Mature miRNAs Research Articles

Behavioural phenotypes of Dicer knockout in the mouse SCN

AbstractThe suprachiasmatic nucleus (SCN) is the master clock that directly dictates behavioural rhythms to anticipate the earth's light/dark cycles. Although post‐transcriptional regulators called microRNAs have been implicated in physiological SCN function, how the absence of the entire mature miRNome impacts SCN output has not yet been explored. To study the behavioural consequences of miRNA depletion in the SCN, we first generated a mouse model in which Dicer is inactivated in the SCN by crossing Syt10Cre mice with Dicerflox mice to study behavioural consequences of miRNA depletion in the SCN. Loss of all mature miRNAs in the SCN shortened the circadian period length by ~37 minutes at the tissue level and by ~45 minutes at the locomotor activity level. Moreover, knockout animals exhibited a reduction in the precision of the circadian rhythm with more variable activity onsets under both LD 12:12 and DD conditions. We also observed that knockouts with higher onset variations were inclined to develop ultradian rhythms under constant light. In a second mouse model, recombination of Dicerflox via Cre delivery specifically in the SCN resulted in loss of behavioural rhythms in some animals depending on the injection efficiency. Together, our observations highlight the importance of microRNAs for a physiological SCN function and their pivotal role in robust circadian oscillations.

Abstract A039: A sensitive tool for the detection of RNA modifications in blood samples

Abstract The epitranscriptome is the set of chemical modifications applied naturally to RNA to regulate seemingly all aspects of its function, including splicing, translation initiation and fidelity, trafficking, RNA folding and stability, and transcript lifetime. Detecting RNA in blood reliably continues to be a challenge due to its labile nature, and detecting RNA modifications is an even greater challenge. For this reason, current liquid biopsy approaches concentrate on determining the mutational burden in cell-free DNA and do not harness the potential of cfRNA. In contrast to cfDNA, cfRNA is a biomarker for dynamic events that are known to occur at early stages of carcinogenesis, such as alterations to transcriptional profiles and other aspects of the regulatory state of cells. In many RNA species, the modification state is linked to the RNA lifecycle, thus providing important insights into cell state. For example, pre-miRNA is modified before miRNA maturation, tRNA cleavage is initiated by changes to the modification pattern and mRNA is decapped for storage in stress granules. AlidaBio is introducing the EpiPlex platform, an assay platform with an integrated bioinformatics solution that is designed for ultra-low RNA input and has been tested with blood samples. In addition to its sensitivity, the EpiPlex platform has the advantage of detecting multiple RNA modifications in the same reaction, enabling users to elucidate how RNA modifications act in concert to fine-tune RNA function. The approach uses targeted, multiplexed barcoding to reveal co-localization of modifications on the same molecule and to provide semi-quantitative data on mod abundance. This sensitive, multiplexed target detection has the potential to advance the field of RNA liquid biopsy with new biomarker discovery. Citation Format: Andrew Price, Zachary Miles, Byron Purse, Gudrun Stengel. A sensitive tool for the detection of RNA modifications in blood samples [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A039.

MicroRNAome profiling of breast cancer unveils hsa-miR-5683 as a tumor suppressor microRNA predicting favorable clinical outcome.

Breast cancer is a heterogeneous disease with diverse molecular subtypes, underscoring a better understanding of its molecular features and underlying regulatory mechanisms. Therefore, identifying novel prognostic biomarkers and therapeutic targets is crucial for advancing the current standard of care for breast cancer patients. Ninety-six formalin-fixed paraffin-embedded (FFPE) breast cancer samples underwent miRNAome profiling using QIAseq microRNA library kit and sequencing on Illumina platform. Mature miRNA quantification was conducted using CLC Genomics Workbench v21.0.5, while Relapse-free survival (RFS) analysis was conducted using RStudio 2023.09.1. Gain-of-function studies were conducted using miRNA mimics, while the effects of miRNA exogenous expression on cancer hallmark were assessed using 2-dimentional (2D) proliferation assay, three-dimensional (3D) organotypic culture, and live-dead staining. TargetScan database and Ingenuity Pathway Analysis (IPA) were used for miRNA target identification. Hierarchical clustering based on miRNA expression revealed distinct patterns in relation to PAM50 classification and identified miRNAs panels associated with luminal, HER2, and basal subtypes. hsa-miR-5683 emerged as a potential prognostic biomarker, showing a favorable correlation with RFS and suppressing tumorigenicity under 2D and 3D conditions in triple-negative breast cancer (TNBC) models. Findings were further extended to the MCF7 hormone receptor positive (HR+) model. Transcriptomic profiling of hsa-miR-5683 overexpressing TNBC cells revealed its potential role in key oncogenic pathways. Integration of downregulated genes and CRISPR-Cas9 perturbational effects identified ACLY, RACGAP1, AK4, MRPL51, CYB5B, MKRN1, TMEM230, NUP54, ANAPC13, PGAM1, and SOD1 as bona fide gene targets for hsa-miR-5683. Our data provides comprehensive miRNA expression atlas in breast cancer subtypes and underscores the prognostic and therapeutic significance of numerous miRNAs, including hsa-miR-5683 in TNBC. The identified gene targets unravel the intricate regulatory network in TNBC progression, suggesting promising avenues for further research and targeted therapeutic interventions.

EPCO-09. DICER1-MUTANT PRIMARY INTRACRANIAL SARCOMA: ASSESSING THE ONCOGENIC ROLE OF MIRNA DYSREGULATION

Abstract Cross-cancer profiling has shown that mutations in human DICER1 are recurrent in diverse cancers including intracranial sarcoma. Due to the rarity of these tumors, treatment options for these patients remain poorly defined. DICER1 encodes an endoribonuclease that processes the hairpin precursor microRNAs (miRNAs) into functionally mature miRNAs. In particular, hotspot mutations in the RNase IIIb domain of DICER1 are predicted to confer an oncogenic role although the precise mechanism is unknown. In a case of a 28-year-old male who presented with multicentric right frontal dural and parenchymal enhancing masses and underwent partial resection, pathology showed a DICER1-mutant primary intracranial sarcoma with a hotspot E1813G mutation in the RNase IIIb domain. Additionally, due to a family history of an unknown fatal brain tumor in his mother, the patient had additional molecular testing which revealed a germline BRCA2 mutation. After surgery, the patient received intensity-modulated radiation therapy followed by 6 cycles of ICE (ifosfamide, carboplatin, etoposide) chemotherapy with significant radiographic response and clinical improvement. He remains progression-free on radiological surveillance for 13 months since diagnosis. The treatment was well tolerated with clinically manageable myelosuppression (including grade 3 thrombocytopenia). This is the first reported case of an adult DICER1-mutant primary intracranial sarcoma patient with a germline BRCA2 mutation although the interaction between these oncogenic mechanisms is unclear. Based on the role of DICER1 in miRNA processing and to test the hypothesize that DICER1 mutations drive primary intracranial sarcoma through dysregulation of miRNA function, we have established a multi-institutional collaboration that is collecting and profiling DICER 1 mutant primary intracranial sarcoma cases from across the United States preliminary results of which will be presented. Identification of a miRNA-driven oncogenic mechanism in these tumors may yield novel targeted approaches beyond intensive chemotherapy for these patients.

The Role of Dicer Phosphorylation in Gemcitabine Resistance of Pancreatic Cancer.

Dicer, a cytoplasmic type III RNase, is essential for the maturation of microRNAs (miRNAs) and is implicated in cancer progression and chemoresistance. Our previous research demonstrated that phosphorylation of Dicer at S1016 alters miRNA maturation and glutamine metabolism, contributing to gemcitabine (GEM) resistance in pancreatic ductal adenocarcinoma (PDAC). In this study, we focused on the role of Dicer phosphorylation at S1728/S1852 in GEM-resistant PDAC cells. Using shRNA to knock down Dicer in GEM-resistant PANC-1 (PANC-1 GR) cells, we examined cell viability through MTT and clonogenic assays. We also expressed phosphomimetic Dicer 2E (S1728E/S1852E) and phosphomutant Dicer 2A (S1728A/S1852A) to evaluate their effects on GEM resistance and metabolism. Our results show that phosphorylation at S1728/S1852 promotes GEM resistance by reprogramming glutamine metabolism. Specifically, phosphomimetic Dicer 2E increased intracellular glutamine, driving pyrimidine synthesis and raising dCTP levels, which compete with gemcitabine's metabolites. This metabolic shift enhanced drug resistance. In contrast, phosphomutant Dicer 2A reduced GEM resistance. These findings highlight the importance of Dicer phosphorylation in regulating metabolism and drug sensitivity, offering insights into potential therapeutic strategies for overcoming GEM resistance in pancreatic cancer.

Rapid downregulation of DICER is a hallmark of adipose tissue upon high-fat diet feeding

Adipose tissue regulates whole-body energy balance and is crucial for metabolic health. With energy surplus, adipose tissue expands, which may lead to local areas of hypoxia and inflammation, and consequently impair whole-body insulin sensitivity. We report that DICER, a key enzyme for miRNA maturation, is significantly lower in abdominal subcutaneous white adipose tissue of men with obesity compared with men with a lean phenotype. Furthermore, DICER is profoundly downregulated in mouse adipose tissue and liver within the first week on a high-fat diet (HFD), and remains low after prolonged HFD feeding. Downregulation of DICER in mice occurs in both mature adipocytes and stromal vascular cells. Mechanistically, chemically induced hypoxia in vitro shows DICER degradation via interaction with hypoxia-inducible factor 1-α (HIF1α). Moreover, DICER and HIF1α interact in brown adipose tissue post-HFD which may signal for DICER degradation. Finally, RNA sequencing reveals a striking time-dependent downregulation of total miRNA content in mouse subcutaneous adipose tissue after HFD feeding. Collectively, HFD in mice reduces adipose tissue DICER, likely due to hypoxia-induced interaction with HIF1α during tissue expansion, and significantly impacts miRNA content.

Integrated Analysis of microRNAs and Transcription Factor Targets in Floral Transition of Pleioblastus pygmaeus.

Bamboo plants have erratic flowering habits with a long vegetative growth and an uncertain flowering cycle. The process of floral transition has always been one of the hot and intriguing topics in bamboo developmental biology. As master modulators of gene expression at the post-transcriptional level, miRNAs play a crucial role in regulating reproductive growth, especially in floral transition of flowering plants. Pleioblastus pygmaeus is a kind of excellent ground cover ornamental bamboo species. In this study, we performed miRNA expression profiling of the shoot buds and flower buds from the bamboo species, to investigate flowering-related miRNAs in bamboo plants. A total of 179 mature miRNAs were identified from P. pygmaeus, including 120 known miRNAs and 59 novel miRNAs, of which 96 (61 known miRNAs and 35 novel miRNAs) were differentially expressed in the shoots at different growth stages. Based on target gene (TG) prediction, a total of 2099 transcription factors (TFs) were annotated to be TGs of the 96 differentially expressed miRNAs (DEMs), corresponding to 839 recordings of DEM-TF pairs. In addition, we identified 23 known DEMs involved in flowering and six known miRNAs related to floral organ development based on previous reports. Among these, there were 11 significantly differentially expressed miRNAs, with 124 TF targets corresponding to 132 DEM-TF pairs in P. pygmaeus. In particular, we focused on the identification of miR156a-SPL (SQUAMOSA Promoter-Binding protein-Like) modules in the age pathway, which are well-known to regulate the vegetative-to-reproductive phase transition in flowering plants. A total of 36 TF targets of miR156a were identified, among which there were 11 SPLs. The Dual-Luciferase transient expression assay indicated miR156a mediated the repression of the PpSPL targets in P. pygmaeus. The integrated analysis of miRNAs and TGs at genome scale in this study provides insight into the essential roles of individual miRNAs in modulating flowering transition through regulating TF targets in bamboo plants.

ΜicroRNA (miRNA) Variants in Male Infertility: Insights from Whole-Genome Sequencing

Background/Objectives: Male infertility is a complex condition with various underlying genetic factors. microRNAs (miRNAs) play a crucial role in gene regulation, and their disruption can significantly impact fertility. This study aimed to identify variants within miRNA genes and elucidate their impact on male infertility. Methods: Whole genome sequencing was performed on blood samples from men with asthenozoospermia, oligozoospermia, and teratozoospermia, compared to normozoospermic controls. The analysis revealed a significant number of unique variants in each infertile group. We subsequently focused on variants in miRNA regions, followed by an in silico analysis to investigate the role of the identified variants and miRNAs in male infertility. Results: Focused analysis on miRNA genes identified 19 exclusive variants in teratozoospermic men, 24 in asthenozoospermic, and 27 in oligozoospermic, all mapping to pre-miRNAs or mature miRNAs. Functional analyses using Gene Ontology (GO) and KEGG pathways highlighted key biological processes and pathways disrupted by these variants and miRNA–mRNA interactions, including transcription regulation, signaling, and cancer-related pathways. Furthermore, six variants (rs17797090, rs1844035, rs7210937, rs451887, rs12233076, and rs6787734) were common across the infertile groups, suggesting their importance in male infertility or their potential as biomarkers. Common variants were also validated in another clinically relevant group of men. Some miRNAs with identified variants, such as hsa-miR-449b and hsa-miR-296, have been previously implicated in male infertility and exhibit differential expression between fertile and infertile men, according to the literature, too. Conclusion: These results provide new insights into the genetic basis of male infertility and open avenues for future research and therapeutic interventions.

Biogenesis and Regulation of the Freeze–Thaw Responsive microRNA Fingerprint in Hepatic Tissue of Rana sylvatica

Background: Freeze-tolerant animals undergo significant physiological and biochemical changes to overcome challenges associated with prolonged whole-body freezing. In wood frog Rana sylvatica (now Lithobates sylvaticus), up to 65% of total body water freezes in extracellular ice masses and, during this state of suspended animation, it is completely immobile and displays no detectable brain, heart, or respirometry activity. To survive such extensive freezing, frogs integrate various regulatory mechanisms to ensure quick and smooth transitions into or out of this hypometabolic state. One such rapid and reversible regulatory molecule capable of coordinating many aspects of biological functions is microRNA. Herein, we present a large-scale analysis of the biogenesis and regulation of microRNAs in wood frog liver over the course of a freeze–thaw cycle (control, 24 h frozen, and 8 h thawed). Methods/Results: Immunoblotting of key microRNA biogenesis factors showed an upregulation and enhancement of microRNA processing capacity during freezing and thawing. This was followed with RT-qPCR analysis of 109 microRNA species, of which 20 were significantly differentially expressed during freezing and thawing, with the majority being upregulated. Downstream bioinformatics analysis of miRNA/mRNA targeting coupled with in silico protein–protein interactions and functional clustering of biological processes suggested that these microRNAs were suppressing pro-growth functions, including DNA replication, mRNA processing and splicing, protein translation and turnover, and carbohydrate metabolism. Conclusions: Our findings suggest that this enhanced miRNA maturation capacity might be one key factor in the vital hepatic miRNA-mediated suppression of energy-expensive processes needed for long-term survival in a frozen state.

The Role of Exogenous microRNAs on Human Health: The Plant-Human Trans-Kingdom Hypothesis.

MicroRNAs (miRNAs) are small, endogenous, single-stranded RNAs that act on gene silencing at the post-transcriptional level by binding to a target messenger RNA (mRNA), leading to its degradation or inhibiting translation into functional proteins. The key role of miRNAs in development, proliferation, differentiation andapoptosis has been deeply investigated, revealing that deregulation in their expression is critical in various diseases, such as metabolic disorders and cancer. Since these small molecules initially evolved as a mechanism of protection against viruses and transposable elements, the fascinating hypothesis that they can move between organisms both of the same or different species has been postulated. Trans-kingdom is the term used to define the migration that occurs between species. This mechanism has been well analyzed between plants and their pests, in order to boost defense and increase pathogenicity, respectively. Intriguingly, in the last decades, the plant-human trans-kingdom migration via food intake hypothesis arose. In particular, various studies highlighted the ability of exogenous miRNAs, abundant in the mainly consumed plant-derived food, to enter the human body affecting gene expression. Notably, plant miRNAs can resist the strict conditions of the gastrointestinal tract through a methylation step that occurs during miRNA maturation, conferring high stability to these small molecules. Recent studies observed the anti-tumoral, immune modulator and anti-inflammatory abilities of trans-kingdom interaction between plant and human. Here, we depict the existing knowledge and discuss the fascinating plant-human trans-kingdom interaction, highlighting first the eventual role of plant miRNAs from foods on our somatic gene identity card and then the potential impact of using plant miRNAs as novel therapeutic avenues.

Distribution Of Microrna Counts Across Human Chromosomes.

microRNAs (miRNAs) are a class of non-coding RNAs that play important roles in gene regulation. miRNAs are transcribed from DNA sequences into primary miRNAs and then processed into precursor miRNAs and mature miRNAs. miRNA gene counts in chromosomes for different species have been studied. Certain chromosomes have higher numbers of miRNA genes in all species, such as the X chromosome, while the Y chromosome has the fewest or no miRNA genes. miRNA counts in different chromosomes might have a positive correlation with coding gene counts in many species. In this study, a regression model was used to find the relationship between the miRNA count and the coding gene count across human chromosomes, and miRNA counts for 23 human chromosomes were predicted based on this regression model. In addition, the chromosome locations for the miRNA biomarkers of major depression, diabetes, Parkinson's disease, and COVID-19 are discussed. The results reveal that miRNA biomarkers of these diseases are located in various chromosomes. The dispersion of miRNA locations across different chromosomes might explain the complication of the pathology of these diseases. Moreover, diabetes and COVID-19 have the largest number of miRNA biomarkers from Chromosome X. As Chromosome X is a sex chromosome, this phenomenon may explain the gender difference in the prevalence or severity of diabetes and COVID-19. The significant gender difference in the prevalence or severity of diabetes and COVID-19 might be due to the regulation function of their miRNA biomarkers from Chromosome X.

Overexpression of lily MicroRNA156-resistant SPL13A stimulates stem elongation and flowering in Lilium formosanum under non-inductive (non-chilling) conditions.

Flowering plants undergo juvenile vegetative, adult vegetative, and reproductive phases. Lily plants (Lilium spp.) develop scaly leaves during their juvenile vegetative phase. Stem elongation occurs in the adult vegetative phase and is followed by floral transition. As the duration of the juvenile vegetative phase is long in lilies, the microRNA156 (miR156) and SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) modules are expected to play a major role in vegetative phase change and flower induction. In the present study, we aimed to explore the functions of lily SLP13A. We evaluated phenotypic changes and gene expression in L. formosanum plants overexpressing miR156-resistant SPL13A (rSPL13A) and examined the accumulation levels of gene transcripts and mature miRNAs in non-transformed L. longiflorum plants. Lily plants overexpressing rSPL13A exhibited stem elongation under non-inductive conditions, and FLOWERING LOCUS T (FT) genes were poorly involved in this stem elongation. Flowering was induced in the transformed plants with elongated stems, and the accumulation of MADS5 (APETALA1) transcripts and mature miR172 was elevated in these plants. In non-transformed lilies, SPL13A transcripts were highly accumulated in the shoot apices of both juvenile and adult plants. As mature miR156 was poorly accumulated in the shoot apices of the adult plants, SPL13A was active enough to stimulate stem elongation and flower induction. In contrast, mature miR156 was reliably detected in shoot apices of the juvenile plants. Because our transient assay using tobacco plants expressing a SPL13A-GFP fusion protein indicated that miR156 repressed SPL13A expression mainly at the translational level, SPL13A activity should be insufficient to stimulate stem elongation in the juvenile plants. In addition, the accumulation of MADS5 transcripts and mature miR172 in the shoot apices increased with plant growth and peaked before the transition to the reproductive phase. Therefore, we conclude that SPL13A regulates stem elongation in the adult vegetative phase, which differs from the mechanisms evaluated in Arabidopsis and rice, wherein stem elongation proceeds in a reproductive phase and FT genes are heavily involved in it, and that SPL13A induces flowering by the activation of genes related to the age pathway underlying floral transition, as APETALA1 and primary-MIR172 are mainly involved in this pathway.

Genome-Wide Transcriptional Response of Avocado to Fusarium sp. Infection.

The avocado crop is relevant for its economic importance and because of its unique evolutionary history. However, there is a lack of information regarding the molecular processes during the defense response against fungal pathogens. Therefore, using a genome-wide approach in this work, we investigated the transcriptional response of the Mexican horticultural race of avocado (Persea americana var. drymifolia), including miRNAs profile and their possible targets. For that, we established an avocado-Fusarium hydroponic pathosystem and studied the response for 21 days. To guarantee robustness in the analysis, first, we improved the avocado genome assembly available for this variety, resulting in 822.49 Mbp in length with 36,200 gene models. Then, using an RNA-seq approach, we identified 13,778 genes differentially expressed in response to the Fusarium infection. According to their expression profile across time, these genes can be clustered into six groups, each associated with specific biological processes. Regarding non-coding RNAs, 8 of the 57 mature miRNAs identified in the avocado genome are responsive to infection caused by Fusarium, and the analysis revealed a total of 569 target genes whose transcript could be post-transcriptionally regulated. This study represents the first research in avocados to comprehensively explore the role of miRNAs in orchestrating defense responses against Fusarium spp. Also, this work provides valuable data about the genes involved in the intricate response of the avocado during fungal infection.

An efficient CRISPR-Cas12a-mediated MicroRNA knockout strategy in plants.

In recent years, the CRISPR-Cas9 nuclease has been used to knock out MicroRNA (miRNA) genes in plants, greatly promoting the study of miRNA function. However, due to its propensity for generating small insertions and deletions, Cas9 is not well-suited for achieving a complete knockout of miRNA genes. By contrast, CRISPR-Cas12a nuclease generates larger deletions, which could significantly disrupt the secondary structure of pre-miRNA and prevent the production of mature miRNAs. Through the case study of OsMIR390 in rice, we confirmed that Cas12a is a more efficient tool than Cas9 in generating knockout mutants of a miRNA gene. To further demonstrate CRISPR-Cas12a-mediated knockout of miRNA genes in rice, we targeted nine OsMIRNA genes that have different spaciotemporal expression and have not been previously investigated via genetic knockout approaches. With CRISPR-Cas12a, up to 100% genome editing efficiency was observed at these miRNA loci. The resulting larger deletions suggest Cas12a robustly generated null alleles of miRNA genes. Transcriptome profiling of the miRNA mutants, as well as phenotypic analysis of the rice grains revealed the function of these miRNAs in controlling gene expression and regulating grain quality and seed development. This study established CRISPR-Cas12a as an efficient tool for genetic knockout of miRNA genes in plants.

New anti-ovarian cancer quinolone derivatives acting by modulating microRNA processing machinery.

MicroRNAs (miRNAs) play a crucial role in ovarian cancer (OC) pathogenesis and miRNA processing can be the object of pharmacological intervention. By exploiting our in-house quinolone library, we combined a cell-based screening with medicinal chemistry efforts, ultimately leading to derivative 33 with anti-OC activity against distinct cell lines (GI50 values 13.52-31.04 μM) and CC50 Wi-38 = 142.9 μM. Compound 33 retained anticancer activity against additional cancer cells and demonstrated a synergistic effect with cisplatin against cisplatin-resistant A2780 cells. Compound 33 bound TRBP by SPR (K D = 4.09 μM) and thermal shift assays and its activity was TRBP-dependent, leading to modulation of siRNA and miRNA maturation. Derivative 33 exhibited augmented potency against OC cells and a stronger binding affinity for TRBP compared to enoxacin, the sole quinolone identified as a modulator of miRNA maturation. Consequently, 33 represents a promising template for developing novel anti-OC agents with a distinctive mechanism of action.

Programmable editing of primary MicroRNA switches stem cell differentiation and improves tissue regeneration

Programmable RNA editing is harnessed for modifying mRNA. Besides mRNA, miRNA also regulates numerous biological activities, but current RNA editors have yet to be exploited for miRNA manipulation. To engineer primary miRNA (pri-miRNA), the miRNA precursor, we present a customizable editor REPRESS (RNA Editing of Pri-miRNA for Efficient Suppression of miRNA) and characterize critical parameters. The optimized REPRESS is distinct from other mRNA editing tools in design rationale, hence enabling editing of pri-miRNAs that are not editable by other RNA editing systems. We edit various pri-miRNAs in different cells including adipose-derived stem cells (ASCs), hence attenuating mature miRNA levels without disturbing host gene expression. We further develop an improved REPRESS (iREPRESS) that enhances and prolongs pri-miR-21 editing for at least 10 days, with minimal perturbation of transcriptome and miRNAome. iREPRESS reprograms ASCs differentiation, promotes in vitro cartilage formation and augments calvarial bone regeneration in rats, thus implicating its potentials for engineering miRNA and applications such as stem cell reprogramming and tissue regeneration.

MicroRNA and Rare Human Diseases

Background: The role of microRNAs (miRNAs) in the pathogenesis of rare genetic disorders has been gradually discovered. MiRNAs, a class of small non-coding RNAs, regulate gene expression by silencing target messenger RNAs (mRNAs). Their biogenesis involves transcription into primary miRNA (pri-miRNA), processing by the DROSHA–DGCR8 (DiGeorge syndrome critical region 8) complex, exportation to the cytoplasm, and further processing by DICER to generate mature miRNAs. These mature miRNAs are incorporated into the RNA-induced silencing complex (RISC), where they modulate gene expression. Methods/Results: The dysregulation of miRNAs is implicated in various Mendelian disorders and familial diseases, including DICER1 syndrome, neurodevelopmental disorders (NDDs), and conditions linked to mutations in miRNA-binding sites. We summarized a few mechanisms how miRNA processing and regulation abnormalities lead to rare genetic disorders. Examples of such genetic diseases include hearing loss associated with MIR96 mutations, eye disorders linked to MIR184 mutations, and skeletal dysplasia involving MIR140 mutations. Conclusions: Understanding these molecular mechanisms is crucial, as miRNA dysregulation is a key factor in the pathogenesis of these conditions, offering significant potential for the diagnosis and potential therapeutic intervention.

MiR472 deficiency enhances Arabidopsis thaliana defense without reducing seed production.

After having co-existed in plant genomes for at least 200 million years, the products of microRNA (miRNA) and Nucleotide-Binding Leucine Rich Repeat protein (NLR) genes formed a regulatory relationship in the common ancestor of modern gymnosperms and angiosperms. From then on, DNA polymorphisms occurring at miRNA target sequences within NLR transcripts must have been compensated by mutations in the corresponding mature miRNA sequence. The potential evolutionary advantage of such regulation remains largely unknown and might be related to two non-exclusive scenarios: miRNA-dependent regulation of NLR levels might prevent defense mis-activation with negative effects on plant growth and reproduction; or reduction of active miRNA levels in response to pathogen derived molecules (PAMPS and silencing suppressors) might rapidly release otherwise silent NLR transcripts for rapid translation and thereby enhance defense. Here, we used Arabidopsis thaliana plants deficient for miR472 function to study the impact of releasing its NLR targets on plant growth and reproduction and on defense against the fungal pathogen Plectospharaella cucumerina. We show that miR472 regulation has a dual role, participating both in the tight regulation of plant defense and growth. MIM472 lines, with reduced active miR472, are more resistant to pathogens and, correlatively, have reduced relative growth compared to wild-type plants although the end of their reproductive phase is delayed, exhibiting higher adult biomass and similar seed yield as the wild-type. Our study highlights how negative consequences of defense activation might be compensated by changes in phenology and that miR472 reduction is an integral part of plant defense responses.

Development of miRNA-based PROTACs targeting Lin28 for breast cancer therapy.

Lin28, a highly conserved carcinogenic protein, plays an important role in the generation of cancer stem cells, contributing to the unfavorable prognosis of cancer patients. This RNA binding protein specifically binds to pri/pre-microRNA (miRNA) lethal-7 (let-7), impeding its miRNA maturation. The reduced expression of tumor suppressor miRNA let-7 fosters development and progression-related traits such as proliferation, invasion, metastasis, and drug resistance. We report a series of miRNA-based Lin28A-miRNA proteolysis-targeting chimeras (Lin28A-miRNA-PROTACs) designed to efficiently degrade Lin28A through a ubiquitin-proteasome-dependent mechanism, resulting in up-regulation of mature let-7 family. The augmented levels of matured let-7 miRNAs further exert inhibitory effects on cancer cell proliferation and migration, and increase its sensitivity to chemotherapy. In a mouse ectopic tumor model, Lin28A-miRNA-PROTAC demonstrates a substantial efficacy in inhibiting tumor growth. When combined with tamoxifen, the tumors exhibit gradual regression. This study displays an effective miRNA-based PROTACs to degrade Lin28A and inhibit tumor growth, providing a promising therapeutic avenue for cancer treatment with miRNA-based therapy.

HASTY-mediated miRNA dynamics modulate nitrogen starvation-induced leaf senescence in Arabidopsis

Nitrogen (N) deficiency responses are essential for plant survival and reproduction. Here, via an expression genome-wide association study (eGWAS), we reveal a mechanism that regulates microRNA (miRNA) dynamics necessary for N deficiency responses in Arabidopsis. Differential expression levels of three NAC transcription factor (TF) genes involved in leaf N deficiency responses among Arabidopsis accessions are most significantly associated with polymorphisms in HASTY (HST), which encodes an importin/exportin family protein responsible for the generation of mature miRNAs. HST acts as a negative regulator of N deficiency-induced leaf senescence, and the disruption and overexpression of HST differently modifies miRNA dynamics in response to N deficiency, altering levels of miRNAs targeting transcripts. Interestingly, N deficiency prevents the interaction of HST with HST-interacting proteins, DCL1 and RAN1, and some miRNAs. This suggests that HST-mediated regulation of miRNA dynamics collectively controls regulations mediated by multiple N deficiency response-associated NAC TFs, thereby being central to the N deficiency response network.