MiRNA inhibition in tissue engineering and regenerative medicine

Abstract

MicroRNAs (miRNAs) are noncoding RNAs that provide an endogenous negative feedback mechanism for translation of messenger RNA (mRNA) into protein. Single miRNAs can regulate hundreds of mRNAs, enabling miRNAs to orchestrate robust biological responses by simultaneously impacting multiple gene networks. MiRNAs can act as master regulators of normal and pathological tissue development, homeostasis, and repair, which has motivated expanding efforts toward the development of technologies for therapeutically modulating miRNA activity for regenerative medicine and tissue engineering applications. This review highlights the tools currently available for miRNA inhibition and their recent therapeutic applications for improving tissue repair.

Keywords: Anti-miR; MiRNA; MiRNA inhibition; Regenerative medicine; Tissue engineering.

Figure 1

Sites of intervention for different anti-miRs along (A) the miRNA biogenesis pathway. Anti-miRNA oligos (AMOs) are typically single stranded oligos that are introduced exogenously into the cell and can bind to (B) pri-miRNA to inhibit Drosha activity or (C) pre-miRNA to inhibit Dicer cleavage. (D) miRNA sponges are expressed as transgenes that contain multiple miRNA binding sites for competitive inhibition of binding to mRNA. (E) AMOs are most commonly designed to bind to and inhibit mature miRNA. (G) Blockmirs are oligonucleotides that block miRNA activity by specifically masking the 3’ UTR of target mRNA. Small molecule miRNA inhibitors act by either (F) inhibiting the formation of active RISC, or (H) preventing expression of miRNA genes into pri-miRNA.

Figure 2

Common oligonucleotide modifications to improve anti-miR activity. (A) The 2’ OH of ribose RNA can be methylated to create (B) OMe-modified RNA, or a methylene bridge can be added between the ribose 2’-O and 4’-C to create (C) locked nucleic acid (LNA). (D) The phosphodiester bonds in the backbone can be replaced with phosphorothiolate (PS) bonds, or a (E) ZEN modifier can be added between phosphate groups near oligo ends. Finally, neutrally-charged, synthetic (F) phosphorodiamidate morpholino oligonucleotide (PMO) and (G) peptide nucleic acid (PNA) chemistries can also be designed strongly inhibit miRNA.

Figure 3

Potential anti-miR targets for regenerative medicine.

Figure 4

Micro-CT sections following implantation of PGS scaffolds without BMSCs (PGS), or seeded with BMSCs transfected with lentiviruses encoding for miR-31 overexpression (BMSCs/miR-31), miR-31 inhibition (BMSCs/anti-miR), or an irrelevant DNA sequence (BMSCs/miR-Neg). These data suggest that miR-31 impairs bone repair, while transfection of BMSCs with anti-miR-31 prior to implantation greatly increases bone regeneration. Reproduced with kind permission from eCM journal (www.ecmjournal.org).

Figure 5

LNA–anti-miR-192 attenuates glomerular growth, mesangial expansion, and TGFβ expression in 17-week diabetic mice. (A-C) Periodic acid–Schiff [PAS] staining of representative kidney sections. (D-F) Masson’s trichrome staining showing glomerular and tubulointerstitial fibrosis. (G-I) TGF-β immunostaining of kidney sections. Reproduced from Putta et al. (2012); with kind permission of the Journal of the American Society of Nephrology.

Figure 6

Histology of heart tissue sections stained with Masson’s trichrome reveals that inhibition of miR-25 reduces fibrosis under conditions that mimic heart failure. Reproduced and adapted with kind permission from Nature Publishing Group.