miR-497 and miR-302b regulate ethanol-induced neuronal cell death through BCL2 protein and cyclin D2

In chronic alcoholism, brain shrinkage and cognitive defects because of neuronal death are well established, although the sequence of molecular events has not been fully explored yet. We explored the role of microRNAs (miRNAs) in ethanol-induced apoptosis of neuronal cells. Ethanol-sensitive miRNAs in SH-SY5Y, a human neuroblastoma cell line, were identified using real-time PCR-based TaqMan low-density arrays. Long-term exposure to ethanol (0.5% v/v for 72 h) produced a maximum increase in expression of miR-497 (474-fold) and miR-302b (322-fold). Similar to SH-SY5Y, long-term exposure to ethanol induced miR-497 and miR-302b in IMR-32, another human neuroblastoma cell line. Using in silico approaches, BCL2 and cyclin D2 (CCND2) were identified as probable target genes of these miRNAs. Cotransfection studies with 3'-UTR of these genes and miRNA mimics have demonstrated that BCL2 is a direct target of miR-497 and that CCND2 is regulated negatively by either miR-302b or miR-497. Overexpression of either miR-497 or miR-302b reduced expression of their identified target genes and increased caspase 3-mediated apoptosis of SH-SY5Y cells. However, overexpression of only miR-497 increased reactive oxygen species formation, disrupted mitochondrial membrane potential, and induced cytochrome c release (mitochondria-related events of apoptosis). Moreover, ethanol induced changes in miRNAs, and their target genes were substantially prevented by pre-exposure to GSK-3B inhibitors. In conclusion, our studies have shown that ethanol-induced neuronal apoptosis follows both the mitochondria-mediated (miR-497- and BCL2-mediated) and non-mitochondria-mediated (miR-302b- and CCND2-mediated) pathway.     

MicroRNA Biogenesis and Hedgehog-Patched Signaling Cooperate to Regulate an Important Developmental Transition in Granule Cell Development

The Dicer1, Dcr-1 homolog (Drosophila) gene encodes a type III ribonuclease required for the canonical maturation and functioning of microRNAs (miRNAs). Subsets of miRNAs are known to regulate normal cerebellar granule cell development, in addition to the growth and progression of medulloblastoma, a neoplasm that often originates from granule cell precursors. Multiple independent studies have also demonstrated that deregulation of Sonic Hedgehog (Shh)-Patched (Ptch) signaling, through miRNAs, is causative of granule cell pathologies. In the present study, we investigated the genetic interplay between miRNA biogenesis and Shh-Ptch signaling in granule cells of the cerebellum by way of the Cre/lox recombination system in genetically engineered models of Mus musculus (mouse). We demonstrate that, although the miRNA biogenesis and Shh-Ptch-signaling pathways, respectively, regulate the opposing growth processes of cerebellar hypoplasia and hyperplasia leading to medulloblastoma, their concurrent deregulation was nonadditive and did not bring the growth phenotypes toward an expected equilibrium. Instead, mice developed either hypoplasia or medulloblastoma, but of a greater severity. Furthermore, some genotypes were bistable, whereby subsets of mice developed hypoplasia or medulloblastoma. This implies that miRNAs and Shh-Ptch signaling regulate an important developmental transition in granule cells of the cerebellum. We also conclusively show that the Dicer1 gene encodes a haploinsufficient tumor suppressor gene for Ptch1-induced medulloblastoma, with the monoallielic loss of Dicer1 more severe than biallelic loss. These findings exemplify how genetic interplay between pathways may produce nonadditive effects with a substantial and unpredictable impact on biology. Furthermore, these findings suggest that the functional dosage of Dicer1 may nonadditively influence a wide range of Shh-Ptch-dependent pathologies.     

Dysregulation of microRNA biogenesis machinery in cancer

MicroRNAs (miRNAs) are integral to the gene regulatory network. A single miRNA is capable of controlling the expression of hundreds of protein coding genes and modulate a wide spectrum of biological functions, such as proliferation, differentiation, stress responses, DNA repair, cell adhesion, motility, inflammation, cell survival, senescence and apoptosis, all of which are fundamental to tumorigenesis. Overexpression, genetic amplification, and gain-of-function mutation of oncogenic miRNAs (“onco-miRs”) as well as genetic deletion and loss-of-function mutation of tumor suppressor miRNAs (“suppressor-miRs”) are linked to human cancer. In addition to the dysregulation of a specific onco-miR or suppressor-miRs, changes in global miRNA levels resulting from a defective miRNA biogenesis pathway play a role in tumorigenesis. The function of individual onco-miRs and suppressor-miRs and their target genes in cancer has been described in many different articles elsewhere. In this review, we primarily focus on the recent development regarding the dysregulation of the miRNA biogenesis pathway and its contribution to cancer.     

A comprehensive analysis of precursor microRNA cleavage by human Dicer

Dicer cleaves double-stranded RNAs (dsRNAs) or precursor microRNAs (pre-miRNAs) to yield ∼22-nt RNA duplexes. The pre-miRNA structure requirement for human Dicer activity is incompletely understood. By large-scale in vitro dicing assays and mutagenesis studies, we showed that human Dicer cleaves most, although not all, of the 161 tested human pre-miRNAs efficiently. The stable association of RNAs with Dicer, as examined by gel shift assays, appears important but is not sufficient for cleavage. Human Dicer tolerates remarkable structural variation in its pre-miRNA substrates, although the dsRNA feature in the stem region and the 2-nt 3′-overhang structure in a pre-miRNA contribute to its binding and cleavage by Dicer, and a large terminal loop further enhances pre-miRNA cleavage. Dicer binding protects the terminal loop from digestion by S1 nuclease, suggesting that Dicer interacts directly with the terminal loop region.     

Inhibition of pre-miRNA-136 processing by Dicer with small molecule BzDANP suggested the formation of ternary complex of pre-miR-136–BzDANP–Dicer

Small-molecule modulators, along with antisense oligonucleotide, would be powerful tools and potential drug candidates for modulating miRNA-related gene expressions. The mechanism of the inhibitory effect of the C-bulge binding small molecule BzDANP for the Dicer processing reaction of pre-miR-136 was discussed on the data obtained by SPR, NMR, and kinetic analysis for Dicer processing. SPR and NMR analysis showed the preference of BzDANP binding to the C-bulge. Michaelis-Menten analysis suggested the formation of a ternary complex pre-miR-136–BzDANP–Dicer during the Dicer-cleavage reaction of pre-miR-136 in the presence of BzDANP. The inhibitory effect of BzDANP is likely attributed to the slower reaction from the ternary complex than that from the binary pre-miR-136–Dicer complex.