microRNA Biogenesis Research Articles

MicroRNA and their implications in dental pulp inflammation: current trends and future perspectives.

MicroRNAs (miRNAs) are short, 19-23 nucleotide non-coding RNA molecules that regulate gene expression by silencing or degrading the target mRNA gene. Since their discovery in the nineties of the last century, they have emerged as key inflammatory regulators. Inflammation induces the synthesis of various miRNAs that modulate the expression of multiple molecules involved in orchestrating the inflammatory response. This review aims to provide an insight into the role of miRNAs as potential biomarkers, mediators, and potential therapeutic targets of dental pulp inflammation. A literature search was conducted using the keywords; biogenesis of microRNA, human dental pulp cells, pulpitis, and inflammation in PubMed and Scopus index databases for all the published articles dealing with the role of miRNAs in pulp inflammation in the last 10years. According to the literature, there is a clear correlation between miRNAs and several physiological events, as well as their role as mediators of innate immune response and inflammation in dental pulp cells. Our narrative review stipulates that numerous miRNAs play a key role in modulating inflammation, delaying or enhancing cell repair, cell differentiation, and survival in dental pulp diseases. However, further studies are required for the validation of miRNAs as reliable biomarkers in dental pulp pathology and their targeted therapy.

Arabidopsis AAR2, a conserved splicing factor in eukaryotes, acts in microRNA biogenesis

MicroRNAs (miRNAs) play an essential role in plant growth and development, and as such, their biogenesis is fine-tuned via regulation of the core microprocessor components. Here, we report that Arabidopsis AAR2, a homolog of a U5 snRNP assembly factor in yeast and humans, not only acts in splicing but also promotes miRNA biogenesis. AAR2 interacts with the microprocessor component hyponastic leaves 1 (HYL1) in the cytoplasm, nucleus, and dicing bodies. In aar2 mutants, abundance of nonphosphorylated HYL1, the active form of HYL1, and the number of HYL1-labeled dicing bodies are reduced. Primary miRNA (pri-miRNA) accumulation is compromised despite normal promoter activities of MIR genes in aar2 mutants. RNA decay assays show that the aar2-1 mutation leads to faster degradation of pri-miRNAs in a HYL1-dependent manner, which reveals a previously unknown and negative role of HYL1 in miRNA biogenesis. Taken together, our findings reveal a dual role of AAR2 in miRNA biogenesis and pre-messenger RNA splicing.

Structural basis of microRNA biogenesis by Dicer-1 and its partner protein Loqs-PB

In animals and plants, Dicer enzymes collaborate with double-stranded RNA-binding domain (dsRBD) proteins to convert precursor-microRNAs (pre-miRNAs) into miRNA duplexes. We report six cryo-EM structures of Drosophila Dicer-1 that show how Dicer-1 and its partner Loqs‑PB cooperate (1) before binding pre-miRNA, (2) after binding and in a catalytically competent state, (3) after nicking one arm of the pre-miRNA, and (4) following complete dicing and initial product release. Our reconstructions suggest that pre-miRNA binds a rare, open conformation of the Dicer‑1⋅Loqs‑PB heterodimer. The Dicer-1 dsRBD and three Loqs‑PB dsRBDs form a tight belt around the pre-miRNA, distorting the RNA helix to place the scissile phosphodiester bonds in the RNase III active sites. Pre-miRNA cleavage shifts the dsRBDs and partially closes Dicer-1, which may promote product release. Our data suggest a model for how the Dicer‑1⋅Loqs‑PB complex affects a complete cycle of pre-miRNA recognition, stepwise endonuclease cleavage, and product release.

Chromatin-associated microprocessor assembly is regulated by the U1 snRNP auxiliary protein PRP40.

In plants, microRNA (miRNA) biogenesis involves cotranscriptional processing of RNA polymerase II (RNAPII)-generated primary transcripts by a multi-protein complex termed the microprocessor. Here, we report that Arabidopsis (Arabidopsis thaliana) PRE-MRNA PROCESSING PROTEIN 40 (PRP40), the U1 snRNP auxiliary protein, positively regulates the recruitment of SERRATE, a core component of the plant microprocessor, to miRNA genes. The association of DICER-LIKE1 (DCL1), the microprocessor endoribonuclease, with chromatin was altered in prp40ab mutant plants. Impaired cotranscriptional microprocessor assembly was accompanied by RNAPII accumulation at miRNA genes and retention of miRNA precursors at their transcription sites in the prp40ab mutant plants. We show that cotranscriptional microprocessor assembly, regulated by AtPRP40, positively affects RNAPII transcription of miRNA genes and is important to reach the correct levels of produced miRNAs.

Miat and interacting protein Metadherin maintain a stem-like niche to promote medulloblastoma tumorigenesis and treatment resistance

Long noncoding RNAs (lncRNAs) play essential roles in the development and progression of many cancers. However, the contributions of lncRNAs to medulloblastoma (MB) remain poorly understood. Here, we identify Miat as an lncRNA enriched in the sonic hedgehog group of MB that is required for maintenance of a treatment-resistant stem-like phenotype in the disease. Loss of Miat results in the differentiation of tumor-initiating, stem-like MB cells and enforces the differentiation of tumorigenic stem-like MB cells into a nontumorigenic state. Miat expression in stem-like MB cells also facilitates treatment resistance by down-regulating p53 signaling and impairing radiation-induced cell death, which can be reversed by therapeutic inhibition of Miat using antisense oligonucleotides. Mechanistically, the RNA binding protein Metadherin (Mtdh), previously linked to resistance to cytotoxic therapy in cancer, binds to Miat in stem-like MB cells. Like the loss of Miat, the loss of Mtdh reduces tumorigenicity and increases sensitivity to radiation-induced death in stem-like MB cells. Moreover, Miat and Mtdh function to regulate the biogenesis of several microRNAs and facilitate tumorigenesis and treatment resistance. Taken together, these data reveal an essential role for the lncRNA Miat in sustaining a treatment-resistant pool of tumorigenic stem-like MB cells.

The OTUD6B‐LIN28B‐MYC axis determines the proliferative state in multiple myeloma

Deubiquitylases (DUBs) are therapeutically amenable components of the ubiquitin machinery that stabilize substrate proteins. Their inhibition can destabilize oncoproteins that may otherwise be undruggable. Here, we screened for DUB vulnerabilities in multiple myeloma, an incurable malignancy with dependency on the ubiquitin proteasome system and identified OTUD6B as an oncogene that drives the G1/S‐transition. LIN28B, a suppressor of microRNA biogenesis, is specified as a bona fide cell cycle‐specific substrate of OTUD6B. Stabilization of LIN28B drives MYC expression at G1/S, which in turn allows for rapid S‐phase entry. Silencing OTUD6B or LIN28B inhibits multiple myeloma outgrowth in vivo and high OTUD6B expression evolves in patients that progress to symptomatic multiple myeloma and results in an adverse outcome of the disease. Thus, we link proteolytic ubiquitylation with post‐transcriptional regulation and nominate OTUD6B as a potential mediator of the MGUS‐multiple myeloma transition, a central regulator of MYC, and an actionable vulnerability in multiple myeloma and other tumors with an activated OTUD6B‐LIN28B axis.

DDX5 and DDX17-multifaceted proteins in the regulation of tumorigenesis and tumor progression.

DEAD-box (DDX)5 and DDX17, which belong to the DEAD-box RNA helicase family, are nuclear and cytoplasmic shuttle proteins. These proteins are expressed in most tissues and cells and participate in the regulation of normal physiological functions; their abnormal expression is closely related to tumorigenesis and tumor progression. DDX5/DDX17 participate in almost all processes of RNA metabolism, such as the alternative splicing of mRNA, biogenesis of microRNAs (miRNAs) and ribosomes, degradation of mRNA, interaction with long noncoding RNAs (lncRNAs) and coregulation of transcriptional activity. Moreover, different posttranslational modifications, such as phosphorylation, acetylation, ubiquitination, and sumoylation, endow DDX5/DDX17 with different functions in tumorigenesis and tumor progression. Indeed, DDX5 and DDX17 also interact with multiple key tumor-promoting molecules and participate in tumorigenesis and tumor progression signaling pathways. When DDX5/DDX17 expression or their posttranslational modification is dysregulated, the normal cellular signaling network collapses, leading to many pathological states, including tumorigenesis and tumor development. This review mainly discusses the molecular structure features and biological functions of DDX5/DDX17 and their effects on tumorigenesis and tumor progression, as well as their potential clinical application for tumor treatment.

APE1 controls DICER1 expression in NSCLC through miR-33a and miR-130b

Increasing evidence suggests different, not completely understood roles of microRNA biogenesis in the development and progression of lung cancer. The overexpression of the DNA repair protein apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) is an important cause of poor chemotherapeutic response in lung cancer and its involvement in onco-miRNAs biogenesis has been recently described. Whether APE1 regulates miRNAs acting as prognostic biomarkers of lung cancer has not been investigated, yet. In this study, we analyzed miRNAs differential expression upon APE1 depletion in the A549 lung cancer cell line using high-throughput methods. We defined a signature of 13 miRNAs that strongly correlate with APE1 expression in human lung cancer: miR-1246, miR-4488, miR-24, miR-183, miR-660, miR-130b, miR-543, miR-200c, miR-376c, miR-218, miR-146a, miR-92b and miR-33a. Functional enrichment analysis of this signature revealed its biological relevance in cancer cell proliferation and survival. We validated DICER1 as a direct functional target of the APE1-regulated miRNA-33a-5p and miR-130b-3p. Importantly, IHC analyses of different human tumors confirmed a negative correlation existing between APE1 and Dicer1 protein levels. DICER1 downregulation represents a prognostic marker of cancer development but the mechanisms at the basis of this phenomenon are still completely unknown. Our findings, suggesting that APE1 modulates DICER1 expression via miR-33a and miR-130b, reveal new mechanistic insights on DICER1 regulation, which are of relevance in lung cancer chemoresistance and cancer invasiveness.

Widespread alterations in microRNA biogenesis in human Huntington’s disease putamen

Altered microRNA (miRNA) expression is a common feature of Huntington’s disease (HD) and could participate in disease onset and progression. However, little is known about the underlying causes of miRNA disruption in HD. We and others have previously shown that mutant Huntingtin binds to Ago2, a central component of miRNA biogenesis, and disrupts mature miRNA levels. In this study, we sought to determine if miRNA maturation per se was compromised in HD. Towards this end, we characterized major miRNA biogenesis pathway components and miRNA maturation products (pri-miRNA, pre-miRNA, and mature) in human HD (N = 41, Vonsattel grades HD2-4) and healthy control (N = 25) subjects. Notably, the striatum (putamen) and cortex (BA39) from the same individuals were analyzed in parallel. We show that Ago2, Drosha, and Dicer were strongly downregulated in human HD at the early stages of the disease. Using a panel of HD-related miRNAs (miR-10b, miR-196b, miR-132, miR-212, miR-127, miR-128), we uncovered various types of maturation defects in the HD brain, the most prominent occurring at the pre-miRNA to mature miRNA maturation step. Consistent with earlier findings, we provide evidence that alterations in autophagy could participate in miRNA maturation defects. Notably, most changes occurred in the striatum, which is more prone to HTT aggregation and neurodegeneration. Likewise, we observed no significant alterations in miRNA biogenesis in human HD cortex and blood, strengthening tissue-specific effects. Overall, these data provide important clues into the underlying mechanisms behind miRNA alterations in HD-susceptible tissues. Further investigations are now required to understand the biological, diagnostic, and therapeutic implications of miRNA/RNAi biogenesis defects in HD and related neurodegenerative disorders.

MicroRNA Processing Pathway-Based Polygenic Score for Clear Cell Renal Cell Carcinoma in the Volga-Ural Region Populations of Eurasian Continent.

The polygenic scores (PGSs) are developed to help clinicians in distinguishing individuals at high risk of developing disease outcomes from the general population. Clear cell renal cell carcinoma (ccRCC) is a complex disorder that involves numerous biological pathways, one of the most important of which is responsible for the microRNA biogenesis machinery. Here, we defined the biological-pathway-specific PGS in a case-control study of ccRCC in the Volga-Ural region of the Eurasia continent. We evaluated 28 DNA SNP variants, located in microRNA biogenesis genes, in 464 individuals with clinically diagnosed ccRCC and 1042 individuals without the disease. Individual genetic risks were defined using the SNP-variant effects derived from the ccRCC association analysis. The final weighted and unweighted PGS models were based on 21 SNPs, and 7 SNPs were excluded due to high LD. In our dataset, microRNA-machinery-weighted PGS revealed 1.69-fold higher odds (95% CI [1.51–1.91]) for ccRCC risk in individuals with ccRCC compared with controls with a p-value of 2.0 × 10−16. The microRNA biogenesis pathway weighted PGS predicted the risk of ccRCC with an area under the curve (AUC) = 0.642 (95%nCI [0.61–0.67]). Our findings indicate that DNA variants of microRNA machinery genes modulate the risk of ccRCC in Volga-Ural populations. Moreover, larger powerful genome-wide association studies are needed to reveal a wider range of genetic variants affecting microRNA processing. Biological-pathway-based PGSs will advance the development of innovative screening systems for future stratified medicine approaches in ccRCC.

MdMYB88/124 modulates apple tree microRNA biogenesis through post-transcription processing and/or transcription pathway

MdMYB88/124 modulates apple tree microRNA biogenesis through post-transcription processing and/or transcription pathway

MicroRNA biogenesis proteins follow tissue-dependent expression during freezing in Dryophytes versicolor

Grey tree frogs (Dryophytes versicolor) have the remarkable ability to endure full-body freezing over the winter, with up to 42% of total body water converted into extracellular ice. Survival is aided by metabolic rate depression that greatly reduces tissue energy costs over the winter. Post-transcriptional controls on gene expression which include miRNA regulation of gene transcripts can aid implementation of the reversible changes required for freeze tolerance, since miRNAs are ideal for facilitating the rapid metabolic reorganization needed for this process. The energy cost for synthesizing new miRNAs is low, and miRNAs' ability to target more than one mRNA transcript (and vice versa) allows a wide versatility in their capability for metabolic restructuring. Western immunoblotting was used to examine protein expression levels of members of the miRNA biogenesis pathway in D. versicolor liver, skeletal muscle, and kidney. Four of these proteins (Dicer, Drosha, Trbp, Xpo5) were upregulated in liver of frozen frogs, suggesting enhanced capacity for miRNA biogenesis, whereas expression of four proteins in frozen muscle (Ago1, Ago2, Dgcr8, Xpo5) and six proteins in kidney (Ago1, Ago2, Ago3, Ago4, Dgcr8, Ran-GTP) were downregulated, indicating an opposite trend. Overall, the data show that miRNA biosynthesis is altered during freezing and differentially regulated across tissues. We suggest that miRNAs are central for the freeze tolerance strategy developed by D. versicolor, and future research will expound upon specific miRNAs and their roles in mediating responses to freezing stress.

Upregulation of β-catenin due to loss of miR-139 contributes to motor neuron death in amyotrophic lateral sclerosis.

SummaryAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of motor neurons (MNs). There are no effective treatments and patients usually die within 2–5 years of diagnosis. Emerging commonalities between familial and sporadic cases of this complex multifactorial disorder include disruption to RNA processing and cytoplasmic inclusion bodies containing TDP-43 and/or FUS protein aggregates. Both TDP-43 and FUS have been implicated in RNA processing functions, including microRNA biogenesis, transcription, and splicing. In this study, we explore the misexpression of microRNAs in an iPSC-based disease model of FUS ALS. We identify the downregulation of miR-139, an MN-enriched microRNA, in FUS and sporadic ALS MN. We discover that miR-139 downregulation leads to the activation of canonical WNT signaling and demonstrate that the WNT transcriptional mediator β-catenin is a major driver of MN degeneration in ALS. Our results highlight the importance of homeostatic RNA networks in ALS.

Argonaute1 and Gawky Are Required for the Development and Reproduction of Melon fly, Zeugodacus cucurbitae.

Argonaute family genes encode a highly conserved group of proteins that have been associated with RNA silencing in both animals and plants. This study investigates the importance of microRNA biogenesis key regulators Argonaute1 (Ago1) and Gawky genes in the post-embryonic and ovarian development of the melon fly, Zeugodacus cucurbitae. The expression levels of these genes were mapped in all developmental stages and different adult tissues. Their roles in development were investigated using RNA interference (RNAi) via two different dsRNA delivery techniques. Embryo microinjection and oral feeding of third instar larvae successfully knocked down and greatly reduced the expression level of the target genes. Additionally, ex vivo essays revealed the stability of dsRNA in food was sufficient for gene silencing, although its integrity was affected in midgut. A wide range of phenotypes were observed on pupation, segmentation, pigmentation, and ovarian development. RNAi-mediated silencing of Gawky caused high mortality and loss of body segmentation, while Ago1 knockdown affected ovarian development and pigmentation. Developmental abnormalities and ovarian malformations caused by silencing these genes suggest that these genes are crucial for viability and reproductive capacity of Z. cucurbitae, and may be used as potential target genes in pest management.

The New Face of a Well-Known Antibiotic: A Review of the Anticancer Activity of Enoxacin and Its Derivatives.

Simple SummaryEnoxacin is a second-generation quinolone with promising anticancer activity. In contrast to other members of the quinolone group, it exhibits an extraordinary cytotoxic mechanism of action. Enoxacin enhances RNA interference and promotes microRNA processing, as well as the production of free radicals. Interestingly, apart from its proapoptotic, cell cycle arresting and cytostatic effects, enoxacin manifests a limitation of cancer invasiveness. The underlying mechanisms are the competitive inhibition of vacuolar H+-ATPase subunits and c-Jun N-terminal kinase signaling pathway suppression. The newly synthesized enoxacin derivatives have shown a magnified cytotoxic effect with an emphasis on prooxidative, proapoptotic and microRNA interference actions. The mentioned mechanisms seem to contribute to a safer, more selective and more effective anticancer therapy. Enoxacin as a second-generation synthetic quinolone is known for its antibacterial action; however, in recent years there have been studies focusing on its anticancer potential. Interestingly, it turns out that compared to other fluoroquinolones, enoxacin exhibits uncommon cytotoxic properties. Besides its influence on apoptosis, the cell cycle and cell growth, it exhibits a regulatory action on microRNA biogenesis. It was revealed that the molecular targets of the enoxacin-mediated inhibition of osteoclastogenesis are vacuolar H+-ATPase subunits and the c-Jun N-terminal kinase signaling pathway, causing a decrease in cell invasiveness. Interestingly, the prooxidative nature of the subjected fluoroquinolone enhanced the cytotoxic effect. Crucial for the anticancer activity were the carboxyl group at the third carbon atom, fluorine at the seventh carbon atom and nitrogen at the eighth position of naphyridine. Modifications of the parent drug improved the induction of oxidative stress, cell cycle arrest and the dysregulation of microRNA. The inhibition of V-ATPase–microfilament binding was also observed. Enoxacin strongly affected various cancer but not normal cells, excluding keratinocytes, which suffered from phototoxicity. It seems to be an underestimated anticancer drug with pleiotropic action. Furthermore, its usage as a safe antibiotic with well-known pharmacokinetics and selectivity will enhance the development of anticancer treatment strategies. This review covers articles published within the years 2000–2021, with a strong focus on the recent years (2016–2021). However, some canonical papers published in twentieth century are also mentioned.

Lost in Translation: Exploring microRNA Biogenesis and Messenger RNA Fate in Anoxia-Tolerant Turtles

Red-eared slider turtles face natural changes in oxygen availability throughout the year. This includes long-term anoxic brumation where they reduce their metabolic rate by ~90% for months at a time, which they survive without apparent tissue damage. This metabolic rate depression (MRD) is underlaid by various regulatory mechanisms, including messenger RNA (mRNA) silencing via microRNA (miRNA), leading to mRNA decay or translational inhibition in processing bodies (P-bodies) and stress granules. Regulation of miRNA biogenesis was assessed in red-eared slider turtle liver and skeletal muscle via immunoblotting. Hepatic miRNA biogenesis was downregulated in early processing steps, while later steps were upregulated. These contradictory findings indicate either overall decreased miRNA biogenesis, or increased biogenesis if sufficient pre-miRNA stores were produced in early anoxia. Conversely, muscle showed clear upregulation of multiple biogenesis steps indicating increased miRNA production. Additionally, immunoblotting indicated that P-bodies may be favoured by the liver for mRNA storage/decay during reoxygenation with a strong suppression of stress granule proteins in anoxia and reoxygenation. Muscle however showed downregulation of P-bodies during anoxia and reoxygenation, and upregulation of stress granules for mRNA storage during reoxygenation. This study advances our understanding of how these champion anaerobes regulate miRNA biogenesis to alter miRNA expression and mRNA fate during prolonged anoxia.

Abstract 2298: OTUD6B is a dependency in multiple myeloma that drives S-phase entry via MYC activation

Abstract Multiple myeloma (MM) is the second most common hematological malignancy and remains incurable, thus demanding for new therapeutic targets. While the pathophysiology of MM is poorly understood, the substantial responsiveness of MM patients to proteasomal inhibitors (PIs) like bortezomib or carfilzomib hints towards a central role of the ubiquitin proteasome system (UPS). Deubiquitylases (DUBs) are therapeutically targetable components of the UPS, whose inhibition can destabilize oncoproteins. However, the identities of oncoprotein-regulating DUBs remain largely elusive. To identify new vulnerabilities in MM, a CRISPR/Cas9 screen targeting all human DUBs was performed. For validated candidates, phenotypical analysis regarding proliferation and cell cycle progression was performed, as well affinity and non-affinity mass spectrometry-based screens to identify substrates. We thereby identified OTUD6B as a novel oncogene that drives G1/S-transition. LIN28B, a suppressor of microRNA biogenesis, was delineated as both a cell cycle-specific deubiquitylation substrate and activator of OTUD6B. RNA-Seq and qPCR analyses of OTUD6B and LIN28B depleted MM cells revealed that the stabilization of LIN28B drives MYC expression and activity at the G1/S transition, which in turn allows for rapid S-phase entry. Thus, silencing of OTUD6B as well as LIN28B inhibited MM outgrowth in xenograft experiments. Analyses of large MM patient cohorts revealed a progressive increase of OTUD6B expression along the transition from normal plasma cells to MGUS to MM and that high expression of OTUD6B was associated with a significantly adverse overall survival. Furthermore, OTUD6B expression was found to strongly correlate with MYC expression and significantly reduced progression-free survival in patients treated with the PI bortezomib. Knockout of OTUD6B in MM cells significantly enhance the anti-myeloma activity of the drug when using sub-lethal doses. Together, these results validate OTUD6B as a new therapeutically targetable oncogene, dependency, and prognostic factor in MM, that eventually serves as a master regulator of MYC activity to drive cell cycle progression. Citation Format: Ria Spallek, Carmen Paulmann, Oleksandra Karpiuk, Jana Zecha, Susan Klaeger, Isabell Schaeffer, Rupert Öllinger, Thomas Engleitner, Jan Krönke, Matthias Wirth, Ullrich Keller, Roland Rad, Bernahrd Kuster, Florian Bassermann. OTUD6B is a dependency in multiple myeloma that drives S-phase entry via MYC activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2298.

Abstract 2504: Next generation sequencing identifies novel potential biomarkers for DICER1 syndrome

Abstract MicroRNAs (miRNAs) are small RNA molecules that play a critical role in regulating gene expression. The DICER1 enzyme is a key component of the microRNA biogenesis pathway. Reflecting this central role, patients who inherit or acquire mutations in the DICER1 gene have an increased risk of developing a spectrum of benign and malignant tumors —thereby earning the recognition as a pediatric cancer predisposition syndrome known as DICER1 syndrome (OMIM 601200). DICER1 syndrome is definitively diagnosed by DNA sequencing demonstrating presence of a loss of function mutation on one copy of DICER1 plus a “hotspot” mutation on the other copy of DICER1. As tumor and germline DNA sequencing can pose logistical and/or cost difficulties, a complementary diagnostic method based on marker expression is valuable for both diagnostics and surveillance. Biomarkers that are candidate therapeutic targets are also appealing as these may offer a more tailored approach for therapy. As there are no approved biomarkers for identification of DICER1-associated tumors, we performed RNAseq and miRNAseq in an engineered murine mesenchymal cell model that incorporates a specific DICER1 hotspot mutation to identify candidates. We validated candidate biomarkers at the RNA (Eya2, Vipr1, miR-139-3p, and miR-30d-3p) and protein (Eya2, and Vipr1) levels in vitro, to provide proof of principle data for their eventual clinical utility. The miRNA candidate biomarkers levels were evaluated in both total and small extracellular vesicle-enriched fractions. These novel data pave the way for additional tools for diagnosis and surveillance in patients with DICER1 syndrome. Citation Format: Mona K. Wu, Raphael D. Lopez, Mikako Warren, Rachana Shah, Paolo Neviani, James F. Amatruda. Next generation sequencing identifies novel potential biomarkers for DICER1 syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2504.

Discovery of a Novel Small-Molecule Inhibitor Disrupting TRBP-Dicer Interaction against Hepatocellular Carcinoma via the Modulation of microRNA Biogenesis.

MicroRNAs (miRNAs) are key players in human hepatocellular carcinoma (HCC) tumorigenesis. Therefore, small molecules targeting components of miRNA biogenesis may provide new therapeutic means for HCC treatment. By a high-throughput screening and structural simplification, we identified a small molecule, CIB-3b, which suppresses the growth and metastasis of HCC in vitro and in vivo by modulating expression profiles of miRNAome and proteome in HCC cells. Mechanistically, CIB-3b physically binds to transactivation response (TAR) RNA-binding protein 2 (TRBP) and disrupts the TRBP-Dicer interaction, thereby altering the activity of Dicer and mature miRNA production. Structure-activity relationship study via the synthesis of 45 CIB-3b derivatives showed that some compounds exhibited a similar inhibitory effect on miRNA biogenesis to CIB-3b. These results support TRBP as a potential therapeutic target in HCC and warrant further development of CIB-3b along with its analogues as a novel therapeutic strategy for the treatment of HCC.

Ascitic fluid shear stress in concert with hepatocyte growth factor drive stemness and chemoresistance of ovarian cancer cells via the c-Met-PI3K/Akt-miR-199a-3p signaling pathway.

Overcoming drug resistance is an inevitable challenge to the success of cancer treatment. Recently, in ovarian cancer, a highly chemoresistant tumor, we demonstrated an important role of shear stress in stem-like phenotype and chemoresistance using a three-dimensional microfluidic device, which most closely mimics tumor behavior. Here, we examined a new mechanosensitive microRNA—miR-199a-3p. Unlike most key microRNA biogenesis in static conditions, we found that Dicer, Drosha, and Exportin 5 were not involved in regulating miR-199a-3p under ascitic fluid shear stress (0.02 dynes/cm2). We further showed that hepatocyte growth factor (HGF), but not other ascitic cytokines/growth factors such as epidermal growth factor and tumor necrosis factor α or hypoxia, could transcriptionally downregulate miR-199a-3p through its primary transcript miR-199a-1 and not miR-199a-2. Shear stress in the presence of HGF resulted in a concerted effect via a specific c-Met/PI3K/Akt signaling axis through a positive feedback loop, thereby driving cancer stemness and drug resistance. We also showed that miR-199a-3p expression was inversely correlated with enhanced drug resistance properties in chemoresistant ovarian cancer lines. Patients with low miR-199a-3p expression were more resistant to platinum with a significantly poor prognosis. miR-199a-3p mimic significantly suppressed ovarian tumor metastasis and its co-targeting in combination with cisplatin or paclitaxel further decreased the peritoneal dissemination of ovarian cancer in mice. These findings unravel how biophysical and biochemical cues regulate miR-199a-3p and is important in chemoresistance. miR-199a-3p mimics may serve as a novel targeted therapy for effective chemosensitization.