ABSTRACT
mRNA therapeutics hold great promise for the treatment of human diseases. While incorporating naturally occurring modified nucleotides during synthesis has greatly increased their potency and safety, challenges in selective expression have hindered clinical applications. MicroRNA (miRNA)-regulated in vitro-transcribed mRNAs, called miRNA switches, have been used to control the expression of exogenous mRNA in a cell-selective manner. However, the effect of nucleotide modifications on miRNA-dependent silencing has not been examined. Here we show that the incorporation of pseudouridine, N1-methylpseudourdine, or pseudouridine and 5-methylcytidine, which increases translation, tends to decrease the regulation of miRNA switches. Moreover, pseudouridine and 5-methylcytidine modification enables one miRNA target site at the 3' UTR to be as effective as four target sites. We also demonstrate that the effects of pseudouridine, pseudouridine and 5-methylcytidine, and N1-methylpseudourdine modification are miRNA switch specific and do not correlate with the proportion of modified nucleotides in the miRNA target site. Furthermore, modified miRNA switches containing seed-complementary target sites are poorly regulated by miRNA. We also show that placing the miRNA target site in the 5' UTR of the miRNA switch abolishes the effect of nucleotide modification on miRNA-dependent silencing. This work provides insights into the influence of nucleotide modifications on miRNA-dependent silencing and informs the design of optimal miRNA switches.
ABSTRACT
OBJECTIVE: Infantile hemangiomas (IHs) are the most common benign vascular neoplasms of infancy, characterized by a rapid growth phase followed by a spontaneous involution, or triggered by propranolol treatment by poorly understood mechanisms. LIN28/let-7 axis plays a central role in the regulation of stem cell self-renewal and tumorigenesis. However, the role of LIN28B/let-7 signaling in IH pathogenesis has not yet been elucidated. APPROACH AND RESULTS: LIN28B is highly expressed in proliferative IH and is less expressed in involuted and in propranolol-treated IH samples as measured by immunofluorescence staining and quantitative RT-PCR. Small RNA sequencing analysis of IH samples revealed a decrease in microRNAs that target LIN28B, including let-7, and an increase in microRNAs in the mir-498(46) cistron. Overexpression of LIN28B in HEK293 cells induced the expression of miR-516b in the mir-498(46) cistron. Propranolol treatment of induced pluripotent stem cells, which express mir-498(46) endogenously, reduced the expression of both LIN28B and mir-498(46) and increased the expression of let-7. Furthermore, propranolol treatment reduced the proliferation of induced pluripotent stem cells and induced epithelial-mesenchymal transition. CONCLUSIONS: This work uncovers the role of the LIN28B/let-7 switch in IH pathogenesis and provides a novel mechanism by which propranolol induces IH involution. Furthermore, it provides therapeutic implications for cancers in which the LIN28/let-7 pathway is imbalanced.
