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

doi:10.3877/cma.j.issn.1674-0807.2024.02.006

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＝1 836.010，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)%和(42.35±0.92)%，组间比较差异无统计学意义(t＝0.713，P＝0.515)；2组的划痕愈合率[(62.33±0.72)%比(31.89±0.29)%，t＝68.150，P<0.001]、迁移细胞数[(225.70±4.50)比(104.70±5.69)，t＝28.880，P<0.001]、侵袭细胞数[(157.00±2.00)比(57.33±4.16)，t＝37.380，P<0.001]比较，差异均有统计学意义。WT(注射未经任何处理的4T1细胞)、NC组和KO(KO2)组这3组小鼠乳腺癌移植瘤体积比较，差异有统计学意义(组间比较，F＝13 859.000，P<0.001；不同时间点比较，F＝3 444.021，P<0.001；交互作用，F＝351.700，P<0.001)，KO组移植瘤的肿瘤体积显著低于WT组与NC组(P均<0.001)。3组的瘤体重量分别为(750.08±21.99)mg、(758.60±18.70)mg和(443.80±14.52)mg，组间比较差异有统计学意义(F＝462.000，P<0.001)。

结论

在三阴性乳腺癌细胞中，SIGLEC-15基因可能和肿瘤细胞体外迁移和侵袭能力有关，其作用机制值得深入研究。

关键词: 乳腺肿瘤, 基因编辑, 异种移植瘤

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. After 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, P<0.001). The mRNA expression levels of SIGLEC-15 in sg-control, sg-SIGLEC-15-1, sg-SIGLEC-15-2 and sg-SIGLEC-15-3 groups were 1.00±0.02, 0.20±0.02, 0.14±0.01 and 0.21±0.01, respectively, indicating a significant difference between groups (F=1 836.010, P<0.001). The expression levels of SIGLEC-15 protein also indicated a significant difference among four groups(t=23.810, 24.370, 23.960, P<0.001). There was no significant difference in the proliferation rate between sg-control and sg-SIGLEC-15-2 groups [(42.88±0.90)% vs (42.35±0.92)%, t=0.713, P=0.515]; however, significant differences were observed in the wound healing rate (62.33±0.72)% vs (31.89±0.29)%, t=68.150, P<0.001), the number of migrating cells (225.70±4.50 vs 104.70±5.69, t=28.880, P<0.001) and the number of invading cells (157.00±2.00 vs 57.33±4.16, t=37.380, P<0.001). There were significant differences in tumor volumes among the WT (injected with untreated 4T1 cells), NC and KO(KO2) groups of mice with breast cancer xenografts (group comparison, F=13 859.000, P<0.001; different time points, F=3444.021, P<0.001; interaction, F=351.700, P<0.001). The tumor volume in the KO group was significantly lower than that in the WT and NC groups (both P<0.001). The tumor weights were (750.00±21.99) mg, (758.60±18.70) mg and (443.80±14.52) mg in the WT, NC and KO groups, respectively, with a significant difference between groups (F=462.000, P<0.001).

Conclusion

In triple negative breast cancer cells, the SIGLEC-15 gene may be associated with the migration and invasion abilities of tumor cells, and its mechanism of action warrants further study.

Key words: Breast neoplasms, Gene editing, Xenograft

表1 人源性SIGLEC-15基因单链引导RNA寡核苷酸序列

编号	sgRNA序列
1	GCCGCGGATCGTCAACATCT
2	GGAGAACTTGCTCAACACAG
3	AACACCTGCGGGCCCGCATA

表1 人源性SIGLEC-15基因单链引导RNA寡核苷酸序列

编号	sgRNA序列
1	GCCGCGGATCGTCAACATCT
2	GGAGAACTTGCTCAACACAG
3	AACACCTGCGGGCCCGCATA

表2 鼠源性SIGLEC-15基因单链引导RNA寡核苷酸序列

编号	sgRNA序列
1	GCGACTCTCATAGCGATCGT
2	TCAGCGGCCCGTCGTAGTGG
3	AACACCTGCGGGCCCGCGTA

表2 鼠源性SIGLEC-15基因单链引导RNA寡核苷酸序列

编号	sgRNA序列
1	GCGACTCTCATAGCGATCGT
2	TCAGCGGCCCGTCGTAGTGG
3	AACACCTGCGGGCCCGCGTA

图1 人源性SIGLEC-15-sgRNA重组质粒测序结果　a、b、c图分别为人源SIGLEC-15-sgRNA1、SIGLEC-15-sgRNA2、SIGLEC-15-sgRNA3质粒的测序结果注：SIGLEC-15为唾液酸结合Ig样凝集素15；sgRNA为单链引导RNA

图2 Western blot检测4组MDA-MB-231单克隆细胞中SIGLEC-15蛋白表达水平注：SIGLEC-15为唾液酸结合Ig样凝集素15；GAPDH为甘油醛-3-磷酸脱氢酶；a为空载体对照sg-control；b、c、d分别为sg-SIGLEC-15-1、sg-SIGLEC-15-2和sg-SIGLEC-15-3基因敲除MDA-MB-231细胞

图3 SIGLEC-15基因敲除对乳腺癌MDA-MB-231细胞增殖能力的影响　a、b、c图分别为sg-control对照组细胞的DAPI染色、EdU染色和合并图；d、e、f图分别为sg-SIGLEC实验组细胞的DAPI染色、EdU染色和合并图注：SIGLEC-15为唾液酸结合Ig样凝集素15；DAPI为4′，6-二脒基-2-苯基吲哚；EdU为：5-乙炔基-2′-脱氧尿嘧啶核苷

图4 SIGLEC-15基因敲除MDA-MB-231细胞划痕愈合实验结果　a、b图分别为sg-control细胞划痕实验开始和24 h后对比图；c、d图分别为sg-SIGLEC-15细胞划痕实验开始和24 h后对比图注：SIGLEC-15为唾液酸结合Ig样凝集素15

图5 SIGLEC-15基因敲除MDA-MB-231细胞Transwell迁移实验结果　a、b图分别为sg-control和sg-SIGLEC-15细胞Transwell迁移实验结果，可见sg-SIGLEC-15细胞穿过Transwell小室的细胞数量明显少于sg-control组

图6 SIGLEC-15敲除的MDA-MB-231细胞Transwell侵袭实验结果　a、b图分别为sg-control和sg-SIGLEC-15细胞Transwell侵袭实验结果，可见sg-SIGLEC-15侵袭细胞明显少于sg-control组注：SIGLEC-15为唾液酸结合Ig样凝集素15

图7 鼠源性SIGLEC-15-sgRNA重组质粒测序结果　a、b、c图分别为鼠源SIGLEC-15-sgRNA1、SIGLEC-15-sgRNA2、SIGLEC-15-sgRNA3质粒的测序结果注：SIGLEC-15为唾液酸结合Ig样凝集素15；sgRNA为单链引导RNA

图8 Western blot检测4组4T1单克隆细胞中SIGLEC-15蛋白表达水平注：a为NC空载体对照；b、c、d分别为KO1、KO2和KO3基因敲除4T1细胞；SIGLEC-15为唾液酸结合Ig样凝集素15；GAPDH为甘油醛-3-磷酸脱氢酶

表3 3组小鼠原位移植瘤体积比较(mm^3,

±s)

组别	只数	第8天	第16天	第24天
WT	5	111.80±1.84	263.60±3.83	409.00±4.22
NC	5	107.40±2.79^a	259.00±3.48^d	414.60±2.78^g
KO	5	95.69±1.54^b，c	179.00±4.52^e，f	266.60±3.62^h，i
F值		76.360	718.700	2 738.000
P值		<0.001	<0.001	<0.001

表3 3组小鼠原位移植瘤体积比较(mm^3,

±s)

组别	只数	第8天	第16天	第24天
WT	5	111.80±1.84	263.60±3.83	409.00±4.22
NC	5	107.40±2.79^a	259.00±3.48^d	414.60±2.78^g
KO	5	95.69±1.54^b，c	179.00±4.52^e，f	266.60±3.62^h，i
F值		76.360	718.700	2 738.000
P值		<0.001	<0.001	<0.001

图9 小鼠乳腺癌移植瘤瘤体图注：SIGLEC-15为唾液酸结合Ig样凝集素15；KO为注射SIGLEC-15基因稳定敲除的KO2组4T1细胞；NC为注射带有阴性载体的4T1细胞；WT为注射未经任何处理的4T1细胞

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

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