Transfer of miR-100 and miR-125b increases 3D growth and invasiveness in recipient cancer cells

Abstract

Aim: Extracellular communication via the transfer of vesicles and nanoparticles is now recognized to play an important role in tumor microenvironment interactions. Cancer cells upregulate and secrete abundant levels of miR-100 and miR-125b that can alter gene expression in donor and recipient cells. In this study, we sought to identify targets of miR-100 and miR-125b and conclusively demonstrate that microRNAs (miRNAs) can be functionally transferred from donor to recipient cells. Methods: To identify targets of miR-100 and miR-125b , we used bioinformatic approaches comparing multiple colorectal cancer (CRC) cell lines, including knockout lines lacking one or both of these miRNAs. We also used spheroid and 3D growth conditions in collagen to test colony growth and invasiveness. We also used Transwell co-culture systems to demonstrate functional miRNA transfer. Results: From an initial list of 96 potential mRNA targets, we identified and tested 15 targets, with 8 showing significant downregulation in the presence of miR-100 and miR-125b . Among these, cingulin (CGN) and protein tyrosine phosphatase receptor type-R (PTPRR) are downregulated in multiple cancers, consistent with regulation by increased levels of miR-100 and miR-125b. We also show that increased cellular levels of miR-100 and miR-125b enhance 3D growth and invasiveness in CRC and glioblastoma cell lines. Lastly, we demonstrate that extracellular transfer of miR-100 and miR-125b can silence both reporter and endogenous mRNA targets in recipient cells and also increase the invasiveness of recipient spheroid colonies when grown under 3D conditions in type I collagen. Conclusion: miR-100 and miR-125b target multiple mRNAs that can regulate 3D cell-autonomous growth and invasiveness. By extracellular transfer, miR-100 and miR-125b can also increase colony growth and invasiveness in recipient cells through non-cell-autonomous mechanisms.