Knockdown of c-Myc expression by RNAi inhibits MCF-7 breast tumor cells growth in vitro and in vivo

Abstract

Introduction: Breast cancer is the leading cause of cancer death in women worldwide. Elevated expression of c-Myc is a frequent genetic abnormality seen in this malignancy. For a better understanding of its role in maintaining the malignant phenotype, we used RNA interference (RNAi) directed against c-Myc in our study. RNAi provides a new, reliable method to investigate gene function and has the potential for gene therapy. The aim of the study was to examine the anti-tumor effects elicited by a decrease in the protein level of c-Myc by RNAi and its possible mechanism of effects in MCF-7 cells.

Method: A plasmid-based polymerase III promoter system was used to deliver and express short interfering RNA (siRNA) targeting c-myc to reduce its expression in MCF-7 cells. Western blot analysis was used to measure the protein level of c-Myc. We assessed the effects of c-Myc silencing on tumor growth by a growth curve, by soft agar assay and by nude mice experiments in vivo. Standard fluorescence-activated cell sorter analysis and TdT-mediated dUTP nick end labelling assay were used to determine apoptosis of the cells.

Results: Our data showed that plasmids expressing siRNA against c-myc markedly and durably reduced its expression in MCF-7 cells by up to 80%, decreased the growth rate of MCF-7 cells, inhibited colony formation in soft agar and significantly reduced tumor growth in nude mice. We also found that depletion of c-Myc in this manner promoted apoptosis of MCF-7 cells upon serum withdrawal.

Conclusion: c-Myc has a pivotal function in the development of breast cancer. Our data show that decreasing the c-Myc protein level in MCF-7 cells by RNAi could significantly inhibit tumor growth both in vitro and in vivo, and imply the therapeutic potential of RNAi on the treatment of breast cancer by targeting overexpression oncogenes such as c-myc, and c-myc might be a potential therapeutic target for human breast cancer.

Figure 1

Schematic drawing of the pSilencer1.0_U6 vector. The U6-RNA promoter was cloned in front of the gene-specific targeting sequence (19-nucleotide sequences from the target transcript separated by a short spacer from the reverse complement of the same sequence) and six thymidines (T₆) as a termination signal. The predicted secondary structure of the pSilencer–c-Myc transcript target c-Myc is shown. The transcript, a short hairpin double-stranded RNA (dsRNA), is believed to be further cleaved by Dicer to generate a 21-nucleotide siRNA that forms dsRNA–endonuclease complexes and will bind and destroy c-myc mRNA.

Figure 2

Time course of the reduction in c-Myc protein levels by pSilencer–c-Myc. Exponentially proliferating MCF-7 cells were transfected with pSilencer–c-Myc or pSilencer and whole cell lysates were prepared at the time points indicated. Total cell lysates were separated by SDS–polyacrylamide-gel electrophoresis and immunoblotted with an antibody against c-Myc; expression levels were normalized for loading by probing for β-actin.

Figure 3

RNAi directed against c-Myc leads to a reduced cellular growth rate. MCF-7 cells were transfected with pSilencer–c-Myc or pSilencer. After 48 hours the cells were trypsinized and replated at a density of 50 cells/mm² in triplicate. Cells were counted every 2 days. The data shown are means and SD from three independent experiments. **P < 0.01.

Figure 4

Knockdown of c-Myc by RNAi reduces colony formation in soft agar. MCF-7 cells were transfected with pSilencer–c-Myc or pSilencer as controls, and seeded in 0.35% agarose containing Dulbecco's modified Eagle's medium with 10% fetal bovine serum. The colony numbers were counted 2 weeks later. (a) Representative wells demonstrating the total number of colonies formed by MCF-7 transfected with the indicated plasmids. (b) The numbers of colonies of pSilencer–c-Myc-treated cells standardized against the control cells (set at 100%). The data shown are means and SD from two independent triplicate experiments. The difference between treatments is statistically significant (P < 0.001).

Figure 5

Apoptosis induced after serum deprivation by depletion of c-Myc in MCF-7 cells. (a) Downregulation of c-Myc promoted apoptosis of MCF-7 cells after serum deprivation. MCF-7 cells were transfected with pSilencer–c-Myc or pSilencer. After 24 hours, cells were deprived of serum for 36 hours. Cells were then collected by trypsinization. The apoptotic cells were determined by flow cytometry. The cell population in sub-G1 is shown. The x and y axes show DNA content and cell number, respectively. (b) TUNEL assay to detect apoptotic cells in situ. MCF-7 cells were grown on coverslips and transfected with pSilencer–c-Myc or pSilencer. After 24 hours, cells were deprived of serum for 36 hours. Cells were then analyzed for apoptosis with the TUNEL assay. Dark-blue staining of nuclei indicates apoptosis. Arrows indicate representative TUNEL-positive cells.