摘要
目的探讨铁死亡相关核心基因基因鸟嘌呤核苷酸结合蛋白亚基α1(GNAI1)在乳腺癌(BC)侵袭及迁移中的作用及机制。方法利用Ualcan在线网站分析GNAI1在BC和正常乳腺组织中的表达,并收集2020年9月至2022年12月郑州大学第一附属医院诊断为BC的病例样本41例,通过qPCR来验证BC较癌旁组织中GNAI1低表达,在MCF-7和MDA-MB-231细胞中敲低GNAI1来通过Transwell等表型实验以及铁死亡相关实验观察GNAI1的表达与铁死亡的关系以及对BC迁移和侵袭能力的影响。其中转染sh-GNAI1的BC细胞为GNAI1沉默组,对照组为转染sh-Control的BC细胞。两组间比较采用独立样本t检验。结果生信分析结果显示GNAI1在BC组织中表达低于正常组织BC患者的病例样本qPCR结果显示GNAI1的mRNA表达(0.006±0.002)低于癌旁正常组织(0.012±0.002,t=6.734,P<0.01),免疫组织化学结果显示病例样本GNAI1蛋白阳性率[(73.487±4.840)%]低于正常组织[(19.601±3.796)%,t=21.236,P<0.01]。Transwell细胞迁移实验结果显示GNAI1沉默后MDA-MB-31细胞和MCF-7细胞迁移能力高于对照组,进入下室的细胞分别为[(123.415±12.342)、(129.354±13.245)个比(30.124±14.985)个,t=17.650、18.675,P<0.01;(147.893±11.380)、(139.796±16.798)个比(33.657±13.964)个,t=25.934、22.236,P<0.01]。铁死亡相关实验显示GNAI1可以降低MDA-MB-231和MCF-7细胞系中Fe^(2+)[(198.325±7.455)%、(193.232±13.663)%比(429.785±23.453)%,t=6.675、5.121,P<0.01;(203.125±12.326)%、(191.745±11.452)%比(417.235±10.833)%,t=7.245、6.983,P<0.01]和脂质过氧化物[(40.313±7.213)%、(47.341±5.751)%比(72.856±5.895)%,t=8.351、5.312,P<0.01;(38.321±3.876)%、(39.685±5.894)%比(73.145±4.978)%,t=9.345、7.983,P<0.01]的比例,并促进超氧化物歧化酶[(423.871±15.313)%、(425.784±18.371)%比(296.193±22.322)%,t=7.233、6.983,P<0.01;(417.341±25.31)%、(429.312±17.685)%比(278.236±13.341)%,t=4.233、7.513,P<0.01]的比例升高。结论沉默GNAI1会抑制BC细胞铁死亡的发生,从而促进BC细胞的增殖,迁移和侵袭。
Objective Exploring the role and mechanism of the core ferroptosis-related gene guanine nucleotide-binding protein subunit alpha-1(GNAI1)in the invasion and migration of breast cancer(BC).Methods The Ualcan online website was used to analyze the expression of GNAI1 in breast cancer and normal breast tissue,and 41 case samples diagnosed with breast cancer at the First Affiliated Hospital of Zhengzhou University from September 2020 to December 2022 were collected.qPCR was used to verify the low expression of GNAI1 in breast cancer compared to adjacent tissues.Then,GNAI1 was knocked down in MCF-7 and MDA-MB-231 cells to observe the relationship between GNAI1 expression and iron death,as well as its impact on the migration and invasion ability of breast cancer cells through phenotype experiments such as cell counting kit-8(CCK-8),clone formation,Transwell as iron death related experiments.Breast cancer(BC)cells transfected with shRNA targeting GNAI1(sh-GNAI1)comprised the knockdown group,with shRNA control(sh-Control)-transfected BC cells as controls.Independent sample t-test is used for comparison between two groups.Results Bioinformatics analysis results showed that GNAI1 expression was significantly lower in BC tissues than in normal tissues.qPCR results of BC patient samples showed that GNAI1 mRNA expression(0.006±0.002)was significantly lower than in adjacent normal tissues(0.012±0.002,t=6.734,P<0.01).Immunohistochemistry results showed that the GNAI1 protein positive rate in patient samples[(73.487±4.840)%]was significantly lower than in normal tissues[(19.601±3.796)%,t=21.236,P<0.01].The results of the Transwell cell migration assay showed that the migration ability of MDA-MB-31 cells and MCF-7 cells was higher after GNAI1 silencing than that of the control group,with the number of cells migrating to the lower chamber being[(123.415±12.342),(129.354±13.245)cells vs.(30.124±14.985)cells,t=17.650,18.675,P<0.01;(147.893±11.380),(139.796±16.798)cells vs.(33.657±13.964)cells,t=25.934,22.236,P<0.01].Ferroptosis-related experiments showed that GNAI1 could reduce the proportion of Fe ^(2+)[(198.325±7.455)%,(193.232±13.663)%vs.(429.785±23.453)%,t=6.675,5.121,P<0.01;(203.125±12.326)%,(191.745±11.452)%vs.(417.235±10.833)%,t=7.245,6.983,P<0.01]and lipid peroxides[(40.313±7.213)%,(47.341±5.751)%vs.(72.856±5.895)%,t=8.351,5.312,P<0.01;(38.321±3.876)%,(39.685±5.894)%vs.(73.145±4.978)%,t=9.345,7.983,P<0.01]in MDA-MB-231 and MCF-7 cell lines,and promote an increase in the proportion of superoxide dismutase[(423.871±15.313)%,(425.784±18.371)%vs.(296.193±22.322)%,t=7.233,6.983,P<0.01;(417.341±25.31)%,(429.312±17.685)%vs.(278.236±13.341)%,t=4.233,7.513,P<0.01].Conclusion Silencing of GNAI1 inhibits the onset of BC cell iron death,thereby promoting BC cell proliferation,migration and invasion.
作者
王新星
洪泽豪
王楠
迟江瑞
孙怡
杜子岩
马泽原
张士博
李林
Wang Xinxing;Hong Zehao;Wang Nan;Chi Jiangrui;Sun Yi;Du Ziyan;Ma Zeyuan;Zhang Shibo;Li Lin(Department of Breast Surgery,First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)
出处
《中华实验外科杂志》
2025年第7期1281-1284,共4页
Chinese Journal of Experimental Surgery
基金
中国博士后科学基金面上项目(2021M702965)
河南省高等学校重点科研项目(22A320021、23A320016)
河南省医学科技攻关省部共建重点项目(SBGJ202102123)
河南省医学科技攻关省部共建青年项目(SBGJ202103070)
河南省自然科学面上项目(252300420102)
河南省科技攻关项目(242102310099)。
