CRISPR/Cas9-mediated knockout of miR-155 for Triple Negative Breast Cancer treatment

Abstract

Introduction Triple Negative Breast Cancer (TNBC) is the most aggressive breast cancer subtype. Lack of effective targeted therapies, significant genetic heterogeneity and poor response to conventional chemotherapies are the major reasons contributing to poor prognosis and high mortality rates. A subset of microRNAs (miRNAs) has been documented as oncogenic miRNAs (oncomiRs) because they have an important role in the regulation of tumour initiation and malignant progression. MiR-155 is among the most upregulated miRNAs in cancers and has been reported as associated with breast cancer progression and drug resistance. Methodology In this study, we demonstrate that miR-155 is significantly upregulated in a panel of TNBC cell lines. Therefore, we generated miR-155 knockdown cells using the CRISPR/Cas9 genome editing system. Streptococcus pyogenes CRISPR-associated nuclease Cas9 (SpCas9) vectors containing single-guide RNAs (sgRNAs) sequences targeting miR-155 gene were transfected into Hs578T cell line. Results and Conclusions We assessed the specificity of the designed sgRNAs through the cleavages at target loci and off-target effects by T7 Endonuclease I assays and DNA sequencing. In addition to demonstrating that CRISPR transient editing can reduce the expression of miR-155, we also confirmed the association between miR-155 and the transcription factor Forkhead box 3 (FOXO3), a negative regulator of Akt signalling pathway which is implicated in drug resistance. The present data suggests that CRISPR-based in vitro genome editing may provide a strategy for TNBC treatment.