027)and decreased the p53-mediated ferroptosis process in MKN45cells under the treatment of ferroptosis inducer Erastin.Conclusion miR-223promotes the occurrence and development of gastric cancer by inhibiting the expression of ALOX12,and the specific mechanism of miR-223and ALOX12participating in ferroptosis needs further study.
作者
高丽珍
王俊青
陈俊林
周立强
GAO Li-zhen;WANG Jun-qing;CHEN Jun-lin;ZHOU Li-qiang(Huanxing Cancer Hospital of Chaoyang District,Beijing100023,China;Department of Oncology,Cancer Hospital Chinese Academy of Medical Sciences,Beijing100021,China)
出处
《中华肿瘤防治杂志》
CAS
北大核心
2021年第23期1805-1811,1825,共8页
Chinese Journal of Cancer Prevention and Treatment
