Galectin-1的4T1乳腺癌过表达细胞的构建及其对增殖和转移的影响

doi:10.13560/j.cnki.biotech.bull.1985.2022-0114

摘要/Abstract

摘要：

探查galectin-1促癌作用的广泛性，并为进一步研究该基因在三阴性乳腺癌中促癌转移的分子作用机制提供必要的实验工具和依据。采用生物信息学方法去分析多种癌症病人样本中该基因的表达情况，及其与乳腺癌病人总体生存率的关系；通过慢病毒转染去构建galectin-1过表达的4T1细胞株，并检测该基因对细胞增殖（CCK8检测法）、迁移和侵袭（Transwell检测法）的影响。研究结果显示，galectin-1在乳腺癌和淋巴癌等多种癌症中呈现高表达状态，且与乳腺癌病人的总体生存率呈负相关。同时，galectin-1过表达三阴性乳腺癌4T1细胞株被成功构建。Galectin-1的过表达能够显著促进4T1肿瘤细胞的增殖、迁移和侵袭。这些都显示了galectin-1是一个对乳腺癌等多种癌症具有促癌作用，并能促进三阴性乳腺癌细胞体外增殖和转移的因子。

关键词: galectin-1, 三阴性乳腺癌, 增殖, 迁移, 侵袭

Abstract:

This work is aimed at assessing the pro-tumor effects of galectin-1（Gal-1）and providing necessary experimental kit and evidences in the further investigation of the molecular mechanism of Gal-1-mediated cell proliferation and metastasis in triple-negative breast cancer（TNBC）. Bioinformatics was applied to analyze the expression of Gal-1 in cancers and its correlation with the overall survival rate of breast cancer patients. A Gal-1 overexpressing murine TNBC cell line 4T1 was constructed by lentiviral transfection, and the effects of Gal-1 overexpression on the cell proliferation, migration and invasion were assessed by CCK8 assay and transwell assays, respectively. The results showed that Gal-1 was highly expressed in many types of cancers, including breast cancer and lymphoma. Gal-1 expression also was negatively correlated with the overall survival rate of breast cancer patients. A Gal-1- overexpressing 4T1 cell line was constructed and characterized by both qPCR and Western Blot. Overexpression of Gal-1 promoted cellular proliferation, migration and invasion. These reveal that Gal-1 presents pro-cancer effects on breast cancer, etc.，and is a factor promoting the proliferation and migration of TNBC cell in vitro.

Key words: galectin-1, triple-negative breast cancer, proliferation, migration, invasion

王树萱, 向钢, 马小京, 于晶. Galectin-1的4T1乳腺癌过表达细胞的构建及其对增殖和转移的影响[J]. 生物技术通报, 2022, 38(11): 97-103.

WANG Shu-xuan, XIANG Gang, MA Xiao-jing, YU Jing. Construction of Galectin-1 Overexpressing 4T1 Mammary Tumor Cells and Its Effects on the Proliferation and Migration[J]. Biotechnology Bulletin, 2022, 38(11): 97-103.

图/表 6

表1 Lgals1和Gapdh引物序列

Table 1 Lgals1 and Gapdh primer sequences

基因 Gene	引物序列 Primer sequence（5'-3'）
Lgals1	F：AACCTGGGGAATGTCTCAAAGT
	R：GGTGATGCACACCTCTGTGA
Gapdh	F：AGGTCGGTGTGAACGGATTTG
	R：TGTAGACCATGTAGTTGAGGTCA

图1 生物信息学数据库分析galectin-1在乳腺癌中的表达及其对乳腺癌患者总体生存率的关联关系 A：Oncomine数据库分析多种癌症中galectin-1的表达（黄色长框表示乳腺癌，数字表示研究样本数量。红色越深代表基因表达越高，蓝色越深代表表达越低）；B：Oncomine数据库中Zhao Breast数据集分析galectin-1在正常组织与乳腺癌肿瘤组织中mRNA的表达差异；C：GEO数据库GSE54140数据集分析的不同乳腺癌亚型中galectin-1基因拷贝数的差异；D-E：Kaplan-Meier分析galectin-1的表达分别与（D）乳腺癌患者和（E）三阴性乳腺癌患者总体生存率的相关性

Fig. 1 Bioinformatics database analysis of the expression of galectin-1 in breast cancer and the correlation of it with overall survival rate of breast cancer patients A：Oncomine database analysis of galectin-1 expression in various cancers（The long yellow box indicates breast cancer；the number indicates the amounts of samples that galectin-1 was analyzed in different cancers. Darker red indicates higher gene expression，darker blue indicates lower expression）. B：Oncomine database analysis of differential mRNA expression of galectin-1 in normal and tumor tissues in Zhao Breast dataset. C：GEO database（GSE54140 data set）analysis of copy number variation of galectin-1 gene in different breast cancer subtypes. D-E：Kaplan-Meier analysis of the correlation between galectin-1 expression and the overall survival rate of（D）breast cancer patients and（E）TNBC patients

图2 Galactin-1表达质粒pCDH-Lgals1构建的酶切及测序分析图 A：琼脂糖凝胶电泳图分析pCDH载体和Lgals1 PCR扩增产物的酶切图。1：15000 DNA 标准品；2：pCDH载体酶切；3：Lgals1 PCR扩增产物酶切；B：构建的表达质粒中galectin-1基因的测序比对结果（红框内序列为6×His的标签）

Fig. 2 Digestion and sequencing analysis of the galactin-1-expressed plasmid pCDH-Lgals1 A：Agarose gel electrophoresis of the digestion of pCDH vector and PCR amplification products of Lgals1. 1：15000 DNA ladder；2：pCDH digestion vector；3：Lgals1 PCR products. B：Alignment of DNA sequencing results of galectin-1 in the constructed expression plasmid（the sequence of 6×His tag is shown in red box）

图3 qPCR和Western Blot检测galectin-1在galectin-1过表达4T1细胞中的过表达水平 A：qPCR检测galectin-1过表达4T1细胞株中galectin-1的mRNA水平；B：Western Blot分析galectin-1过表达4T1细胞株中galectin-1的蛋白水平；C：B图的定量图

Fig. 3 qPCR and Western Blot analysis of galectin-1 in galectin-1 overexpressing 4T1 cells A：qPCR analysis of mRNA levels of galectin-1 in galectin-1 overexpressing 4T1 cell line. B：Western Blot analysis of protein levels of galectin-1 in galectin-1 overexpressing 4T1 cells. C：Quantitative diagram of the B chart

图4 过表达galectin-1对三阴性乳腺癌4T1细胞增殖的促进作用

Fig. 4 Overexpression of galectin-1 promotes the prolifer-ation of 4T1 triple-negative breast cancer cells

图5 过表达galectin-1对三阴性乳腺癌4T1细胞迁移和侵袭的促进作用 A：细胞迁移实验；B：细胞侵袭实验（左边两图为显微镜镜检结果，右图为镜检定量图）

Fig. 5 Overexpression of galectin-1 promotes the migration and invasion of triple-negative breast cancer 4T1 cells A：Transwell migration assay. B：Transwell invasion assay

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