Publications

TRIM71 is an important RNA-binding protein in development and disease, yet its direct targets have not been investigated globally. Here we describe a number of disease and developmentally-relevant TRIM71 RNA targets such as the MBNL family, LIN28B, MDM2, and TCF7L2. We describe a new role for TRIM71 as capable of positive or negative RNA regulation depending on the RNA target. We found that TRIM71 co-precipitated with IMP1 which could explain its multiple mechanisms of RNA regulation, as IMP1 is typically thought to stabilize RNAs. Deletion of the NHL domain of TRIM71 impacted its ability to bind to RNA and RNAs bound by congenital hydrocephalus-associated point mutations in the RNA-binding NHL domain of TRIM71 clustered closely with RNAs bound by the NHL deletion mutant. Our work expands the possible mechanisms by which TRIM71 may regulate RNAs and elucidates further potential RNA targets.

Peptide nucleic acids (PNAs) have emerged as one of the most versatile tools with a wide range of biomedical applications including antisense, antimiR, antigene, as well as site-specific gene editing. The application and potential of PNAs has been limited due to low solubility and poor cellular uptake. Several strategies have been employed to overcome the aforementioned challenges like conjugation to cationic peptides or nanotechnology to achieve superior transfection efficiency ex vivo and in vivo. Here, we report a detailed procedure optimized in our lab for synthesis of short cationic PNA probes, which exhibit high purity and yield in comparison to full-length PNA oligomers. We also provide step-by-step details of encapsulating short cationic PNA probes in poly (lactic-co-glycolic acid) nanoparticles by double emulsion solvent evaporation technique. 1.Detailed procedure for synthesis of short cationic PNAs with or without fluorophore (dye) conjugation while ensuring high yield and purity.2.Step-by-step details for encapsulation of short cationic PNAs in PLGA nanoparticles via double emulsion solvent evaporation technique.

Selective inhibition of microRNAs (miRNAs) offers a new avenue for cancer therapeutics. While most of the current anti-miRNA (antimiR) reagents target full length miRNAs, here we investigate novel nanoparticle-delivered short PNA probes containing cationic domains targeting the seed region of the miRNA for effective antimiR therapy. For proof of concept, we tested PNAs targeting miRNA-155 and employed poly(lactic-co-glycolic acid) (PLGA)-based nanoparticle formulation for delivery. A comprehensive evaluation of PLGA nanoparticles (NPs) containing short PNA probes showed significantly superior loading, release profile, and uniform size distribution, compared to conventional non-cationic PNA probes. Confocal microscopy and flow cytometry analyses showed efficient transfection efficiency and uniform distribution of PLGA NPs containing short PNA probes in the cytoplasm. Functional analysis also confirmed efficient miRNA-155 inhibition including an effect on its downstream target proteins. Further, reduced tumor growth was observed after systemic delivery of PLGA nanoparticles containing short PNA probes in vivo in a xenograft mouse model following inhibition of miR-155. There was no evidence of acute or chronic toxicity associated with systemic delivery of PLGA NPs containing short PNA probes in the mice. Overall, in this paper we present a novel antimiR strategy based on PLGA nanoparticle delivered short PNA probes for potential cancer therapy.

Cancer cells subvert host immune surveillance by altering immune checkpoint (IC) proteins. Some Epstein-Barr virus (EBV)-associated tumors have higher Programmed Cell Death Ligand, PD-L1 expression. However, it is not known how EBV alters ICs in the context of its preferred host, the B lymphocyte and in derived lymphomas. Here, we found that latency III-expressing Burkitt lymphoma (BL), diffuse large B-cell lymphomas (DLBCL) or their EBNA2-transfected derivatives express high PD-L1. In a DLBCL model, EBNA2 but not LMP1 is sufficient to induce PD-L1. Latency III-expressing DLBCL biopsies showed high levels of PD-L1. The PD-L1 targeting oncosuppressor microRNA miR-34a was downregulated in EBNA2-transfected lymphoma cells. We identified early B-cell factor 1 (EBF1) as a repressor of miR-34a transcription. Short hairpin RNA (shRNA)-mediated knockdown of EBF1 was sufficient to induce miR-34a transcription, which in turn reduced PD-L1. MiR-34a reconstitution in EBNA2-transfected DLBCL reduced PD-L1 expression and increased its immunogenicity in mixed lymphocyte reactions (MLR) and in three-dimensional biomimetic microfluidic chips. Given the importance of PD-L1 inhibition in immunotherapy and miR-34a dysregulation in cancers, our findings may have important implications for combinatorial immunotherapy, which include IC inhibiting antibodies and miR-34a, for EBV-associated cancers.

The accurate inheritance of genetic material is a basic necessity in all domains of life and an unexpectedly large number of RNA processing factors are required for mitotic progression and genome stability. NRDE2 (nuclear RNAi defective-2) is an evolutionarily conserved protein originally discovered for its role in nuclear RNA interference (RNAi) and heritable gene silencing in Caenorhabditis elegans (C. elegans). The function of the human NRDE2 gene remains poorly understood. Here we show that human NRDE2 is an essential protein required for suppressing intron retention in a subset of pre-mRNAs containing short, GC-rich introns with relatively weak 5' and 3' splice sites. NRDE2 preferentially interacts with components of the U5 small nuclear ribonucleoprotein (snRNP), the exon junction complex, and the RNA exosome. Interestingly, NRDE2-depleted cells exhibit greatly increased levels of genomic instability and DNA damage, as well as defects in centrosome maturation and mitotic progression. We identify the essential centriolar satellite protein, CEP131, as a direct NRDE2-regulated target. NRDE2 specifically binds to and promotes the efficient splicing of CEP131 pre-mRNA, and depleting NRDE2 dramatically reduces CEP131 protein expression, contributing to impaired recruitment of critical centrosomal proteins (e.g., γ-tubulin and Aurora Kinase A) to the spindle poles during mitosis. Our work establishes a conserved role for human NRDE2 in RNA splicing, characterizes the severe genomic instability phenotypes observed upon loss of NRDE2, and highlights the direct regulation of CEP131 splicing as one of multiple mechanisms through which such phenotypes might be explained.

MicroRNAs (miRNAs) have emerged as crucial players in the development and maintenance of disease. miR-155 is an inflammation-associated, oncogenic miRNA, frequently overexpressed in hematological malignancies and solid tumors. However, the mechanism of oncogenesis by miR-155 is not well characterized, and research has focused primarily on individual, direct targets, which does not recapitulate the complexities of cancer. Using a powerful, inducible transgenic mouse model that overexpresses miR-155 and develops miR-155-addicted hematological malignancy, we describe here a multi-step process of oncogenesis by miR-155, which involves cooperation between miR-155, its direct targets, and other oncogenes. miR-155 is known to target DNA-repair proteins, leading to a mutator phenotype, and we find that over 93% of tumors in our miR-155 overexpressing mice contain activating mutations in a single oncogene, c-Kit. Treating mice with dasatinib or imatinib, which target c-Kit, resulted in complete tumor regression, indicating that c-Kit activity is crucial in the oncogenic process. Interestingly, c-Kit expression is high when miR-155 is overexpressed, indicating further cooperation between miR-155 and c-Kit. Our findings support a multi-step model of oncogenesis by miR-155 in which miR-155 promotes both a mutator phenotype and a cellular environment particularly susceptible to mutations in a given oncogene.

BACKGROUND: There is an urgent need to identify factors that increase vulnerability to opioid addiction to help stem the opioid epidemic and develop more efficient pharmacotherapeutics. MicroRNAs are small non-coding RNAs that regulate gene expression at a posttranscriptional level and have been implicated in chronic drug-taking in humans and in rodent models. Recent evidence has shown that chronic opioid treatment regulates the microRNA miR-9. The present study was designed to test the hypothesis that miR-9 in the nucleus accumbens potentiates oxycodone addictive-like behavior.

METHODS: We utilized adeno-associated virus (AAV) to overexpress miR-9 in the nucleus accumbens of male rats and tested the effects on intravenous self-administration of the highly abused prescription opioid, oxycodone, in 1-hour short-access followed by 6-h long-access sessions, the latter of which leads to escalation of drug intake. In separate rats, we assessed the effects of nucleus accumbens miR-9 overexpression on mRNA targets including RE1-silencing transcription factor (REST) and dopamine D2 receptor (DRD2), which have been shown to be regulated by drugs of abuse.

RESULTS: Overexpression of miR-9 in the nucleus accumbens significantly increased oxycodone self-administration compared with rats expressing a control, scrambled microRNA. Analysis of the pattern of oxycodone intake revealed that miR-9 overexpression increased "burst" episodes of intake and decreased the inter-infusion interval. Furthermore, miR-9 overexpression decreased the expression of REST and increased DRD2 in the nucleus accumbens at time points that coincided with behavioral effects.

CONCLUSIONS: These results suggest that nucleus accumbens miR-9 regulates oxycodone addictive-like behavior as well as the expression of genes that are involved in drug addiction.

Developing new targeted therapy for pancreatic cancer is one of the major current challenges in cancer research. KRAS mutations and miRNA dysregulation (e.g. miR-21-5p oncomiR) play key roles in Pancreatic Ductal Adenocarcinoma (PDAC), leading to rapid progression of the disease. As the KRAS mutation is a main driver of PDAC, anti-KRAS strategies remain a major therapeutic approach for PDAC treatment. Previously, utilization of either siKRAS or small chemically modified single-stranded RNA molecules that specifically disable miR-21 (anti-miR-21) were effective in slowing PDAC tumor growth in various tumor models when packaged in an innovative delivery system (TPN) required for efficient drug delivery to the PDAC tumor site. Here we have tested the utility of targeting the KRAS pathway through multiple mechanisms and via dual targeting of a PDAC oncomiR and oncogene. Initially we found that miR-217, which has been shown to directly regulate KRAS expression, is downregulated in our PDAC samples, thus we tested the benefits of anti-miR-21, miR-217 mimic or siKRAS loaded into the tumor-penetrating nanoparticles (TPN) that we had previously shown to potently target the largely impenetrable PDAC tumors, and found an enhanced anti-tumoral response upon dual treatments in KRAS-mutated PDAC models.

Drug-tolerance is an acute defense response prior to a fully drug-resistant state and tumor relapse, however there are few therapeutic agents targeting drug-tolerance in the clinic. Here we show that miR-147b initiates a reversible tolerant-state to the EGFR inhibitor osimertinib in non-small cell lung cancer. With miRNA-seq analysis we find that miR-147b is the most upregulated microRNA in osimertinib-tolerant and EGFR mutated lung cancer cells. Whole transcriptome analysis of single-cell derived clones reveals a link between osimertinib-tolerance and pseudohypoxia responses irrespective of oxygen levels. Further metabolomics and genetic studies demonstrate that osimertinib-tolerance is driven by miR-147b repression of VHL and succinate dehydrogenase linked to the tricarboxylic acid cycle and pseudohypoxia pathways. Finally, pretreatment with a miR-147b inhibitor delays osimertinib-associated drug tolerance in patient-derived three-dimensional (3D) structures. This link between miR-147b and tricarboxylic acid cycle may provide promising targets for preventing tumor relapse.