胍丁胺酶对乳腺癌细胞增殖、迁移及侵袭能力的影响  

Effect of Agmatinase on the proliferation,migration and invasion of breast cancer cell

作　　者：高永昌 马超[1] 姚庆娟[1] Gao Yongchang;Ma Chao;Yao Qingjuan(Department of General Surgery,General Hospital of Tianjin Medical University,Tianjin Institute of General Surgery,Tianjin 300052,China)

机构地区：[1]天津医科大学总医院普通外科、天津市普通外科研究所,天津300052

出　　处：《国际外科学杂志》2024年第6期388-393,I0010-I0012,共9页International Journal of Surgery

基　　金：天津市自然科学基金(应用基础研究多元投入基金青年项目)(21JCQNJC01660);天津市卫生健康科技项目(人才培育项目)(KJ20180)。

摘　　要：目的探讨胍丁胺酶(AGMAT)在乳腺癌中的表达及其对乳腺癌细胞系的增殖、迁移及侵袭等生物学行为的影响。方法分析癌症基因组图谱(TCGA)数据库中1094份乳腺癌及癌旁组织样本中AGMAT的表达水平。并验证其在乳腺癌细胞系MDA-MB-231、MCF-7、HCC-1937和T-47D中的表达程度。应用短发夹RNA(shRNA)敲低乳腺癌细胞AGMAT表达,采用实时荧光定量聚合酶链反应(RT-qPCR)检测AGMAT mRNA的表达水平,Western blotting法检测其蛋白表达水平,细胞计数和MTT法检测细胞增殖情况,流式细胞仪检测细胞凋亡和周期变化,细胞划痕实验检测细胞迁移能力,Transwell实验检测细胞侵袭能力,Western blotting法检测细胞内相关蛋白表达水平。计量资料以均数±标准差(x±s)表示,多组间比较采用方差分析,成对比较采用独立样本t检验,Tukey做事后多重检验。结果分析TCGA数据库中乳腺癌样本数据,结果显示,癌组织比癌旁组织中AGMAT的表达水平明显增高(FC=10.54,P<0.001)。流式细胞术检测结果显示,敲低AGMAT表达抑制了乳腺癌细胞增殖,诱导细胞凋亡[(3.20±0.10)%比(6.83±0.06)%,t=62.35,P<0.001],并导致G_(1)期细胞周期停滞[(49.51±2.22)%比(31.44±1.67)%,t=42.56,P=0.001]。细胞划痕实验、Transwell实验检测结果显示,降低GMAT表达能够降低细胞迁移能力[(34.27±1.67)%比(57.97±0.58)%,t=33.52,P<0.001]、侵袭能力(163.00±1.77比61.00±0.74,t=52.50,P<0.001)。Western blotting法检测结果显示,扭曲蛋白(Twist)、波形蛋白(Vimentin)、基质金属蛋白酶-2(MMP-2)和基质金属蛋白酶-9(MMP-9)蛋白表达水平降低,而E钙黏蛋白(E-cadherin)表达水平升高,AGMAT参与了乳腺癌细胞上皮间质转化过程。结论AGMAT可作为影响乳腺癌侵袭、转移和治疗靶点的前瞻性生物学标志物。Objective To investigate the expression of Agmatinase(AGMAT)in breast cancer and its effect on the proliferation,migration and invasion of breast cancer cell lines.Methods The expression levels of AGMAT in cancer and adjacent tissues of 1094 breast cancer samples in The Cancer Genome Atlas(TCGA)database were analyzed.And its expression degree was verified in breast cancer cell lines MDA-MB-231,MCF-7,HCC-1937 and T-47D.The expression of AGMAT in breast cancer cells was knocked down by shRNA,and the expression level of AGMAT mRNA was detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR),and the protein expression level was detected by Western blotting assay.Cell count and MTT assay were used to detect cell proliferation.Cell apoptosis and cell cycle changes were detected by flow cytometry.Cell migration ability was detected by cell scratch assay,cell invasion ability was detected by Transwell assay,and the expression level of related proteins in cells was detected by Western blotting assay.Measurement data were expressed as mean±standard deviation(x±s),comparisons between multiple groups were performed using ANOVA,pairwise comparisons were performed using independent samples t-test,and Tukey′s post hoc multiple test was used.Results Analyzing the data of breast cancer samples in TCGA database,it was found that the expression level of AGMAT in cancer tissues was significantly higher than that in adjacent tissues(FC=10.537,P<0.001).Flow cytometry showed that knocking down the expression of AGMAT inhibited the proliferation of breast cancer cells,and induced cell apoptosis[(3.20±0.10)%vs(6.83±0.06)%,t=62.35,P<0.001],and caused G_(1) cell cycle arrest[(49.51±2.22)%vs(31.44±1.67)%,t=42.56,P=0.001].The results of cell scratch assay and Transwell assay showed that decreased GMAT expression could reduce the cell migration ability[(34.27±1.67)%vs(57.97±0.58)%,t=33.52,P<0.001],invasive ability(163.00±1.77 vs 61.00±0.74,t=52.50,P<0.001).The results of Western blotting assay showed that

关 键 词：乳腺肿瘤 细胞增殖 细胞侵袭 细胞迁移分析 胍丁胺酶 生物标志物 上皮间质转化 

分 类 号：R737.9[医药卫生—肿瘤]