CRISPR/Cas9-Mediated Knockout of LXRα and LXRß in MCF-7 Breast Cancer Cells, Revealing Their Roles in Target Gene Regulation and Cell Proliferation

Background Liver X receptors (LXRs), including LXRα and LXRß, are ligand-activated nuclear receptors that regulate cholesterol homeostasis and lipid metabolism. Increasing evidence suggests that LXRs also influence cancer cell proliferation; however, the individual and combined contributions of LXRα and LXRß in breast cancer cells remain incompletely defined. This study aimed to establish a comprehensive CRISPR/Cas9 knockout model of LXRα and LXRβ in MCF-7 breast cancer cells to elucidate their roles in target gene regulation and cell proliferation. Methods In this study, we employed the CRISPR/Cas9 D10A nickase system to generate LXRα (NR1H3) and LXRβ (NR1H2) knockout cell lines, as well as a dual LXRα/β knockout cell line, in MCF-7 human breast adenocarcinoma cells. Genome editing efficiency was assessed using Surveyor nuclease assay and Sanger sequencing. Clonal knockout cell populations were established by fluorescence-activated single-cell sorting and validated by Western blot analysis and quantitative PCR. Functional consequences of LXR disruption were evaluated by measuring expression of canonical LXR target genes (ABCA1, ABCG1) and by assessing cell viability following treatment with the LXR agonist (GW3965) and IGF-1 stimulation. Results CRISPR/Cas9-mediated editing efficiently disrupted the LXRα and LXRß loci, with knockout clones exhibiting markedly reduced expression of both receptors at the mRNA and protein levels. In wild-type MCF-7 cells, GW3965 treatment induced robust upregulation of ABCA1 and ABCG1, whereas this response was abolished in LXRα/ß-deficient cells, which exhibited increased basal proliferation and reduced sensitivity to GW3965-mediated growth inhibition compared with wild-type controls. Conclusion This study established CRISPR/Cas9-mediated LXRα, LXRß, and dual LXRα/ß knockout models in MCF-7 breast cancer cells and demonstrated that loss of LXR function disrupted target gene regulation and altered cellular proliferation responses. These findings support a role of LXRs in regulating gene expression and growth in ER-positive breast cancer cells and provide a useful platform for further investigation of LXR-associated pathways.