唾液酸结合Ig样凝集素15对三阴性乳腺癌细胞增殖、迁移和侵袭的影响  

Effect of sialic acid-binding Ig-like lectin 15 on proliferation,migration and invasion of triple negative breastcancer cells

作　　者：唐璐 徐静 樊俊 张哲 唐莉娟 罗文田 徐琰 Tang Lu;Xu Jing;Fan Jun;Zhang Zhe;Tang Lijuan;Luo Wentian;Xu Yan(Department of Breast and Thyroid Surgery,Army Medical Center,Army Medical University,Chongqing 400042,China)

机构地区：[1]陆军军医大学附属陆军特色医学中心乳腺甲状腺外科,重庆400042

出　　处：《中华乳腺病杂志（电子版）》2024年第2期93-101,共9页Chinese Journal of Breast Disease(Electronic Edition)

摘　　要：目的:探讨唾液酸结合Ig样凝集素15(SIGLEC-15)基因对三阴性乳腺癌细胞增殖、迁移和侵袭过程的影响。方法:分别设计特异性靶向人源性和鼠源性SIGLEC-15基因的3个单链引导RNA(sgRNA)序列,利用CRISPR/Cas9技术获得稳定敲除SIGLEC-15基因的MDA-MB-231单克隆细胞[sg-control(对照组)、sg-SIGLEC-15-1、sg-SIGLEC-15-2和sg-SIGLEC-15-3共4组]和稳定敲除SIGLEC-15基因的小鼠三阴性乳腺癌4T1细胞[NC(对照组)、KO1、KO2和KO3共4组]。采用qRT-PCR、Western blot实验检测8组单克隆细胞SIGLEC-15基因mRNA和蛋白表达水平,以评估SIGLEC-15基因的敲除效率。通过EdU增殖实验、划痕愈合实验、Transwell迁移实验和侵袭实验来评估SIGLEC-15基因敲除对MDA-MB-231细胞增殖、迁移侵袭功能的影响。将SIGLEC-15基因敲除的4T1细胞注射到BALB/c雌性小鼠皮下脂肪垫中,每2天1次评估小鼠的肿瘤体积,24 d后处死小鼠,测量肿瘤的体积和重量。SIGLEC-15的mRNA表达量、蛋白表达量、细胞增殖率、细胞划痕愈合率、迁移细胞数、侵袭细胞数、移植瘤体积和重量的2组间比较采用独立样本t检验,多组间比较采用单因素方差分析和重复测量方差分析,多组间两两比较采用Tukey法。结果:NC、KO1、KO2和KO3这4组单克隆细胞株中SIGLEC-15基因的mRNA表达量分别为1.00±0.06、0.19±0.02、0.15±0.01和0.16±0.02,组间比较差异有统计学意义(F=405.807,P<0.001)。KO1、KO2和KO3这3组中SIGLEC-15蛋白的表达量也明显低于NC组(t=74.832、103.210、71.850,P<0.001)。sg-control、sg-SIGLEC-15-1、sg-SIGLEC-15-2和sg-SIGLEC-15-3这4组单克隆细胞中SIGLEC-15基因的mRNA表达量分别为1.00±0.02、0.20±0.02、0.14±0.01和0.21±0.01,组间比较差异有统计学意义(F=1836.010,P<0.001)。SIGLEC-15基因敲除的这3组细胞中SIGLEC-15蛋白的表达量也明显低于sg-control组(t=23.810、24.370、23.960,P<0.001)。sg-control和sg-SIGLEC-15-2组的细胞增殖率分别为(42.88±0.90)%Objective To investigate the effect of Sialic acid-binding Ig-like lectin 15(SIGLEC-15)gene on the proliferation,migration and invasion of triple negative breast cancer(TNBC)cells.Methods Three single-guide RNA(sgRNA)sequences specifically targeting human and murine SIGLEC-15 genes were designed.Stable SIGLEC-15 knockout MDA-MB-231 monoclonal cells(sg-control,sg-SIGLEC-15-1,sg-SIGLEC-15-2,and sg-SIGLEC-15-3 groups)and stable SIGLEC-15 knockout mouse TNBC 4T1 cells[normal control(NC group),KO1,KO2,and KO3 groups] were obtained using the CRISPR/Cas9 technology.The qRT-PCR and Western blot analysis were conducted to detect the mRNA and protein expression levels of SIGLEC-15 gene in eight monoclonal cell groups to evaluate the efficiency of SIGLEC-15 gene knockout.The effect of SIGLEC-15 gene knockout on the proliferation,migration and invasion abilities of MDA-MB-231 cells were assessed by EdU proliferation assay,wound healing assay,Transwell migration and invasion assay.The SIGLEC-15 gene-knocked-out 4T1 cells were injected subcutaneously into the adipose pads of female BALB/c mice,and the tumor volume was assessed every two days.Afier 24 days,the mice were euthanized,and the tumor volume and weight were measured.For quantitative data such as mRNA expression levels,protein expression levels,cell proliferation rates,cell scratch healing rates,number of migrating cells,number of invasive cells,volume and weight of xenograft tumors,comparisons between two groups were conducted using independent sample t-tests.Comparisons among multiple groups were performed using one-way ANOVA and repeated measures ANOVA,with pairwise comparisons among multiple groups conducted using Tukey's method.Results The mRNA expression levels of SIGLEC-15 in the NC,KO1,KO2 and KO3 groups were 1.00±0.06,0.19±0.02,0.15±0.01 and 0.16±0.02,respectively,indicating a significant difference between groups(F=405.807,P<0.001).The expression levels of SIGLEC-15 protein also indicated a significant difference among four groups(t=74.832,103.210,71.850,

关 键 词：乳腺肿瘤 基因编辑 异种移植瘤 

分 类 号：R737.9[医药卫生—肿瘤] Q789[医药卫生—临床医学]