Production of recombinant lentiviruses expressing miR-328 to overcome of breast cancer stem-like cells drug resistance

Introduction & Aim: Drug resistance is a major cause of recurrence and progression of breast cancer. Breast cancer stem cells (BCSCs) were identified as a rare population which involve in resistance to anticancer therapeutics. One of the mechanisms of chemo-resistance of CSCs is the overexpression of ATPbindingcassette (ABC) efflux transporters such as breast cancer resistance protein (BCRP/ABCG2). Since miR-328 regulate post-transcriptionally ABCG2 expression, we will develop a miR-328 based lentiviral strategy to sensitize BCSC to chemotherapeutic agents. Methods: A DNA fragment that contained the miR-328 precursor was cloned in a lentiviral plasmid.Recombinant lentiviral particles were produced by transient calcium phosphate co- transfection of HEK293T cells with the major and two helper plasmids. Viral supernatants were harvested and concentrated by ultracentrifuge. Virus titration was determined by quantitative real-time PCR (qPCR). Altered expression levels of miR-328 and its target ABCG2 were evaluated by qPCR Results: The identity of DNA was established by colony-PCR, enzymatic digestion of positive clones, and DNA sequencing. The determined virus titer by qPCR was 1 × 108 TU/ml. Real-time PCR assay showed that miR-328 expression levels significantly increased and its target mRNA ABCG2 was downregulated intransduced cells compared with the control group. Conclusion: This lentivirus expression system could be considered as a tool for efficient delivery of produced miRNAs to cells. Also, these recombinant lentivirus structures that contained the miR-328 precursor maybe sensitize breast cancer stem cells to chemotherapeutic agents by modulating ABCG2 expression