Construction of Galectin-1 Overexpressing 4T1 Mammary Tumor Cells and Its Effects on the Proliferation and Migration

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

Abstract

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

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.

Figures/Tables 6

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

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

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）

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

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

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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