The potential ability of microrna-targeted human adenovirus type 5 to replicate and lyse breast cancer cells

Mohammad Shayestehpour,1,* Shaghayegh yazdani,2 Talat mokhtari-azad,3

1. Autoimmune Diseases Research Center, Kashan University of Medical Sciences, Kashan, I.R. Iran
2. Department of Microbiology, Faculty of Advanced Science & Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran
3. Department of Virology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

Abstract

Introduction

Microrna (mir)-targeting method is the newest approach to decrease cytotoxicity of oncolytic viruses in normal cells. in this strategy, mir binding sites are inserted into the viral genome so that viral replication can be regulated by concentration of cellular microrna. microrna-145 is reportedly downregulated in breast tumors but expressed frequently in normal cells. this study was aimed to evaluate the ability of two mir145-5p-targeted adenovirus 5 to replicate and lyse the breast cancer cells, but not normal cells.

Methods

Control and recombinant adenoviruses carrying five or ten copies of mir145-5p binding sites were generated using pad/v5-dest and pentr11 vectors according to gateway cloning technology. the viruses inoculated at an moi of 1.0 into normal human mammary epithelial cells (hmepc), bt-20, mda-mb-453 and mcf-7 breast cancer cell lines. the viral titers were measured 12, 24, 36 and 48 h postinfection using tissue culture infectious dose 50 (tcid50) assay, and adenoviral genomes at 48 h after infection were measured using quantitative real-time pcr. at 3 and 6 days post-infection (moi: 2.0), cell viability was estimated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (mtt) assay.

Results

Titer of control virus and adenoviruses carrying mir145-5p binding sites in breast cancer cell lines (bt-20, mda-mb-453, mcf-7) - with a low level of mir145- exponentially increased within 48 h. hmepc had a high level of mir145; therefore, the replication of adenoviruses carrying mir binding sites in hmepc was inhibited by endogenous mir145-5p at all-time points after infection. dna copy number of adenovirus carrying 10mir sites in hmepc was approximately 22 times lower than that of adenovirus carrying 5mir sites. six days after infection with adenovirus carrying mir binding sites, viability of bt-20, mda-mb-453 and mcf-7 breast cancer cell lines was in the range of 35-45%, while over 80% of normal breast cells (hmepcs) retained their viability.

Conclusion

Microrna145-5p-targeted adenovirus can potentially replicate and selectively lyse breast cancer cells with low cytotoxicity in normal cells. increasing the number of microrna binding sites within the adenoviral genome confers more selectivity for viral replication in cancer cells.

Keywords

Adenovirus, breast cancer, microrna