Small RNA Research Articles

TRNA-Derived Fragments as Biomarkers in Bladder Cancer.

Bladder cancer (BC) diagnosis is reliant on cystoscopy, an invasive procedure associated with urinary tract infections. This has sparked interest in identifying noninvasive biomarkers in body fluids such as blood and urine. A source of biomarkers in these biofluids are extracellular vesicles (EVs), nanosized vesicles that contain a wide array of molecular cargo, including small noncoding RNA such as transfer RNA-derived fragments (tRF) and microRNA. Here, we performed small-RNA next-generation sequencing from EVs from urine and serum, as well as from serum supernatant. RNA was extracted from 15 non-cancer patients (NCPs) with benign findings in cystoscopy and 41 patients with non-muscle invasive BC. Urine and serum were collected before transurethral resection of bladder tumors (TUR-b) and at routine post-surgery check-ups. We compared levels of tRFs in pre-surgery samples to samples from NCPs and post-surgery check-ups. To further verify our findings, samples from 10 patients with stage T1 disease were resequenced. When comparing tRF expression in urine EVs between T1 stage BC patients and NCPs, 14 differentially expressed tRFs (DEtRFs) were identified. In serum supernatant, six DEtRFs were identified among stage T1 patients when comparing pre-surgery to post-surgery samples and four DEtRFs were found when comparing pre-surgery samples to NCPs. By performing a blast search, we found that sequences of DEtRFs aligned with genomic sequences pertaining to processes relevant to cancer development, such as enhancers, regulatory elements and CpG islands. Our findings display a number of tRFs that may hold potential as biomarkers for the diagnosis and recurrence-free survival of BC.

Novel Blood Biomarkers for Response Prediction and Monitoring of Stereotactic Ablative Radiotherapy and Immunotherapy in Metastatic Oligoprogressive Lung Cancer.

Up to 80% of patients under immune checkpoint inhibitors (ICI) face resistance. In this context, stereotactic ablative radiotherapy (SABR) can induce an immune or abscopal response. However, its molecular determinants remain unknown. We present early results of a translational study assessing biomarkers of response to combined ICI and SABR (I-SABR) in liquid biopsy from oligoprogressive patients in a prospective observational multicenter study. Cohort A includes metastatic patients in oligoprogression to ICI maintaining the same ICI due to clinical benefit and who receive concomitant SABR. B is a comparative group of oligometastatic patients receiving only SABR. Blood samples are extracted at baseline (T1), after the first (T2) and last (T3) fraction, two months post-SABR (T4) and at further progression (TP). Response is evaluated by iRECIST and defined by the objective response rate (ORR)-complete and partial responses. We assess peripheral blood mononuclear cells (PBMCs), circulating cell-free DNA (cfDNA) and small RNA from extracellular vesicles. Twenty-seven patients could be analyzed (cohort A: n = 19; B: n = 8). Most were males with non-small cell lung cancer and one progressing lesion. With a median follow-up of 6 months, the last ORR was 63% (26% complete and 37% partial response). A decrease in cfDNA from T2 to T3 correlated with a good response. At T2, CD8+PD1+ and CD8+PDL1+ cells were increased in non-responders and responders, respectively. At T2, 27 microRNAs were differentially expressed. These are potential biomarkers of response to I-SABR in oligoprogressive disease.

MicroRNA (miRNA) PROFILING IN PROSTATE CANCER CARCINOGENESIS: EXPLORATORY RESEARCH

Future research will continue to be carried out in terms of looking for molecular markers involved in the early processes of carcinogenesis, metastasis and therapeutic targets in prostate cancer patients. MicroRNAs (miRNAs) are small noncoding RNAs that contribute to prostate cancer progression by controlling genes involved in the androgen receptor (AR) signaling pathway, ectopic expression of proteins involved in the cell cycle and apoptosis, epithelial-mesenchymal transition (EMT) and metastasis of cancer stem cells (CSCs). This study was an exploratory study, the aim was to assess miRNA expression in low histopathological grade prostate cancer compared with high histopathological grade prostate cancer. The novelty of this research is to assess the 25 most increased (up-regulated) and 25 lowest (down-regulated) miRNAs from 800 types of miRNA. Next, we will analyze the mechanisms and confirm the carcinogenesis of prostate cancer. The research results can be used for further research data, especially for targeted therapy in prostate cancer patients.

High glucose-induced p66Shc mitochondrial translocation regulates autophagy initiation and autophagosome formation in syncytiotrophoblast and extravillous trophoblast.

p66Shc, as a redox enzyme, regulates reactive oxygen species (ROS) production in mitochondria and autophagy. However, the mechanisms by which p66Shc affects autophagosome formation are not fully understood. p66Shc expression and its location in the trophoblast cells were detected in vivo and in vitro. Small hairpin RNAs or CRISPR/Cas9, RNA sequencing, and confocal laser scanning microscope were used to clarify p66Shc's role in regulating autophagic flux and STING activation. In addition, p66Shc affects mitochondrial-associated endoplasmic reticulum membranes (MAMs) formation were observed by transmission electron microscopy (TEM). Mitochondrial function was evaluated by detected cytoplastic mitochondrial DNA (mtDNA) and mitochondrial membrane potential (MMP). High glucose induces the expression and mitochondrial translocation of p66Shc, which promotes MAMs formation and stimulates PINK1-PRKN-mediated mitophagy. Moreover, mitochondrial localized p66Shc reduces MMP and triggers cytosolic mtDNA release, thus activates cGAS/STING signaling and ultimately leads to enhanced autophagy and cellular senescence. Specially, we found p66Shc is required for the interaction between STING and LC3II, as well as between STING and ATG5, thereby regulates cGAS/STING-mediated autophagy. We also identified hundreds of genes associated several biological processes including aging are co-regulated by p66Shc and ATG5, deletion either of which results in diminished cellular senescence. p66Shc is not only implicated in the initiation of autophagy by promoting MAMs formation, but also helps stabilizing active autophagic flux by activating cGAS/STING pathway in trophoblast.

Serum tRF-33-RZYQHQ9M739P0J as a novel biomarker for auxiliary diagnosis and disease course monitoring of hepatocellular carcinoma

ObjectiveIn most cases, patients with hepatocellular carcinoma (HCC) develop advanced disease when diagnosed. Finding new molecules to combine with traditional biomarkers is crucial for HCC early diagnosis. In cancer development, tRNA-derived small RNAs (tsRNA) play a crucial role. Here, we aimed to identify a novel biomarker among tsRNAs that can facilitate HCC diagnosis and monitor its prognosis. MethodsWe screened candidate tsRNAs in 3 pairs of HCC and adjacent tissues through high-throughput sequencing. tRF-33-RZYQQ9M739P0J was screened in tissues, sera, and cells through quantitative real-time polymerase chain reaction (qRT-PCR) for further analysis. tRF-33-RZYQHQ9M739P0J was characterized using agarose gel electrophoresis, Sanger sequencing, and nuclear and cytoplasmic RNA isolation. Experiments at room temperature and repeated freeze-thaw cycles were conducted to evaluate the detection performance of tRF-33-RZYQHQ9M739P0J. We measured the levels of differential expression of tRF-33-RZYQHQ9M739P0J in sera using qRT-PCR. We applied the chi-square test to evaluate the correlation between tRF-33-RZYQHQ9M739P0J expression levels and clinicopathological features, and assessed its prognostic value by plotting Kaplan-Meier curves. The diagnostic efficacy of tRF-33-RZYQHQ9M739P0J was evaluated using the receiver operating characteristic (ROC) curve. Finally, the downstream genes related to tRF-33-RZYQHQ9M739P0J were explored through bioinformatics prediction. ResultstRF-33-RZYQHQ9M739P0J was highly expressed in HCC tissues and sera, and its expression was correlated with metastasis, TNM stage, BCLC stage, and vein invasion. Expression of tRF-33-RZYQHQ9M739P0J were decreased after surgery in patients with HCC. High serum tRF-33-RZYQHQ9M739P0J levels are associated with low survival rates, and they can predict survival times in patients with HCC according to the Kaplan-Meier analysis. Combining tRF-33-RZYQHQ9M739P0J with serum alpha-fetoprotein and prothrombin induced by vitamin K absence II can improve the diagnostic efficiency of HCC, suggesting its potential as a biomarker for HCC. ConclusiontRF-33-RZYQHQ9M739P0J may not only be a promising non-invasive marker for early diagnosis, but also a predictor of liver cancer progression.

New Genera and Species of Caulobacter and Brevundimonas Bacteriophages Provide Insights into Phage Genome Evolution.

Previous studies have identified diverse bacteriophages that infect Caulobacter vibrioides strain CB15 ranging from small RNA phages to four genera of jumbo phages. In this study, we focus on 20 bacteriophages whose genomes range from 40 to 60 kb in length. Genome comparisons indicated that these diverse phages represent six Caulobacter phage genera and one additional genus that includes both Caulobacter and Brevundimonas phages. Within species, comparisons revealed that both single base changes and inserted or deleted genetic material cause the genomes of closely related phages to diverge. Among genera, the basic gene order and the orientation of key genes were retained with most of the observed variation occurring at ends of the genomes. We hypothesize that the nucleotide sequences of the ends of these phage genomes are less important than the need to maintain the size of the genome and the stability of the corresponding mRNAs.

SUMO protease FUG1, histone reader AL3 and chromodomain protein LHP1 are integral to repeat expansion-induced gene silencing in Arabidopsis thaliana.

Epigenetic gene silencing induced by expanded repeats can cause diverse phenotypes ranging from severe growth defects in plants to genetic diseases such as Friedreich's ataxia in humans. The molecular mechanisms underlying repeat expansion-induced epigenetic silencing remain largely unknown. Using a plant model with a temperature-sensitive phenotype, we have previously shown that expanded repeats can induce small RNAs, which in turn can lead to epigenetic silencing through the RNA-dependent DNA methylation pathway. Here, using a genetic suppressor screen and yeast two-hybrid assays, we identified novel components required for epigenetic silencing caused by expanded repeats. We show that FOURTH ULP GENE CLASS 1 (FUG1)-an uncharacterized SUMO protease with no known role in gene silencing-is required for epigenetic silencing caused by expanded repeats. In addition, we demonstrate that FUG1 physically interacts with ALFIN-LIKE 3 (AL3)-a histone reader that is known to bind to active histone mark H3K4me2/3. Loss of function of AL3 abolishes epigenetic silencing caused by expanded repeats. AL3 physically interacts with the chromodomain protein LIKE HETEROCHROMATIN 1 (LHP1)-known to be associated with the spread of the repressive histone mark H3K27me3 to cause repeat expansion-induced epigenetic silencing. Loss of any of these components suppresses repeat expansion-associated phenotypes coupled with an increase in IIL1 expression with the reversal of gene silencing and associated change in epigenetic marks. Our findings suggest that the FUG1-AL3-LHP1 module is essential to confer repeat expansion-associated epigenetic silencing and highlight the importance of post-translational modifiers and histone readers in epigenetic silencing.

Comparative miRNome and transcriptome analyses reveal the expression of novel miRNAs in the panicle of rice implicated in sustained agronomic performance under terminal drought stress.

Differential expression of 128 known and 111 novel miRNAs in the panicle of Nagina 22 under terminal drought stress targeting transcription factors, stress-associated genes, etc., enhances drought tolerance and helps sustain agronomic performance under terminal drought stress. Drought tolerance is a complex multigenic trait, wherein the genes are fine-tuned by coding and non-coding components in mitigating deleterious effects. MicroRNA (miRNA) controls gene expression at post-transcriptional level either by cleaving mRNA (transcript) or by suppressing its translation. miRNAs are known to control developmental processes and abiotic stress tolerance in plants. To identify terminal drought-responsive novel miRNA in contrasting rice cultivars, we constructed small RNA (sRNA) libraries from immature panicles of drought-tolerant rice [Nagina 22 (N 22)] and drought-sensitive (IR 64) cultivars grown under control and terminal drought stress. Our analysis of sRNA-seq data resulted in the identification of 169 known and 148 novel miRNAs in the rice cultivars. Among the novel miRNAs, 68 were up-regulated while 43 were down-regulated in the panicle of N 22 under stress. Interestingly, 31 novel miRNAs up-regulated in N 22 were down-regulated in IR 64, whereas 4 miRNAs down-regulated in N 22 were up-regulated in IR 64 under stress. To detect the effects of miRNA on mRNA expression level, transcriptome analysis was performed, while differential expression of miRNAs and their target genes was validated by RT-qPCR. Targets of the differentially expressed miRNAs include transcription factors and stress-associated genes involved in cellular/metabolic/developmental processes, response to abiotic stress, programmed cell death, photosynthesis, panicle/seed development, and grain yield. Differential expression of the miRNAs could be validated in an independent set of the samples. The findings might be useful in genetic improvement of drought-tolerant rice.

Identifying piRNAs that regulate BaP-induced lung injuries: A bottom-up approach from toxicity pathway investigation to animal validation

PIWI-interacting RNAs (piRNAs) is an emerging class of small non-coding RNAs that has been recently reported to have functions in infertility, tumorigenesis, and multiple diseases in humans. Previously, 5 toxicity pathways were proposed from hundreds of toxicological studies that underlie BaP-induced lung injuries, and a “Bottom-up” approach was established to identify small non-coding RNAs that drive BaP-induced pulmonary effects by investigating the activation of these pathways in vitro, and the expression of the candidate microRNAs were validated in tissues of patients with lung diseases from publications. Here in this study, we employed the “Bottom-up” approach to identifying the roles of piRNAs and further validated the mechanisms in vivo using mouse acute lung injury model. Specifically, by non-coding RNA profiling in in vitro BaP exposure, a total of 3 suppressed piRNAs that regulate 5 toxicity pathways were proposed, including piR-004153 targeting CYP1A1, FGFR1, ITGA5, IL6R, NGRF, and SDHA, piR-020326 targeting CDK6, and piR-020388 targeting RASD1. Animal experiments demonstrated that tail vein injection of respective formulated agomir-piRNAs prior to BaP exposure could all alleviate acute lung injury that was shown by histopathological and biochemical evidences. Immunohistochemical evaluation focusing on NF-kB and Bcl-2 levels showed that exogenous piRNAs protect against BaP-induced inflammation and apoptosis, which further support that the inhibition of the 3 piRNAs had an important impact on BaP-induced lung injuries. This mechanism-driven, endpoint-supported result once again confirmed the plausibility and efficiency of the approach integrating in silico, in vitro, and in vivo evidences for the purpose of identifying key molecules.

SARS-CoV-2 remodels the landscape of small non-coding RNAs with infection time and symptom severity

The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 has significantly impacted global health, stressing the necessity of basic understanding of the host response to this viral infection. In this study, we investigated how SARS-CoV-2 remodels the landscape of small non-coding RNAs (sncRNA) from a large collection of nasopharyngeal swab samples taken at various time points from patients with distinct symptom severity. High-throughput RNA sequencing analysis revealed a global alteration of the sncRNA landscape, with abundance peaks related to species of 21-23 and 32-33 nucleotides. Host-derived sncRNAs, including microRNAs (miRNAs), transfer RNA-derived small RNAs (tsRNAs), and small nucleolar RNA-derived small RNAs (sdRNAs) exhibited significant differential expression in infected patients compared to controls. Importantly, miRNA expression was predominantly down-regulated in response to SARS-CoV-2 infection, especially in patients with severe symptoms. Furthermore, we identified specific tsRNAs derived from Glu- and Gly-tRNAs as major altered elements upon infection, with 5’ tRNA halves being the most abundant species and suggesting their potential as biomarkers for viral presence and disease severity prediction. Additionally, down-regulation of C/D-box sdRNAs and altered expression of tinyRNAs (tyRNAs) were observed in infected patients. These findings provide valuable insights into the host sncRNA response to SARS-CoV-2 infection and may contribute to the development of further diagnostic and therapeutic strategies in the clinic.

LDLR-Mediated Targeting and Productive Uptake of siRNA-Peptide Ligand Conjugates In Vitro and In Vivo.

Small RNA molecules such as microRNA and small interfering RNA (siRNA) have become promising therapeutic agents because of their specificity and their potential to modulate gene expression. Any gene of interest can be potentially up- or down-regulated, making RNA-based technology the healthcare breakthrough of our era. However, the functional and specific delivery of siRNAs into tissues of interest and into the cytosol of target cells remains highly challenging, mainly due to the lack of efficient and selective delivery systems. Among the variety of carriers for siRNA delivery, peptides have become essential candidates because of their high selectivity, stability, and conjugation versatility. Here, we describe the development of molecules encompassing siRNAs against SOD1, conjugated to peptides that target the low-density lipoprotein receptor (LDLR), and their biological evaluation both in vitro and in vivo.

Overexpression of Let-7a mitigates diploidization in mouse androgenetic haploid embryonic stem cells

Mouse androgenetic haploid embryonic stem cells (mAG-haESCs) can be utilized to uncover gene functions, especially those of genes with recessive effects, and to produce semicloned mice when injected into mature oocytes. However, mouse haploid cells undergo rapid diploidization during long-term culture invitro and subsequently lose the advantages of haploidy, and the factors that drive diploidization are poorly understood. In this study, we compared the small RNAs (sRNAs) of mAG-haESCs, normal embryonic stem cells (ESCs), and mouse round spermatids by high-throughput sequencing and identified distinct sRNA profiles. Several let-7 family members and miR-290-295 cluster microRNAs (miRNAs) were found significantly differentially transcribed. Knockdown and overexpression experiments showed that let-7a and let-7g suppress diploidization while miR-290a facilitates diploidization. Our study revealed the unique sRNA profile of mAG-haESCs and demonstrated that let-7a overexpression can mitigate diploidization in mAG-haESCs. These findings will help us to better understand mAG-haESCs and utilize them as tools in the future.

Mimicking bacterial infection in male mice changes sperm small RNA profiles and multigenerationally alters offspring behavior and physiology

Paternal pre-conceptual exposures, including stress, diet, substance abuse, parasite infection, and viral immune activation via Poly I:C, have been reported to influence the brains and behavior of offspring through sperm epigenetic changes. However, the effects of paternal (F0) pre-conceptual exposure to bacterial-induced immune activation on the behavior and physiology of F1 and F2 generations remain unexplored. We examined this using C57BL/6J mice. Eight-week-old males (F0) received a single intraperitoneal injection of the bacterial mimetic lipopolysaccharide (LPS: 5 mg/kg) or 0.9 % saline (vehicle control) before mating with naïve females at four weeks post-injection. Comprehensive behavioral assessments were conducted to investigate anxiety, social behaviors, depressive-like behaviors and cognition in both the F1 and F2 generations within the age range of 8 to 14 weeks. Results demonstrated that only female offspring of LPS-exposed fathers exhibited reduced anxiety levels in the light/dark box, large open field, and novelty-suppressed feeding test. These F1 female offspring also exhibited heightened sociability in the 3-chambered social interaction test and a reduced preference for saccharin in the saccharin preference test. Additionally, the F1 male offspring of LPS-challenged males demonstrated an increased total distance traveled in the light/dark box and a longer distance covered in the light zone. They also exhibited diminished preference for social novelty in the 3-chambered social interaction test and an elevated novel arm preference index in the Y-maze. In the F2 generation, male descendants of LPS-treated fathers showed reduced latency to feed in the novelty-suppressed feeding test. Additionally, the F2 generation of LPS-challenged fathers, but not the F1 generation, displayed enhanced immune response in both sexes after an acute LPS immune challenge (5 mg/kg). Analysis of sperm small noncoding RNA profiles from LPS-treated F0 mice revealed significant changes at 4 weeks after administration of LPS. These changes included three microRNAs, eight PIWI-interacting RNAs, and two transfer RNAs, exhibiting significant upregulation (mmu-miR-146a-5p, mmu-piR-27082 and mmu-piR-29102) or downregulation (mmu-miR-5110, mmu-miR-467e-3p, mmu-piR-22583, mmu-piR-23548, mmu-piR-36341, mmu-piR-50293, mmu-piR-16583, mmu-piR-36507, Mus_musculus_tRNA-Ile-AAT-2-1 and Mus_musculus_tRNA-Tyr-GTA-1-1). Additionally, we detected 52 upregulated small noncoding RNAs (including 9 miRNAs, 41 piRNAs, and 2 tRNAs) and 7 downregulated small noncoding RNAs (3 miRNAs, 3 piRNAs, and 1 tRNA) in the sperm of F1 offspring from LPS-treated males. These findings provide compelling evidence for the involvement of epigenetic mechanisms in the modulation of brain function and immunity, and associated behavioral and immunological traits, across generations, in response to bacterial infection.

Identification of an active RNAi pathway in Candida albicans

RNA interference (RNAi) is a fundamental regulatory pathway with a wide range of functions, including regulation of gene expression and maintenance of genome stability. Although RNAi is widespread in the fungal kingdom, well-known species, such as the model yeast Saccharomyces cerevisiae, have lost the RNAi pathway. Until now evidence has been lacking for a fully functional RNAi pathway in Candida albicans, a human fungal pathogen considered critically important by the World Health Organization. Here, we demonstrated that the widely used C. albicans reference strain (SC5314) contains an inactivating missense mutation in the gene encoding for the central RNAi component Argonaute. In contrast, most other C. albicans isolates contain a canonical Argonaute protein predicted to be functional and RNAi-active. Indeed, using high-throughput small and long RNA sequencing combined with seamless CRISPR/Cas9-based gene editing, we demonstrate that an active C. albicans RNAi machinery represses expression of subtelomeric gene families. Thus, an intact and functional RNAi pathway exists in C. albicans, highlighting the importance of using multiple reference strains when studying this dangerous pathogen.

Iron starvation increases the production of the Pseudomonas aeruginosa RsmY and RsmZ sRNAs in static conditions.

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that induces virulence gene expression in response to host-mediated iron starvation. Recently, our laboratory showed that some virulence factors are responsive to iron limitation in static but not shaking growth conditions. One of these is the HSI-2-type six secretion system (T6SS), which is also induced during chronic infection. Iron regulation of T6SS was partially impacted by the iron-responsive PrrF sRNA and completely dependent upon the Pseudomonas quinolone signal (PQS) biosynthetic gene pqsA. Here, we analyzed the impact of iron on the expression of two small regulatory RNAs (sRNAs), RsmY and RsmZ, that activate the expression of T6SS by sequestering the RsmA translation inhibitor. Our results demonstrate that iron starvation induces the expression of RsmY and RsmZ in static but not shaking cultures. We further show that this induction occurs through the rsmY and rsmZ promoters and is dependent upon PqsA. Disruption of either the pqsR gene also eliminated iron-dependent regulation of rsmY and rsmZ promoter activity. Taken together, our results show novel targets of iron regulation that are specific to static growth, highlighting the importance of studying regulatory mechanisms in static communities that may be more representative of growth during chronic infection.IMPORTANCEIron is a central component of various bacterial metabolic pathways making it an important host-acquired nutrient for pathogens to establish infection. Previous iron regulatory studies primarily relied on shaking bacterial cultures; while these ensure cultural homogeneity, they do not reflect growth conditions during infection. We recently showed that static growth of Pseudomonas aeruginosa promotes iron-dependent regulation of a type six secretion system (T6SS), a virulence factor that is induced during chronic infections. In the current study, we found that static growth also promotes iron-dependent regulation of the RsmY and RsmZ sRNAs, which are global regulators that affect T6SS during chronic P. aeruginosa lung infection. Hence, our work demonstrates the Rsm sRNAs as potential effectors of iron regulation during static growth that may also be relevant in chronic infection.

BMSC-derived Exosomes Ameliorate Peritoneal Dialysis-associated Peritoneal Fibrosis via the Mir-27a-3p/TP53 Pathway.

Peritoneal fibrosis (PF) is the main cause of declining efficiency and ultrafiltration failure of the peritoneum, which restricts the long-term application of peritoneal dialysis (PD). This study aimed to investigate the therapeutic effects and mechanisms of bone marrow mesenchymal stem cells-derived exosomes (BMSC-Exos) on PF in response to PD. Small RNA sequencing analysis of BMSC-Exos was performed by second-generation sequencing. C57BL/6J mice were infused with 4.25% glucose-based peritoneal dialysis fluid (PDF) for 6 consecutive weeks to establish a PF model. A total of 36 mice were randomly divided into 6 groups: control group, 1.5% PDF group, 2.5% PDF group, 4.25% PDF group, BMSC-Exos treatment group, and BMSC-Exos+TP53 treatment group. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed to measure the expression level of miR-27a-3p in BMSC-Exos and peritoneum of mice treated with different concentrations of PDF. HE and Masson staining were performed to evaluate the extent of PF. The therapeutic potential of BMSC-Exos for PF was examined through pathological examination, RT-qPCR, Western blotting, and peritoneal function analyses. Epithelial-mesenchymal transition (EMT) of HMrSV5 was induced with 4.25% PDF. Cells were divided into control group, 4.25% PDF group, BMSC-Exos treatment group, and BMSC-Exos+TP53 treatment group. Cell Counting Kit-8 assay was used to measure cell viability, and transwell migration assay was used to verify the capacity of BMSC-Exos to inhibit EMT in HMrSV5 cells. Small RNA sequencing analysis showed that miR-27a-3p was highly expressed in BMSC-derived exosomes compared to BMSCs. The RT-qPCR results showed that the expression of miR-27a-3p was upregulated in BMSC-Exos, but decreased in PD mice. We found that PF was glucose concentration-dependently enhanced in the peritoneum of the PD mice. Compared with the control mice, the PD mice showed high solute transport and decreased ultrafiltration volume as well as an obvious fibroproliferative response, with markedly increased peritoneal thickness and higher expression of α-SMA, collagen-I, fibronectin, and ECM1. The mice with PD showed decreased miR-27a-3p. Peritoneal structural and functional damage was significantly attenuated after BMSC-Exos treatment, while PF and mesothelial damage were significantly ameliorated. Additionally, markers of fibrosis (α-SMA, collagen-I, fibronectin, ECM1) and profibrotic cytokines (TGF-β1, PDGF) were downregulated at the mRNA and protein levels after BMSC-Exos treatment. In HMrSV5 cells, BMSC-Exos reversed the decrease in cell viability and the increase in cell migratory capacity caused by high-glucose PDF. Western blotting and RT-qPCR analysis revealed that BMSC-Exos treatment resulted in increased expression of E-cadherin (epithelial marker) and decreased expression of α-SMA, Snail, and vimentin (mesenchymal markers) compared to those of the 4.25% PDF-treated cells. Importantly, a dual-luciferase reporter assay showed that TP53 was a target gene of miR-27a-3p. TP53 overexpression significantly reversed the decreases in PF and EMT progression induced by BMSC-Exos. The present results demonstrate that BMSC-Exos showed an obvious protective effect on PD-related PF and suggest that BMSC-derived exosomal miR-27a-3p may exert its inhibitory effect on PF and EMT progression by targeting TP53.

MicroRNAs as potential biomarkers in ruminant, avian and porcine

In recent years, studies on microRNAs have increased considerably. miRNAs are small RNA molecules, ranging from 19 to 25 nucleotides in length, that control the suppression of target genes after transcription. MiRNAs serve as fine-tuning factors that influence the expression of up to 60% of all mammalian protein-coding genes. Unlike proteins, miRNA sequences are widely conserved across species. This conservation strongly suggests that miRNAs emerged early in evolution and maintain their functional importance. It has been revealed that these small structures containing a small number of nucleotides can act as critical points in the organism. While traditional cancer biomarkers are mainly produced by tumor tissues or normal embryo tissues, they are absent or present in small amounts in tissue organs and the blood of healthy adults. MiRNAs can be easily detected in the blood, making them selectable candidates as biomarkers for disease. The ruminant family, one of the most diverse subspecies of terrestrial mammals, lives in a wide variety of environments worldwide and is known to have a major impact on various ecosystems and industries, including agriculture, daily activities, and cultures. MiRNAs have a significant impact on the physiology of farm animals, biological development, and cell differentiation. In this review, we will examine miRNAs that have been identified as candidates or potential candidates for the diagnosis and treatment of diseases seen in ruminants, pigs, and avians in recent years. In this way, we will provide a perspective to prevent diseases that can cause great economic losses in veterinary medicine and the production industry.

Small RNA SmsR1 modulates acidogenicity and cariogenic virulence by affecting protein acetylation in Streptococcus mutans.

Post-transcriptional regulation by small RNAs and post-translational modifications (PTM) such as lysine acetylation play fundamental roles in physiological circuits, offering rapid responses to environmental signals with low energy consumption. Yet, the interplay between these regulatory systems remains underexplored. Here, we unveil the cross-talk between sRNAs and lysine acetylation in Streptococcus mutans, a primary cariogenic pathogen known for its potent acidogenic virulence. Through systematic overexpression of sRNAs in S. mutans, we identified sRNA SmsR1 as a critical player in modulating acidogenicity, a key cariogenic virulence feature in S. mutans. Furthermore, combined with the analysis of predicted target mRNA and transcriptome results, potential target genes were identified and experimentally verified. A direct interaction between SmsR1 and 5'-UTR region of pdhC gene was determined by in vitro binding assays. Importantly, we found that overexpression of SmsR1 reduced the expression of pdhC mRNA and increased the intracellular concentration of acetyl-CoA, resulting in global changes in protein acetylation levels. This was verified by acetyl-proteomics in S. mutans, along with an increase in acetylation level and decreased activity of LDH. Our study unravels a novel regulatory paradigm where sRNA bridges post-transcriptional regulation with post-translational modification, underscoring bacterial adeptness in fine-tuning responses to environmental stress.

The role of MicroRNAs in the diagnosis and treatment of oral premalignant disorders.

Oral premalignant disorders (OPMDs) are a group of potentially malignant conditions that pose a significant health burden globally. MicroRNAs (miRNAs), small non-coding RNA molecules, have emerged as crucial regulators of gene expression and have been implicated in various biological processes, including carcinogenesis. This review synthesizes existing knowledge to provide a comprehensive understanding of the molecular mechanisms underlying OPMDs and to highlight the potential of miRNAs as promising biomarkers and therapeutic targets. Additionally, this review seeks to explore the potential of miRNA-based diagnostic biomarkers for early detection of OPMDs in the current literature on miRNAs in OPMDs, examining their involvement in disease pathogenesis, diagnostic potential, and therapeutic implications. Dysregulated miRNAs can target genes involved in critical cellular processes, such as cell cycle regulation, apoptosis, and DNA repair, leading to disease progression. Notably, miR-21, miR-31, miR-135b, and miR-486-5p have shown promise as potential biomarkers for early detection of oral premalignant lesions. Furthermore, the paper discusses the therapeutic implications of miRNAs in OPMDs. Preclinical studies have demonstrated the efficacy of miRNA-targeted therapies, such as miRNA mimics and inhibitors, in suppressing the growth of oral premalignant lesions. Early-phase clinical trials have shown promising results, indicating the potential for personalized treatment approaches. The findings underscore the importance of understanding the molecular mechanisms underlying these disorders and provide insights for the development of improved diagnostic and therapeutic strategies. However, they pose certain limitations given their intrinsic variability in expression profiles, the need for optimized isolation and detection methods, and potential hurdles in transitioning from preclinical success to clinical applications. Thus, future clinical studies are warranted to fully exploit the potential of miRNAs in the management of OPMDs.

The evaluation of miR-1181 and miR-4314 as serum microRNA biomarkers for epithelial ovarian cancer diagnosis and prognosis.

Epithelial ovarian cancer (EOC) is the most ominous tumor of gynecological cancers due to its poor early detection rate and unfavorable prognosis. To date, there is no reliable screening method for the diagnosis of ovarian cancer at an early stage. MiRNAs are small non-coding RNA molecules, and their main function is to regulate gene expression. The present study compared the serum miR-1181 and miR-4314 levels in patients with EOC and healthy controls to measure the diagnostic and prognostic value as candidate biomarkers. We collected serum samples from a total of 135 participants (69 patients with EOC and 66 healthy controls). Relative expressions of miR-1181 and miR-4314 were measured by quantitative real-time polymerase chain reaction assay (qPCR). The present study revealed that both serum miR-1181 and miR-4314 levels in patients with EOC were significantly increased compared to healthy controls for each marker. In addition, there was a significant relationship between miR-1181 and miR-4314 overexpressions and the stage and prognosis of the disease. Finally, patients with high expression levels of miR-1181 and miR-4314 had significantly shorter survival rates than those with low expression levels. The current study proposed that serum miR-1181 and miR-4314 could discriminate the EOC patients from healthy controls. In addition, both miR-1181 and miR-4314 may be predictive biomarkers for ovarian cancer prognosis. Further studies are needed to confirm the findings of the present study.