Effects of 5-fluorouracil Drug Treatment on the Expression Profile of MicroRNAs in MCF7 Breast Cancer Cells

Abstract

Breast cancer is one of the leading causes of deaths in women worldwide. 5-flourouracil (5-FU) is a classic chemotherapeutic drug that has been widely used in the treatment of breast cancer patients. In this study, using several biochemical techniques, we studied the global effects of 5-FU treatment on MCF7 breast cancer cells. The dose-response curve obtained after the treatment of MCF7 cells with 23 different 5-FU concentrations for 48 hours showed an atypical bimodal or biphasic curve, thus indicating a plausible dual mechanism of action for 5-FU. After 48 hours of treatment with 5-FU, the cells were found to be apoptotic, with a distinct reduction in the cell size, compromised anchorage ability but no significant alteration in the cell cycle progression. These findings provided evidence of the global inhibitory effects of 5-FU on human breast cancer cells in vitro and warranted further evaluation to study the molecular basis of aberrant expression of protein-coding genes previously reported after 5-FU treatment. We hypothesized that microRNAs (miRNAs), the newly identified class of small regulatory RNAs, might play a mediator role in inducing the cytotoxicity of 5-FU, by regulating the expression of its target genes. Using a combined advanced microarray and quantitative real time PCR (qRT-PCR) technology, we found for the first time that 5-FU significantly altered the global expression profile of miRNAs in MCF7 breast cancer cells. After 48 hours of treatment with a low dose (0.01µM), 42 miRNAs were differentially expressed in MCF7 cells (23 up-regulated, 19 down-regulated). A majority of these miRNAs have been previously associated with cancer development, and were predicted to potentially target many oncogenes and tumor suppressor genes. To further understand the connection between miRNA dysregulation and 5-FU therapy, we investigated the dose- and time-dependent modification in the miRNA expression levels after 5-FU treatment. Eleven miRNAs (let-7g, miR-10b, miR-15a, miR-16, miR-21, miR-27a, miR-365, miR-374b, miR-483-5p, miR-574-3p and miR-575) previously identified in the microarray as differentially expressed after treatment were selected to analyze their responsiveness to eight different 5-FU dosages (0.001, 0.005, 0.01, 0.1, 0.7, 1, 5 and 10µM). Of these, miR-10b, miR-21, miR-365 and miR-483-5p were shown to be significantly regulated in a beneficial way. Time-response data was also generated for miR-10b, miR-21, miR-483-5p, miR-574-3p and miR-575 following 12, 24, 36, 48, 60 and 72 hours treatment with 0.1, 0.7 and 10µM 5-FU. The data obtained suggested that miRNA expression in MCF7 cells is sensitive to 5-FU therapy at low doses and shorter treatment durations. The down-regulation of an important oncomir, miR-21; and alteration in the expression of three new miRNAs with no previous breast cancer association, miR-483-5p, miR-574-3p and miR-575 indicates that miRNA might play an important role in 5-FU therapy. In conclusion, miRNAs were shown to play an important regulatory role in 5-FU induced cytotoxicity and fit in perfectly in the intricate network of 5-FU activity.