Small RNA Research Articles

Small nucleolar RNA host gene 5 plays a role in orthodontic tooth movement by inhibiting osteoclast differentiation.

The alveolar bone remodelling promoted by reasonable mechanical force triggers orthodontic tooth movement (OTM). The generation of osteoclasts is essential in this process. However, the mechanism of mechanical force mediating osteoclast differentiation remains elusive. Small nucleolar RNA host gene 5 (SNHG5), which was reported to mediate the osteogenic differentiation of bone marrow mesenchymal stem cells in our previous study, was downregulated in human periodontal ligament cells (hPDLCs) under mechanical force. At the same time, the RANKL/OPG ratio increased. Based on this, we probed into the role of SNHG5 in osteoclast formation during OTM and the relevant mechanism. SNHG5 and the RANKL/OPG ratio under different compressive forces were detected by western blotting (WB) and qRT-PCR. Impact of overexpression or knockdown of SNHG5 on osteoclast differentiation was detected by qRT-PCR, WB and transwell experiments. The combination of SNHG5 and C/EBPβ was verified by RNA immunoprecipitation and RNA pull-down assays. The expression of SNHG5 and osteoclast markers in gingiva were analysed by qRT-PCR and the paraffin sections of periodontal tissues were used for histological analysis. Compressive force downregulated SNHG5 and upregulated the RANKL/OPG ratio in hPDLCs. Overexpression of SNHG5 inhibited RANKL's expression and osteoclast differentiation. SNHG5 combined with C/EBPβ, a regulator of osteoclast. The expression of SNHG5 in periodontal tissue decreased during OTM. SNHG5 inhibited osteoclast differentiation during OTM, achieved by affecting RANKL secretion, which may provide a new idea to interfere with bone resorption during orthodontic treatment.

The evolution and characterization of the RNA interference pathways in Lophotrochozoa.

In animals, three main RNA interference mechanisms have been described so far, which respectively maturate three types of small noncoding RNAs (sncRNAs): miRNAs, piRNAs and endo-siRNAs. The diversification of these mechanisms is deeply linked with the evolution of the Argonaute gene superfamily since each type of sncRNA is typically loaded by a specific Argonaute homolog. Moreover, other protein families play pivotal roles in the maturation of sncRNAs, like the DICER ribonuclease family, whose DICER1 and DICER2 paralogs maturate respectively miRNAs and endo-siRNAs. Within Metazoa, the distribution of these families has been only studied in major groups, and there are very few data for clades like Lophotrochozoa. Thus, we here inferred the evolutionary history of the animal Argonaute and DICER families including 43 lophotrochozoan species. Phylogenetic analyses along with newly sequenced sncRNA libraries suggested that in all Trochozoa the proteins related to the endo-siRNA pathway have been lost, a part of them in some phyla (i.e., Nemertea, Bryozoa, Entoprocta), while all of them in all the others. On the contrary, early diverging phyla, Platyhelminthes and Syndermata, showed a complete endo-siRNA pathway. On the other hand, miRNAs were revealed the most conserved and ubiquitous mechanism of the metazoan RNA interference machinery, confirming their pivotal role in animal cell regulation.

Pathologic characteristics of histiocytic and dendritic cell neoplasms.

Histiocytic and dendritic cell neoplasms comprise diverse tumors originating from the mononuclear phagocytic system, which includes monocytes, macrophages, and dendritic cells. The 5th edition of the World Health Organization (WHO) classification updating the categorization of these tumors, reflecting a deeper understanding of their pathogenesis.In this updated classification system, tumors are categorized as Langerhans cell and other dendritic cell neoplasms, histiocyte/macrophage neoplasms, and plasmacytoid dendritic cell neoplasms. Follicular dendritic cell neoplasms are classified as mesenchymal dendritic cell neoplasms within the stroma-derived neoplasms of lymphoid tissues.Each subtype of histiocytic and dendritic cell neoplasms exhibits distinct morphological characteristics. They also show a characteristic immunophenotypic profile marked by various markers such as CD1a, CD207/langerin, S100, CD68, CD163, CD4, CD123, CD21, CD23, CD35, and ALK, and hematolymphoid markers such as CD45 and CD43. In situ hybridization for EBV-encoded small RNA (EBER) identifies a particular subtype. Immunoprofiling plays a critical role in determining the cell of origin and identifying the specific subtype of tumors. There are frequent genomic alterations in these neoplasms, especially in the mitogen-activated protein kinase pathway, including BRAF (notably BRAF V600E), MAP2K1, KRAS, and NRAS mutations, and ALK gene translocation.This review aims to offer a comprehensive and updated overview of histiocytic and dendritic cell neoplasms, focusing on their ontogeny, morphological aspects, immunophenotypic profiles, and molecular genetics. This comprehensive approach is essential for accurately differentiating and classifying neoplasms according to the updated WHO classification.

Possibilities for Differential Diagnosis of Thyroid Follicular Neoplasia by Evaluation of Small Noncoding RNA

Possibilities for Differential Diagnosis of Thyroid Follicular Neoplasia by Evaluation of Small Noncoding RNA

Unveiling microRNA-like small RNAs implicated in the initial infection of Fusarium oxysporum f. sp. cubense through small RNA sequencing

ABSTRACT Banana Fusarium wilt (BFW), caused by Fusarium oxysporum f. sp. cubense (Foc), poses a major challenge to the worldwide banana industry. Fungal microRNA-like small RNAs (milRNAs) play crucial roles in regulating fungal growth, conidiation, development, and pathogenicity. However, the milRNAs and their functions in the pathogenesis of Foc remain poorly understood. In this study, we employed high-throughput sequencing and bioinformatics to profile Foc sRNAs during both pure culture and early infection stages. Our analysis identified six milRNAs exhibiting significantly upregulated expression at the initial Foc infection. Of these, milR106’s biogenesis was found to be Dicer-dependent, whereas milR87, milR133, milR138, and milR148 were associated with Dicer and Argonaute proteins. Genetic manipulation and phenotype analysis confirmed that milR106 is crucial for Foc virulence by regulating conidiation, hydrogen peroxide sensitivity, and infective growth. Gene Ontology analysis of milRNA targets in the banana genome revealed enrichment in defence response to fungus and cellular response to hypoxia, implying the importance of these target genes in response to pathogen infection. In conclusion, our sRNA profiling of Foc identified several infection-induced milRNAs. The corresponding results provide valuable molecular targets for the development of an efficient strategy to control BFW.

MiR-3653-3p Expression in PBMCs: Unveiling the Diagnostic Potential for Ovarian Cancer.

Ovarian cancer is typically diagnosed at an advanced stage, recurs early and often, and currently lacks effective treatment. Therefore, overall survival and progression-free survival are relatively short for this disease. Sensitive and specific biomarkers for early diagnosis and follow-up for effective treatment of the disease are currently lacking. MicroRNA (miRNA/miR) expression studies are widely used in cancer research. Disruption or malfunction of miRNAs, a class of noncoding small RNAs, has been implicated in cancer progression in several publications. Of note, the expression of a series of miRNAs is known to differ in ovarian cancer. In cancer research, it is crucial to analyze expression patterns in both cancer patients and healthy individuals to identify cancer-specific biological markers and to understand their role in cancer. In the present study, the expression levels of miR-3653-3p in the peripheral blood mononuclear cells (PBMCs) of 150 patients with high-risk ovarian cancer were determined, including those with a family history of cancer or an early-age diagnosis of ovarian cancer, as well as 100 healthy individuals. The results were then compared between the two groups. The expression level of miR-3653-3p in the PBMCs of patients with ovarian cancer was determined to be 9.49-fold higher than that in the healthy control group, and this result was statistically significant (P < 0.001). In addition, receiver-operating characteristic curve analysis of PBMC showed statistical significance of miR-3653-3p in discriminating ovarian cancer patients from healthy subjects (P < 0.001). These results suggest that miR-3653-3p detected in peripheral blood may be used as a non-invasive biomarker for ovarian cancer.

Synopsis of the species of OrtholineaShulman, 1962 (Cnidaria: Myxosporea: Ortholineidae)

A synopsis of Ortholinea Shulman, 1962 (Cnidaria: Myxosporea: Ortholineidae) is presented and identifies 26 nominal species presently allocated within this genus. Species morphological and morphometric features, tissue tropism, type-host, and type-locality are provided from original descriptions. Data from subsequent redescriptions and reports is also given. Accession numbers to sequences deposited in GenBank are indicated when available, and the myxospores were redrawn based on original descriptions. The information gathered shows that Ortholinea infect a wide taxonomic variety of freshwater and marine fish. Nonetheless, the broad host specificity reported for several species is not fully supported by morphological descriptions and requires molecular corroboration. The members of this genus are coelozoic and mainly parasitize the urinary system, with few species occurring in the gallbladder. Ortholinea visakhapatnamensis is the only exception, being histozoic in the visceral peritoneum. Molecular data of the small subunit ribosomal RNA gene (SSU rDNA) is available for about one third of Ortholinea species, with genetic interspecific variation ranging between 1.65% and 29.1%. Phylogenetic analyses reveal Ortholinea to be polyphyletic, with available SSU rDNA sequences clustering within the subclades of the highly heterogenous freshwater urinary clade of the oligochaete-infecting lineage. The life cycles of two Ortholinea species have been clarified based on molecular inferences and identify triactinomyxon actinospores as counterparts, and marine oligochaetes of the family Naididae as permissive hosts to this genus.

Highly sensitive detection of Epstein-Barr virus-infected cells by EBER flow FISH.

When Epstein-Barr virus (EBV) infection is suspected, identification of infected cells is important to understand the pathogenesis, determinine the treatment strategy, and predict the prognosis. We used the PrimeFlow™ RNA Assay Kit with a probe to detect EBV-encoded small RNAs (EBERs) and multiple surface markers, to identify EBV-infected cells by flow cytometry. We analyzed a total of 24 patients [11 with chronic active EBV disease (CAEBV), 3 with hydroa vacciniforme lymphoproliferative disorder, 2 with X-linked lymphoproliferative disease type 1 (XLP1), 2 with EBV-associated hemophagocytic lymphohistiocytosis, and 6 with posttransplant lymphoproliferative disorder (PTLD)]. We compared infected cells using conventional quantitative PCR methods and confirmed that infected cell types were identical in most patients. Patients with CAEBV had widespread infection in T and NK cells, but a small amount of B cells were also infected, and infection in patients with XLP1 and PTLD was not limited to B cells. EBV-associated diseases are believed to be complex pathologies caused by EBV infecting a variety of cells other than B cells. We also demonstrated that infected cells were positive for HLA-DR in patients with CAEBV. EBER flow FISH can identify EBV-infected cells with high sensitivity and is useful for elucidating the pathogenesis.

Abstract PO4-23-11: Resistance Mechanisms to CDK4/6 Inhibitors and/or Tamoxifen Using Comprehensive Thermostable Group II Intron Reverse Transcriptase Sequencing

Abstract Background Understanding the mechanisms of resistance to CDK4/6 inhibitors (CDK4/6i) and endocrine therapy (ET) is pivotal in exploring new therapeutic strategies for hormone receptor-positive (HR+), HER2-negative metastatic breast cancer. To decipher these resistance mechanisms, we analyzed the alterations in comprehensive RNA-seq (coding and non-coding RNAs) of HR+ HER2-negative BC cell lines via thermostable group II intron reverse transcriptase sequencing (TGIRT-seq). Methods We established Tamoxifen-resistant (TMR), Abemaciclib-resistant (ACR), Palbociclib-resistant (PCR), Tamoxifen/Abemaciclib double-resistant (TMR-ACR), and Tamoxifen/Palbociclib double-resistant (TMR-PCR) BC cell lines from MCF7 and T47D HR+/HER2- BC cell lines through stepwise dose-escalation continuous drug exposure. We performed a TGIRT-seq transcriptomic analysis using a protocol that allows the sequencing of both long and short non-coding and protein-coding RNAs in a single library. All libraries were sequenced using paired-end 150 bp on the Novaseq platform, resulting in an average of 50 million reads per library. For analysis, raw reads underwent adapter trimming, small RNA mapping, whole genome mapping, and the generation of annotated genes' read count. We used raw counts to detect differentially expressed genes (DEGs) with DESeq2 in R using the cut-off (log2 [fold change] &amp;gt; 1, FDR &amp;lt; 1e-3). We then used the selected DEGs for Gene Set Enrichment Analysis (GSEA) and Kaplan-Meier survival analysis from the TCGA database. Results The TGIRT-seq analysis identified 1171 to 3472 DEGs in different drug resistance and cell line combinations. Most DEGs in resistant cells were consistently downregulated compared to parent cells. Seventy-five percent of DEGs were protein-coding genes, with the rest being non-coding RNAs, such as small nucleolar RNAs, microRNAs, long non-coding RNAs, and transposable element RNAs, which conventional RNA-seq poorly detects. In principal component analysis (PCA) plots, replicates per drug resistance clustered clearly in both cell line backgrounds. PCA also indicated distinctive pathways for acquiring drug resistance in MCF7 and T47D cells. The estrogen receptor (ER) expression decreased, while HER2 increased in resistant cell lines. DEGs identified were enriched in ESR1-related pathways in all MCF7 and T47D cell lines resistant models. Several ER-regulating genes like IL-1R1 and RET were upregulated, while ADCY1 was consistently downregulated across different resistance types. A significant overlap with single-resistance DEGs was observed among the DEGs in double-resistance cell lines, but 20-50% of up/down-regulated DEGs in double-resistance cell lines were uniquely altered. Several previously identified DEGs (e.g., SALL4, TOP2A) and 21 novel candidate genes correlated with poor survival outcomes. Conclusion The analysis identified unique DEGs in double resistance cell lines, suggesting that double resistance mechanisms may not merely be a cumulative effect of single resistance mechanisms, necessitating further investigation and validation. CDK4/6i and/or ET-resistant BC cell lines displayed significant transcriptome reprogramming during the development of drug resistance. In previously published research, ESR1-related pathway alterations were proposed in tamoxifen resistance cell lines. Here we find that CDK4/6i-resistant cells also modify these ESR1-related pathways. Additionally, we identified several targetable genes, such as IL-1R1 and RET, involved in ESR1-related pathways that could pave the way for developing new treatment strategies. Citation Format: Toshiaki Iwase, Hengyi Xu, Nakyung Oh, Jangsoon Lee, Alan M Lambowitz, Naoto Ueno. Resistance Mechanisms to CDK4/6 Inhibitors and/or Tamoxifen Using Comprehensive Thermostable Group II Intron Reverse Transcriptase Sequencing [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-23-11.

Abstract PO2-13-08: Cell-free orphan noncoding RNAs and AI enable early detection of invasive breast cancer and ductal carcinoma in-situ

Abstract Background Earlier detection of breast cancer through mammography screening has reduced disease-specific mortality; however, confounding issues such as technical challenges, breast density, and tumor size can result in false negatives and ultimately later stage diagnosis. Next generation liquid biopsy has the potential to complement mammography and enable earlier detection for more women. We have previously demonstrated high sensitivity and specificity for early detection of invasive breast cancer (IBC) by utilizing a novel category of cancer-associated small RNAs, termed orphan noncoding RNAs (oncRNAs), through a liquid biopsy platform. Here, we further improve the ability to detect breast cancer in a larger, multi-source cohort through an AI-driven approach and demonstrate potential for detection of ductal carcinoma in-situ (DCIS). Methods We utilized The Cancer Genome Atlas (TCGA) small RNA-seq database to discover a library of 20,538 oncRNAs, through a female-specific analysis, that were significantly enriched among 1,103 breast tumors compared to 349 normal tissue samples spanning multiple tissue sites. The diagnostic performance of these oncRNAs were assessed in an independent cohort of archived serum samples from 663 female individuals, sourced from Indivumed (Hamburg, Germany), Proteogenex (Inglewood, CA), and MT Group (Los Angeles, CA), including 279 breast cancer patients of various stages (221 IBC and 58 DCIS; mean age: 57.0 ± 13.8 years; ever-smoker: 25.8%) and 304 age-matched controls (mean age: 58.5 ± 13.9 years; ever-smoker: 23.4%) without breast cancer. All samples were collected between 2010–2022 at time of diagnosis for breast cancer patients. We sequenced the small RNA content of these samples at an average depth of 25.28 ± 9.37 million 50-bp single-end reads. We detected 18,025 (87.8%) unique breast cancer-specific oncRNA species within at least one sample from the study cohort. We then trained a generative AI model using 5-fold cross-validation to predict cancer status for all samples. Results Our oncRNA-based model achieved an overall AUC of 0.95 (95% CI, 0.93–0.97) for prediction of IBC versus cancer-free controls with a sensitivity of 0.87 (0.82–0.91) at 90% specificity. We observed high sensitivities, also at 90% specificity, across all tumor stages and tumor sizes (Table 1). Sensitivities for the earliest stage and smallest tumor size were 0.87 (0.78–0.93) and 0.81 (0.61–0.93) for Stage I (n=83) and T1a–b ( &amp;gt;1mm to ≤10mm; n=26), respectively. Additionally, in a small single-source cohort, we also saw high model accuracy and sensitivity for DCIS, which we aim to confirm in additional cohorts. While our overall cancer cohort primarily consisted of individuals with luminal breast cancer, our model had high sensitivities across all breast cancer subtypes at 0.90 (0.84–0.94), 0.73 (0.59–0.85), and 0.86 (0.42–1.0) for luminal (n=181), HER2 positive (n=49), and triple negative (n=7), respectively. Conclusions We further demonstrate the potential utility of oncRNAs as a blood-based biomarker using an AI algorithm for sensitive and accurate early detection of breast cancer in a large cohort. Additionally, we have shown that this oncRNA-based assay performs well in detecting small, early-stage invasive breast tumors, with potential to detect precursors of breast cancer. Table 1: Model sensitivity in breast cancer by tumor stage and size For each tumor stage and size, as defined by the AJCC 7th Edition breast cancer staging system, sensitivity and 95% Pearson-Clopper confidence intervals (CI) are reported at 90% specificity for the number of samples (N). Citation Format: Noura Tbeileh, Taylor Cavazos, Mehran Karimzadeh, Jeffrey Wang, Alice Huang, Dung Ngoc Lam, Seda Kilinc, Jieyang Wang, Xuan Zhao, Andy Pohl, Helen Li, Lisa Fish, Kimberly Chau, Marra Francis, Lee Schwartzberg, Patrick Arensdorf, Hani Goodarzi, Fereydoun Hormozdiari, Babak Alipanahi. Cell-free orphan noncoding RNAs and AI enable early detection of invasive breast cancer and ductal carcinoma in-situ [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-13-08.

Abstract PO3-26-12: Targeting Lipocalin-2 in HER2+ Inflammatory Breast Cancer Using Herceptin-Conjugated Liposomes

Abstract Inflammatory breast cancer (IBC) is a rare and aggressive subtype of BC that accounts for 1 to 5% of all types of BC. Due to its inflammatory characteristics and the absence of a palpable mass, IBC is usually confounded with a mastitis. Unfortunately, once properly diagnosed, IBC has already metastasized. A high portion of IBC tumors overexpress human epidermal growth factor receptor 2 (HER2). These IBC patients (HER2+) are treated with Trastuzumab, a monoclonal antibody. However, many IBCs become resistant to this therapy. Thus, there is not an optimal treatment against this horrendous disease. One potential target against IBC is Lipocalin-2 (LCN2), a secreted protein involved in iron homeostasis, immune responses, transport of siderophores, and epithelial cell differentiation. In many aggressive tumors, high levels of LCN2 have been associated with increased cancer cell motility, proliferation, angiogenesis, invasion, and metastasis. LCN2 is aberrantly abundant in IBCs compared with non-IBC patients and cell lines. This information indicates that LCN2 could be a target for IBC therapy. Our research team showed that small interference RNAs (siRNAs) targeting LCN2 effectively reduced the cell proliferation and invasion ability of IBC cells. To systemically deliver siRNA specifically to HER2+ IBC cells, we prepared Herceptin-conjugated liposome loaded with siRNA against LCN2. Trastuzumab conjugated liposomes werecharacterized by using dynamic light scattering to measure the size distribution and the zeta potential. Our formulation displayed sizes of 18.3 and 73.7 nm which could correspond to free DSPE-PEG-(2000) trastuzumab and the trastuzumab-liposome, respectively. To determine the optimal timepoint of liposome internalization into IBC cell lines, we incubated HER2+ IBC3 cells with liposomes for different time-points. Optimal internalization was observed at 24 and 48-hr. To further understand the downstream effectors of LCN2 in IBC cells, we performed RNAseq in siRNA-mediated LCN2 knockdown cells. Further bioinformatics with the RNAseq data, revealed 138 dysregulated genes following LCN2 knockdown as compared with a negative control (NC) siRNA. Particularly, increased STAT1 levels has been associated with to distant metastasis and poor clinical prognosis in cancer. In our study, STAT1 levels decreased after LCN2 silencing. Ingenuity Pathway Analysis (IPA) showed 25 canonical pathways altered in the LCN2 knockdown cells as compared with the NC-siRNA. These results indicate that LCN2 activate molecular pathways involved in cell proliferation, invasion and metastasis in IBC cells. Citation Format: Marienid Flores-Colón, Pablo Vivas-Rivera, Pablo E. Vivas-Mejía. Targeting Lipocalin-2 in HER2+ Inflammatory Breast Cancer Using Herceptin-Conjugated Liposomes [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-26-12.

Venlafaxine exposure alters mitochondrial respiration and mitomiR abundance in zebrafish brains.

Wastewater treatment plant (WWTP) effluent often releases pharmaceuticals like venlafaxine (a serotonin-norephinephrine reuptake inhibitor antidepressant) to freshwater ecosystems at levels causing adverse metabolic effects on fish. Changes to fish metabolism can be regulated by epigenetic mechanisms like microRNA (small RNA molecules that regulate mRNA translation), including regulating mitochondrial mRNAs. Nuclear-encoded microRNAs regulate mitochondrial gene expression in mammals, and have predicted effects in fish. We aimed to identify whether venlafaxine exposure changed mitochondrial respiration and resulted in differentially abundant mitochondrial microRNA (mitomiRs) in zebrafish brains. In vitro exposure of brain homogenate to below environmentally relevant concentrations of venlafaxine (<1 µg/L) caused a decrease in mitochondrial respiration, although this was not driven by changes to mitochondrial Complex I or II function. To identify whether these effects occur in vivo, zebrafish were exposed to 1 µg/L venlafaxine for 0, 1, 6, 12, 24, and 96 h. In vivo, venlafaxine exposure had no significant effects on brain mitochondrial respiration; however, select mitomiRs (dre-miR-301a-5p, dre-miR-301b-3p, and dre-miR-301c-3p) were also measured, because they were bioinformatically predicted to regulate mitochondrial cytochrome c oxidase subunit I (COI) abundance. These mitomiRs were differentially regulated based on venlafaxine exposure (with miR-301c-3p abundance differing during the day and miR-301b-3p being lower in exposed fish at night), and with respect to sex and time sampled. Overall, the results demonstrated that in vitro venlafaxine exposure to zebrafish brain caused a decrease in mitochondrial respiration, but these effects were not seen after acute in vivo exposure. Results may have differed because in vivo exposure allows for fish to mitigate effects through mechanisms that could include mitomiR regulation, and because fish were only acutely exposed. Environ Toxicol Chem 2024;00:1-14. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Abstract PO2-14-02: Clinical significance of urinary microRNA expression in primary breast cancer

Abstract Backgrounds: MicroRNAs (miRNAs) are small functional nucleic acids that regulate homeostasis by maintaining a balance of regulatory functions within cells. In humans, more than 2,000 miRNAs have been discovered, and each type of cancer has its own unique pattern of miRNA expression. Like circulating tumor DNA, miRNAs circulate in the blood in the form of exosomes, and some of them are excreted in the urine. Therefore, miRNAs in blood and urine are considered potential biomarkers in cancer. We examined miRNAs in urine, which can be collected without invasive procedures. Aims: This study aimed to investigate the clinical significance of urinary microRNA in primary breast cancer. Patients: Two hundred patients with stage 0-III primary breast cancer treated in our hospital (breast cancer group) and 105 healthy volunteers (control group) were included in the study. Methods: Small RNA seq was performed using a next-generation sequencer, and urinary miRNA profiles were obtained and analyzed. Student’s t-tests were conducted between breast cancer group and the control group, and miRNAs with p-values p-values &amp;lt; 0.05 and Log2FC&amp;gt;0.5 were differentially expressed-miRNAs(DEMs). In the present study, miEAA API version2.0 and miRDIP were used to predict the target genes of the functional DEMs. The functional enrichment of the target genes of the DEMs was assessed using KEGG signalling pathway analyses. A machine learning model for binary classification of breast cancer and control group was created and its performance was evaluated using cross-validation method. Results: The median age was 50.3 years old in the breast cancer group and 46.1 years old in the control group. Of the breast cancer group, 13.5% were at clinical stage 0. Approximately 80% of the patients had HR+HER2- tumors. Twenty-eight miRNAs were identified to differentiate between the breast cancer group and the control group. Of the 28 miRNAs, fifteen miRNAs were up-regulated and thirteen miRNAs were down-regulated in the breast cancer group and compared to the control group. Pathway analysis revealed that miRNAs upregulated in the breast cancer group were associated with the PI3K-Akt signaling pathway and the MAPK signaling pathway. No association was observed between expression patterns of the identified miRNAs and clinicopathological factors other than nodal status and lymphatic invasion, including tumor size and hormone receptor/HER2 expressions. MicroRNA-486-5p, miRNA-486-3p and miRNA-126-3p expressions were decreased in patients with lymph node metastasis or lymphatic invasion. A machine learning model using urinary miRNAs identified breast cancer with an accuracy of AUC=0.83. The model maintained accuracy to diagnose breast cancer at any stage including ductal carcinoma in situ (DCIS), suggesting its usefulness in screening for early-stage cancers, including DCIS. Discussion: Previous reports have shown that miRNA-486-5p and miRNA-486-3p in breast cancer tissues are downregulated in patients with lymph node metastasis, and miRNA-126-3p is downregulated in patients with positive sentinel lymph nodes, which were consistent with the present study urine. This is the same trend as in the present study, which was conducted in using urine miRNAs. Conclusion: We identified miRNAs in urine to differentiate primary breast cancer from healthy controls. The miRNA-486 may correlate with lymph node metastasis and lymphatic invasion. Citation Format: Yuka Inoue, Natsue Uehiro, Nami Yamashita, Hiroki Yamaguchi, Sakura Maezono, Mariko Palfalvi, Yoriko Ando, Yumi Nishiyama, Mika Mizunuma, Yuki Ichikawa, Yuki Matsunaga, Asumi Iesato, Yukinori Ozaki, Tetsuyo Maeda, Yoko Takahashi, Fumitaka Hara, Takayuki Kobayashi, Tomo Osako, Takehiko Sakai, Takayuki Ueno, Shinji Ohno. Clinical significance of urinary microRNA expression in primary breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-14-02.

Effects of arboviral infections on transposable element transcript levels in Aedes aegypti.

Transposable elements (TEs) are mobile repeated sequences found in all genomes. TEs are controlled by RNA interference pathways in most organisms, and this control involves the piRNA pathway and the siRNA pathway, which is also known to be the first line of antiviral defense in invertebrates. Using Drosophila, we recently showed that viral infections result in the modulation of TE transcript levels through modulation of the small RNA repertoire. The Aedes aegypti mosquito is of particular interest because almost half of its genome is made of TEs, and it is described as a major vector of viruses (such as the dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) arboviruses). Moreover, Aedes mosquitoes are unique among insects in that the piRNA pathway is also involved in the somatic antiviral response, in addition to the TE control and piRNA pathway genes expanded in the mosquito genome. For these reasons, we studied the impacts of viral infections on TE transcript levels in Ae. aegypti samples. We retrieved public datasets corresponding to RNA-seq data obtained from viral infections by DENV, ZIKV and CHIKV in various tissues. We found that TE transcripts are moderately modulated following viral infection and that the direction of the modulation varies greatly across tissues and viruses. These results highlight the need for an in-depth investigation of the tightly intertwined interactions between TEs and viruses.

Transcriptional landscape of small non-coding RNAs reveals diversity of categories and functions in molluscs

ABSTRACT Small non-coding RNAs (sncRNAs) are non-coding RNA molecules that play various roles in metazoans. Among the sncRNAs, microRNAs (miRNAs) guide post-translational gene regulation during cellular development, proliferation, apoptosis, and differentiation, while PIWI-interacting RNAs (piRNAs) suppress transposon activity to safeguard the genome from detrimental insertion mutagenesis. While an increasing number of piRNAs are being identified in the soma and germlines of various organisms, they are scarcely reported in molluscs. To unravel the small RNA (sRNA) expression patterns and genomic function in molluscs, we generated a comprehensive sRNA dataset by sRNA sequencing (sRNA-seq) of eight mollusc species. Abundant miRNAs were identified and characterized in all investigated molluscs, and ubiquitous piRNAs were discovered in both somatic and gonadal tissues in six of the investigated molluscs, which are more closely associated with transposon silencing. Tens of piRNA clusters were also identified based on the genomic mapping results, which varied among different tissues and species. Our dataset serves as important reference data for future genomic and genetic studies on sRNAs in these molluscs and related species, especially in elucidating the ancestral state of piRNAs in bilaterians.

Role of small nucleolar RNAs in alternative splicing of the doublesex gene in the silkworm, Bombyx mori.

More and more evidence shows that small noncoding RNAs (ncRNAs) play diverse roles in development, stress response and other cellular processes, but functional study of intermediate-size ncRNAs is still rare. Here, the expression profile of 16 intermediate-size ncRNAs in ovary and testis of silkworm Bombyx mori were analyzed. Twelve ncRNAs, including 5 small nucleolar RNAs (snoRNAs) and 7 unclassified ncRNAs, accumulated more in the testis than in the ovary of silkworm, especially Bm-163, Bm-51 and Bm-68. Four ncRNAs (including three orphan snoRNAs and one unclassified ncRNA) had higher expression level in the ovary than in the testis, especially Bm-86. Overexpression of the testis-enriched snoRNA Bm-68 in the female led to the accumulation of male-specific isoform of doublesex (BmdsxM) and increased the expression ratio of BmdsxM: BmdsxF. While overexpression of ovary-enriched snoRNA Bm-86 in the male decreased the expression ratio of BmdsxM: BmdsxF, indicating the roles of the two snoRNAs played in the alternative splicing of Bmdsx of silkworm, which will provide new clues for the functional study of snoRNAs in insects.

The transcriptional landscape of coding and non-coding RNAs in recurrent and non-recurrent colon cancer

A considerable number of colon cancer patients with local or local advanced disease suffer from recurrence and there is an urgent need for better prognostic biomarkers in this setting. Here, the transcriptomic landscape of messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), small Cajal body-specific RNAs (scaRNAs), pseudogenes and circular RNAs (circRNAs), as well as RNAs denoted as miscellaneous RNAs, was profiled by total RNA sequencing. In addition to well-known coding and non-coding RNAs, differential expression analysis also uncovered transcripts, which have not previously been implicated in colon cancer, such as RNA5SP149, RNU4-2 and SNORD3A. Moreover, there was a profound global upregulation of snRNA pseudogenes, snoRNAs and rRNA pseudogenes in more advanced tumours. A global downregulation of circRNAs in tumours relative to normal tissues was observed, while only few were differentially expressed between tumour stages. Many previously undescribed transcripts, including RNU6-620P, RNU2-20P, VTRNA1-3 and RNA5SP60 indicated strong prognostic biomarker potential in ROC analyses. In summary, this study unveiled numerous differentially expressed RNAs across various classes between recurrent and non-recurrent colon cancer. Notably, there was a significant global upregulation of snRNA pseudogenes, snoRNAs and rRNA pseudogenes in advanced tumours. Many of these newly discovered candidates demonstrated a strong prognostic potential for stage II colon cancer.

Transfer RNA‑derived small RNAs: A class of potential biomarkers in multiple cancers (Review)

Transfer RNA‑derived small RNAs: A class of potential biomarkers in multiple cancers (Review)

Co-localization and analysis of miR477b with fiber length quantitative trait loci in cotton.

Cotton is an important cash crop for the textile industry. However, the understanding of natural genetic variation of fiber elongation in relation to miRNA is lacking. A miRNA gene (miR477b) was found to co-localize with a previously mapped fiber length (FL) quantitative trait locus (QTL). The miR477b was differentially expressed during fiber elongation between two backcross inbred lines (BILs) differing in FL and its precursor sequences. Bioinformatics and qRT-PCR analysis were further used to analyse the miRNA genes, which could produce mature miR477b. Cotton plants with virus-induced gene silencing (VIGS) constructs to over-express the allele of miR477b from the BIL with longer fibers had significantly longer fibers as compared with negative control plants, while the VIGS plants with suppressed miRNA expression had significantly shorter fibers. The expression level of the target gene (DELLA) and related genes (RDL1 and EXPA1 for DELLA through HOX3 protein) in the two BILs and/or the VIGS plants were generally congruent, as expected. This report represents one of the first comprehensive studies to integrate QTL linkage mapping and physical mapping of small RNAs with both small and mRNA transcriptome analysis, followed by VIGS, to identify candidate small RNA genes affecting the natural variation of fiber elongation in cotton.

Smartphone-based device for rapid and single-step detection of piRNA–651 using anti-DNA:RNA hybrid antibody and enzymatic signal amplification

P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs/piRs) are a class of small noncoding RNAs that play a crucial role in regulating various biological processes, including carcinogenesis. One specific piRNA, piR–651, has been reported to be overexpressed in both human blood serum and solid cancer tissues, that can be used a viable biomarker in cancer diagnosis. Early diagnosis of cancer can help reduce the burden of the disease and improve survival rates. In the present work, we report for the first time a smartphone-based colorimetric biosensor for highly sensitive and specific detection of piR–651 thanks to an enzymatic signal amplification, which yielded high colorimetric intensities. Indeed, a heteroduplex DNA:RNA was formed in the presence of piR–651 with the capture DNA probe immobilized on the magnetic beads for easy magnetic separation. Then, a HRP tethered to anti-DNA:RNA (S9.6) was used to reveal the DNA-RNA heteroduplex formed by catalyzing the oxidation of TMB substrate into colorimetric TMBox, which absorbs at 630 nm. The absorbance is positively proportional to the piR–651 concentrations. On the other hand, the colorimetric product of the assay can be photographed with a smartphone camera and analyzed using ImageJ software. Using a smartphone and under optimal conditions, the biosensor responded linearly to the logarithm of piRNA–651 from 8 fM to 100 pM with a detection limit of 2.3 fM and discriminates against other piRNAs. It was also successfully applied to the determination of piRNA–651 levels in spiked human serum.