Restoration of miRNA-149 Expression by TmPyP4 Induced Unfolding of Quadruplex within Its Precursor.

ABSTRACT

Noncoding RNAs are functional RNA molecules that get transcribed from DNA but are not translated into proteins; yet, they can regulate gene expression at transcriptional and post-transcriptional levels. Secondary structures present within these RNAs play a major role in determining their nature of function. In the case of miRNAs, the precursor miRNA have a hairpin stem loop structure which is required for Dicer recognition and further maturation. Alternately, it might assume a G-quadruplex structure. The transition from hairpin to G-quadruplex depends upon the nucleotide sequence as well as the cellular microenvironment, and this might affect the miRNA maturation and other downstream activity. Formation of the G-quadruplex within precursor miRNA-149 has been shown to inhibit Dicer processing activity followed by suppression of miRNA-149 maturation in cancer cells. In this report, we show that suppression of cell proliferation by the upregulated miRNA-149 could be rescued by unfolding the G-quadruplex present in pre-miRNA-149 by TmPyP4 (Porphyrin) treatment. Using UV-visible spectroscopy, circular dichroism, and isothermal titration calorimetry, we observed that TmPyP4 binds strongly to G-quadruplex and unfolds it, which was further verified by NMR spectroscopy. In cellulo, qRT-PCR measurements of miRNA-149 in MCF-7 breast cancer cells showed concentration dependent enhancement of mature miRNA-149 upon treatment of TmPyP4. As a consequence of enhanced miRNA-149 activity, we also observe the reduction in miRNA-149 target protein ZBTB2 that eventually leads to reduced cell proliferation.