MDA-MB-231 ko cell line | 10x Efficiency | Ubigene
The MDA-MB-231 cell line is a human breast cell line that was established from a pleural effusion of a 51-year-old caucasian female with metastatic mammary adenocarcinoma. This epithelial cell line is one of the most widely used breast cancer cell lines in medical research laboratories, especially those involve CRISPR/Cas9 gene knockout and gene knockdown.
As a result of lacking ER and PR expression and HER2 amplification, the cell line was initially classed as a "basal" breast cancer cell line. However, it is now recognized as belonging to the claudin-low molecular subtype as it exhibits down-regulation of claudin-3 and claudinin-4, low expression of the Ki-67 proliferation marker, enrichment for markers associated with the epithelial-mesenchymal transition and the expression of features associated with mammary cancer stem cells (CSCs), such as the CD44+CD24-/low phenotype. That’s why many studies had been focusing on the gene-editing MDA-MB-231 cell line.
1. CRISPR/Cas9 mutagenesis invalidates a putative cancer dependency targeted in MDA-MB-231 cells.
Cancer cells require the expression of certain genes that encode proteins necessary for tumor growth. Silencing the expression of these genes or blocking the activity of the proteins can trigger cell death and durable tumor regression. So identifying and characterizing cancer dependencies is a key goal of pre-clinical cancer research. MELK has been implicated as a cancer dependency and putative drug target in multiple cancer types, one of which is the MDA-MB-231 cell line. MELK is over-expressed in breast cancer cells, which is associated with poor patient prognosis. Moreover, knockdown of MELK in MDA-MB-231 cells using RNAi has been reported to block cancer cell proliferation and trigger cell cycle arrest or mitotic catastrophe.
Researchers designed seven guide RNAs (gRNAs) against MELK and cloned each guide RNA into a GFP-expressing vector and transduced the guides into three Cas9-expressing cell lines: the triple-negative breast cancer cell line MDA-MB-231 reported to be addicted to MELK expression. Western blot analysis of MELK protein levels in overexpression MDA-MB-231 cells confirmed that the CRISPR system can effectively ablate MELK expression.
2. Distinct effects of β1 integrin on cell proliferation and cellular signaling in MDA-MB-231 breast cancer cells
To explore the functional significance of β1 integrin, researchers established β1-KO MDA-MB-231 cells via a CRISPR/Cas9-based approach. Western blot and FACS analyses were performed to confirm the effective knockout of β1 in the MDA-MB-231 cell line. Surprisingly, the expression of β1 exhibited opposite effects on cell proliferation. These effects were dependent on cell densities and they showed an up-regulation of cell proliferation when cells were cultured under sparse conditions and a down-regulation of cell growth under dense conditions. By comparison with WT cells, the phosphorylation levels of ERK in KO MDA-MB-231 cells were consistently suppressed under sparse culture conditions but consistently up-regulated under dense culture conditions. The phosphorylation levels of EGFR were increased in the KO MDA-MB-231 cells. By contrast, the phosphorylation levels of AKT were decreased in the MDA-MB-231 KO cells. The abilities for both colony and tumor formation were significantly suppressed in the MDA-MB-231 knockout cells, suggesting that β1 plays an important role in cell survival signaling for tumorigenesis. These aberrant phenotypes in the KO cells were rescued in the Res cells. Taken together, these results clearly showed the distinct roles of β1 in cancer cells: the inhibition of cell growth and the promotion of cell survival, which may shed light.
3. Knockdown of BCL2L12 leads to cisplatin resistance in MDA-MB-231 breast cancer cells
Gene BCL2L12 was found to be highly expressed in normal breast tissues and was associated with favorable prognosis in breast cancer patients. Researchers reported that the mRNA levels of BCL2L12 and its transcript variant BCL2L12A could be upregulated upon cisplatin treatment in MDA-MB-231 breast cancer cells. The knockdown of BCL2L12 and BCL2L12A dramatically inhibited cisplatin-induced apoptosis. In contrast, ectopic expressions of each of the proteins promoted cisplatin-induced apoptosis. These results indicated that decreased expressions or loss of BCL2L12 and BCL2L12A may contribute to the cisplatin resistance in breast cancer patients. Furthermore, we found that cisplatin-induced downregulation of β-catenin was partially suppressed in BCL2L12- and BCL2L12A-knocked down MDA-MB-231 cells, which indicated that knockdown of these two proteins may stabilize β-catenin in cisplatin-induced apoptosis. In short, we proposed that BCL2L12 and BCL2L12A may play an important role in cisplatin-induced apoptosis in MDA-MB-231 breast cancer cells.
CRISPR/Cas9 knockout MDA-MB-231 cell line strategies
Workflow of knockout MDA-MB-231 cell line
1. Christopher J Giuliano, Ann Lin, Joan C Smith, Ann C Palladino, Jason M Sheltzer. MELK expression correlates with tumor mitotic activity but is not required for cancer growth. eLife 2018;7:e32838 DOI: 10.7554/eLife.32838.
2. Ann Lin, Christopher J Giuliano, Nicole M Sayles, Jason M Sheltzer. CRISPR/Cas9 mutagenesis invalidates a putative cancer dependency targeted in on-going clinical trials. eLife 2017;6:e24179 DOI: 10.7554/eLife.24179.
3. Yi Hong, Junwu Yang, Weibing Wu, Wenzong Wang, Xiangfei Kong, Yanlin Wang, Xiaojing Yun, Hongliang Zong, Yuanyan Wei, Si Zhang, Jianxing Gu. The knockdown of BCL2L12 leads to cisplatin resistance in MDA-MB-231 breast cancer cells. Volume 1782, Issue 11, November 2008, Pages 649-657.
4. Hou, S, Isaji, T, Hang, Q. et al. Distinct effects of β1 integrin on cell proliferation and cellular signaling in MDA-MB-231 breast cancer cells. Sci Rep 6, 18430 (2016). https://doi.org/10.1038/srep18430.