Non-coding miRNAs Research Articles

Serum microRNA Biomarker Expression in HIV and TB: A Concise Overview.

Non-coding RNAs (ncRNAs), specifically MicroRNAs or miRNAs, are now under-stood to be essential regulators in the complex field of gene expression. By selectively bind-ing to certain mRNA targets, these tiny RNA molecules control the expression of genes., leading to mRNA degradation or translational repression. The discovery of miRNAs has sig-nificantly advanced biomedical research, particularly in elucidating the molecular mecha-nisms underlying various diseases and exploring innovative therapeutic approaches. Recent progress in miRNA research has provided insights into their biogenesis, functional roles, and potential clinical applications. Despite the absence of established methodologies for clinical implementation, miRNAs show great promise as diagnostic and therapeutic agents for a wide array of diseases. Their distinctive attributes, such as high specificity, sensitivity, and accessibility, position them as ideal candidates for biomarker development and targeted therapy. Achieving a comprehensive understanding of miRNA biology and functionality is crucial to fully harnessing their potential in medicine. Ongoing research efforts aim to un-ravel the intricate mechanisms of miRNA-mediated gene regulation and to develop novel approaches for utilizing miRNAs in disease diagnosis, prognosis, and treatment. This review provides a comprehensive analysis of current knowledge on miRNAs, focusing on their bio-genesis, regulatory mechanisms, and potential clinical applications. By synthesizing existing evidence and highlighting key research findings, this review aims to inspire further explora-tion into the diverse roles of miRNAs in health and disease. Ultimately, this endeavour could result in the development of innovative miRNA-based diagnostic and therapeutic strategies.

MiR-98 and miR-629 can be used as a potential biomarker on relapsing-remitting multiple sclerosis patients

IntroductionMultiple Sclerosis (MS) is an autoimmune disease that affects the central nervous system (CNS), characterized by chronic inflammation, demyelination, and neurodegeneration. In recent years, among non-coding RNAs miRNA have emerged as key regulators of different biological processes and it has been suggested that they play an important role in the mechanisms underlying MS pathogenesis. Through in silico methods, miR-629-5p and miR-98-5p were identified as significant factors in MS pathology. The aim of this study was to examine the levels of expression of miR-629-5p and miR-98-5p in blood samples obtained from patients with relapsing–remitting MS. Methods: Total blood were recruited from RRMS and control group and qPCR analysis was used for evaluating of target miRNAs expression. The mirDIP database was utilized to identify the target genes. Hub genes were identified with the Cytoscape and target pathways were identified using the STRING. Results: Expression analysis revealed a significant upregulation of miR-629-5p and miR-98-5p (FC ≥ 1.5 and p < 0.05) in patients with RRMS. PI3K-Akt, Rap1, MAPK and FoxO signaling pathways were found as target. Discussion: The discovery of these miRNAs suggests that they may have a notable impact on MS patients by intervening on pathways involved in both the immune and nervous systems.

Identification and Characterization of Evolutionary Conserved Muskmelon Non-coding miRNAs and Their Target Proteins.

MicroRNAs (miRNAs) are short, endogenously encoded small RNAs, 18-26 nucleotides in length, which can posttranscriptionally regulate gene expression through translation inhibition or endonucleolytic cleavage. The muskmelon is one of the most widely cultivated fruits in the Cucurbitaceae family. Despite its significance, only 120 miRNAs from different families have been reported in muskmelon. In this study, we aimed to expand this knowledge base by predicting 40 new miRNAs in muskmelon using a spectrum of genomic-based tools. Precursor and mature sequences were obtained from microRNA registry database as reference and analyzed via the basic local alignment search tool (Blastn) for ESTs identification. After removing the non-coding sequences, the remaining candidate sequences were analyzed using MFOLD to generate secondary structures for the newly predicted miRNAs. Additionally, the predicted muskmelon miRNAs were validated using a set of five randomly chosen primers and RT-PCR. Through gene ontology (GO) analysis, we identified 89 targets associated with newly predicted muskmelon miRNAs. Transcription factor-coding genes play a crucial role in plant growth and development. Additionally, the miR4249 has been found to have the same targets in muskmelon that have been linked to cell signaling and transcription factors. The identified targets are integral for diverse biological processes including plant growth, development, metabolism, aging, disease resistance, and resistance to environmental stresses, such as salt, cold, and oxidative stress. As a result, the outcomes of this study demonstrate that this mechanism not only contributes to the production of a higher quality crop but also enhances overall production.

Comparative analysis of the profile of circulating microRNAs in the blood plasma of patients with gliomas

Against the background of modest successes in the development of new diagnostic and therapeutic tools to improve the survival of patients with glial brain tumors, early diagnosis of this pathology remains relevant. Endogenous noncoding miRNAs that regulate the expression of target mRNAs have become attractive targets for the development of circulating biomarker-based assays, because sample acquisition does not require invasive sampling such as biopsy.Purpose of the study. To determine the levels of circulating microRNAs in the blood plasma of patients with glial tumors, meningiomas and apparently healthy donors, using high-output sequencing.Material and methods. 26 blood plasma samples were selected from the biobank data base of the National Medical Research Center for Oncology, and the total RNA was studied using the NGS sequencing method. The sample included: 2 cases of oligodendroglioma (grades 2–3), 6 – astrocytomas of 2–4 degrees of malignancy, 7 – glioblastomas of 4 degrees of malignancy, 7 – benign neoplasms (meningiomas), 4 – control (conditionally healthy donors).Results. During the primary analysis, a pool of 71 differentially expressed microRNAs was identified, the expression of which was tumor-specific: 20 microRNAs for glioblastoma, 4 microRNAs for astrocytoma, 23 microRNAs for oligodendroglioma, 24 microRNAs for meningioma. At the same time, 47 microRNAs showed increased levels in the blood plasma compared to the control group, 15 showed a corresponding decrease in levels. A comparative analysis identified microRNAs that specifically differentiate each tumor type.Conclusion. The results obtained seem promising and set the vector for further research, which will include expanding the sample and validating the identified biomarkers to determine their diagnostic value.

Crosstalk between long non-coding RNAs and miRNAs regulates transferase activities in response to salt stress in Pistachio (Pistacia vera L.)

Long non-coding RNA (lncRNA) molecules are important regulators of a broad spectrum of biological processes, including adaptation to diverse stresses in plants. However, how plant lncRNAs function during salt stress conditions is largely unknown. In our study, we identified 48 conserved mature Pistacia vera L. (P. vera) miRNAs among pistachio miRNA precursors and used RNA sequencing data to predict and validate the interaction relationship between lncRNAs and miRNAs. We indicated that the regulatory networks of pve-miRNA-lncRNA-mRNAs were more complex in the salt-tolerant (Ghazvini) compared to the salt-sensitive (Sarakhs) pistachio cultivar under NaCl treatment. We also found that 7 and 8 salt-responsive lncRNAs play a role as competing endogenous target mimics (eTMs) for 7 pve-miRNA families in Ghazvini (mir164, mir169, mir397, and mir827) and Sarakhs (mir156, mir159, mir169, and mir482), respectively. The GO-term enrichment analysis showed that protein-coding target genes of pve-miRNAs that regulated with target mimic relationship mainly enriched in transferase activity include: 7-deoxyloganetin glucosyltransferase, Arginine N-methyltransferase, Glutathione transferase, Flavonol sulfotransferase, and Beta-glucuronosyltransferase. Their activity regulates alkaloids and polysulfated flavonols biosynthesis, sequesters sodium, improves antioxidant capacity and controls epigenetic elements in Ghazvini under salt treatment. In Sarakhs, the analysis of GO-term enrichment in pve-miRNAs protein-coding target genes of lncRNA-related mimic relationship enriched in response to wounding, cell death, and jasmonic acid biosynthetic process. Compared to Sarakhs, Ghazvini had a more effective ceRNA network for regulating transferase enzymes and specific transcription factors including MYB, WRKY, and ABR1 in response to salt stress, which led to more salt tolerance in the Ghazvini cultivar. The present study provides new information about the complex lncRNA-miRNA-mRNA regulatory networks in response to plant stress conditions in pistachio. Furthermore, our findings can be used for different breeding programs and the introduction of salt-tolerant pistachio rootstocks.

Retroelements in targeted antitumor therapy (review)

Background: Pathological expression of retroelements is determined in most malignant tumors, which reflects the pathogenesis of tumors, since activated retrotransposons cause genomic instability due to a cascade of mutual regulatory influence events between transposons, tumor suppressors and oncogenes. These relationships are reflected in the expression profiles of certain long non-coding RNAs and miRNAs in specific tumor tissues, since retroelements are key sources of non-coding RNAs. Since retroelements serve as drivers for the initiation and progression of carcinogenesis, it is promising to use drugs aimed at inhibiting their expression. The aim of the study: To determine the available methods of influencing retroelements in antitumor therapy, to evaluate the ways of their further improvement for the effective treatment of malignant neoplasms. Materials and methods: The databases Scopus, WoS, PubMed were used to analyze the role of retroelements in the development of tumors, methods of influencing them in the treatment of neoplasms. Results: Reverse transcriptase inhibitors have proven to be effective. Silencing of retrotransposons by acting on histone modifiers can lead to a weakening of the antitumor immune response; therefore, this approach requires RNA guides that direct the formation of heterochromatin in the area where strictly specific retroelements are located. A promising method is viral mimicry aimed at activating the interferon and T-cell response to artificially activated endogenous retroviruses. For this purpose, inhibitors of histone methyltransferases, DNA methyltransferases, and histone deacetylases are used. Their joint use is most effective, especially with PD-1 checkpoint blockers. The arrangement of retroelements in gene introns has become the basis for the development of spliceosomal targeted therapy, which also initiates an antitumor immune response to double-stranded RNA transcripts. We suggest the possibility of using antisense oligonucleotides targeting retroelements in anticancer therapy. Such approach is used to treat age-related macular degeneration, Fukuyama muscular dystrophy, amyotrophic lateral sclerosis. Antisense oligonucleotides targeting microRNAs, proto-oncogenes, and oncosuppressors are used in anticancer therapy and are closely related to retroelements, which could become promising targets for antisense oligonucleotides. Conclusion: In antitumor therapy, it is possible to use two diametrically opposed strategies for influencing retroelements: inhibition and activation. The most promising way is a combination of these methods with specific silencing of retrotransposons using guides (complementary RNAs) and stimulation of the expression of elements not involved in carcinogenesis, but activating the immune response.

Gastric Cancer in the Era of Epigenetics.

Gastric cancer (GC) remains a significant contributor to cancer-related mortality. Novel high-throughput techniques have enlightened the epigenetic mechanisms governing gene-expression regulation. Epigenetic characteristics contribute to molecular taxonomy and give rise to cancer-specific epigenetic patterns. Helicobacter pylori (Hp) infection has an impact on aberrant DNA methylation either through its pathogenic CagA protein or by inducing chronic inflammation. The hypomethylation of specific repetitive elements generates an epigenetic field effect early in tumorigenesis. Epstein-Barr virus (EBV) infection triggers DNA methylation by dysregulating DNA methyltransferases (DNMT) enzyme activity, while persistent Hp-EBV co-infection leads to aggressive tumor behavior. Distinct histone modifications are also responsible for oncogene upregulation and tumor-suppressor gene silencing in gastric carcinomas. While histone methylation and acetylation processes have been extensively studied, other less prevalent alterations contribute to the development and migration of gastric cancer via a complex network of interactions. Enzymes, such as Nicotinamide N-methyltransferase (NNMT), which is involved in tumor's metabolic reprogramming, interact with methyltransferases and modify gene expression. Non-coding RNA molecules, including long non-coding RNAs, circular RNAs, and miRNAs serve as epigenetic regulators contributing to GC development, metastasis, poor outcomes and therapy resistance. Serum RNA molecules hold the potential to serve as non-invasive biomarkers for diagnostic, prognostic or therapeutic applications. Gastric fluids represent a valuable source to identify potential biomarkers with diagnostic use in terms of liquid biopsy. Ongoing clinical trials are currently evaluating the efficacy of next-generation epigenetic drugs, displaying promising outcomes. Various approaches including multiple miRNA inhibitors or targeted nanoparticles carrying epigenetic drugs are being designed to enhance existing treatment efficacy and overcome treatment resistance.

Impacts of non-coding RNAs in the pathogenesis of varicocele.

Two classes of non-coding RNAs, namely lncRNAs and miRNAs have been reported to be involved in the pathogenesis of varicocele. MIR210HG, MLLT4-AS1, gadd7, and SLC7A11-AS1 are among lncRNAs whose expression has been changed in patients with varicocele in association with the sperm quality. Animal studies have also suggested contribution of NONRATG001060, NONRATG002949, NONRATG013271, NONRATG027523 and NONRATG023747 lncRNAs in this pathology. Meanwhile, expression of some miRNAs, such as miR-210-3p, miR-21, miR-34a, miR-122a, miR-181a, miR-34c and miR-192a has been altered in this condition. Some of these transcripts have the potential to predict the sperm quality. We summarize the impacts of lncRNAs and miRNAs in the pathogenesis of varicocele.

Advances in RNA Interference for Plant Functional Genomics: Unveiling Traits, Mechanisms, and Future Directions.

RNA interference (RNAi) is a conserved molecular mechanism that plays a critical role in post-transcriptional gene silencing across diverse organisms. This review delves into the role of RNAi in plant functional genomics and its applications in crop improvement, highlighting its mechanistic insights and practical implications. The review begins with the foundational discovery of RNAi's mechanism, tracing its origins from petunias to its widespread presence in various organisms. Various classes of regulatory non-coding small RNAs, including siRNAs, miRNAs, and phasiRNAs, have been uncovered, expanding the scope of RNAi-mediated gene regulation beyond conventional understanding. These RNA classes participate in intricate post-transcriptional and epigenetic processes that influence gene expression. In the context of crop enhancement, RNAi has emerged as a powerful tool for understanding gene functions. It has proven effective in deciphering gene roles related to stress resistance, metabolic pathways, and more. Additionally, RNAi-based approaches hold promise for integrated pest management and sustainable agriculture, contributing to global efforts in food security. This review discusses RNAi's diverse applications, such as modifying plant architecture, extending shelf life, and enhancing nutritional content in crops. The challenges and future prospects of RNAi technology, including delivery methods and biosafety concerns, are also explored. The global landscape of RNAi research is highlighted, with significant contributions from regions such as China, Europe, and North America. In conclusion, RNAi remains a versatile and pivotal tool in modern plant research, offering novel avenues for understanding gene functions and improving crop traits. Its integration with other biotechnological approaches such as gene editing holds the potential to shape the future of agriculture and sustainable food production.

Delivery of miR-214 via extracellular vesicles downregulates Xbp1 expression and pro-inflammatory cytokine genes in macrophages.

Tumor-infiltrating macrophages are tumor-promoting and show activation of the unfolded protein response (UPR). The transcription factor X-box binding protein 1 (XBP1) is a conserved element of the UPR. Upon activation, the UPR mediates the transcriptional activation of pro-inflammatory cytokines and immune suppressive factors, hence contributing to immune dysregulation in the tumor microenvironment (TME). miR-214 is a short non-coding miRNA that targets the 3'-UTR of the Xbp1 transcript. Here, we tested a new method to efficiently deliver miR-214 to macrophages as a potential new therapeutic approach. We generated miR-214-laden extracellular vesicles (iEV-214) in a murine B cell and demonstrated that iEV-214 were enriched in miR-214 between 1,500 - 2,000 fold relative to control iEVs. Bone marrow-derived macrophages (BMDM) treated with iEV-214 for 24 h underwent a specific enrichment in miR-214, suggesting transfer of the miR-214 payload from the iEVs to macrophages. iEV-214 treatment of BMDM markedly reduced (> 50%) Xbp1 transcription under endoplasmic reticulum stress conditions compared to controls. Immune-related genes downstream of XBP1s (Il-6, Il-23p19, and Arg1) were also reduced by 69%, 51%, and 34%, respectively. Together, these data permit to conclude that iEV-214 are an efficient strategy to downregulate the expression of Xbp1 mRNA and downstream genes in macrophages. We propose miRNA-laden iEVs are a new approach to target macrophages and control immune dysregulation in the TME.

Manipulating epigenetic diversity in crop plants: Techniques, challenges and opportunities

Epigenetic modifications act as conductors of inheritable alterations in gene expression, all while keeping the DNA sequence intact, thereby playing a pivotal role in shaping plant growth and development. This review article presents an overview of techniques employed to investigate and manipulate epigenetic diversity in crop plants, focusing on both naturally occurring and artificially induced epialleles. The significance of epigenetic modifications in facilitating adaptive responses is explored through the examination of how various biotic and abiotic stresses impact them. Further, environmental chemicals are explored for their role in inducing epigenetic changes, particularly focusing on inhibitors of DNA methylation like 5-AzaC and zebularine, as well as inhibitors of histone deacetylation including trichostatin A and sodium butyrate. The review delves into various approaches for generating epialleles, including tissue culture techniques, mutagenesis, and grafting, elucidating their potential to induce heritable epigenetic modifications in plants. In addition, the ground breaking CRISPR/Cas is emphasized for its accuracy in targeting specific epigenetic changes. This presents a potent tools for deciphering the intricacies of epigenetic mechanisms. Furthermore, the intricate relationship between epigenetic modifications and non-coding RNA expression, including siRNAs and miRNAs, is investigated. The emerging role of exo-RNAi in epigenetic regulation is also introduced, unveiling its promising potential for future applications. The article concludes by addressing the opportunities and challenges presented by these techniques, emphasizing their implications for crop improvement. Conclusively, this extensive review provides valuable insights into the intricate realm of epigenetic changes, illuminating their significance in phenotypic plasticity and their potential in advancing crop improvement.

Construction of ceRNA network and identification of hub differentially expressed genes associated with breast cancer using reanalysis of microarray dataset: A systems biology approach

The interaction between long non-coding RNAs (lncRNAs), miRNAs and mRNAs has implications in the pathogenesis of different cancer, including breast cancer. In the current study, we developed an in-silico approach to ascertain the competing endogenous RNA (ceRNA) network in breast cancer. Our approach led to identification of 1816 differentially expressed (DE) mRNAs, including 1039 downregulated DEmRNAs (such as LEP and ADIPOQ) and 777 upregulated DEmRNAs (such as COL11A1 and COL10A1), 19 DElncRNAs, including 15 downregulated DElncRNAs (such as CARMN and COPG2IT1) and 4 upregulated DElncRNAs (such as MALAT1 and NRAV) and 27 DEmiRNAs, including 15 downregulated DEmiRNAs (such as MIR452 and MIR224) and 12 upregulated DEmiRNAs (such as MIR6787 and MIR21). Pathway analysis revealed down-regulation of PPAR, Fatty acid metabolism, Adipocytokine, Vascular smooth muscle contraction and Metabolism of xenobiotics by cytochrome P450, while up-regulation of Pyrimidine metabolism, p53 signaling pathway, Cell cycle, Oocyte meiosis and RNA transport pathways in breast cancer. Finally, we constructed an lncRNA/miRNA/mRNA ceRNA network consisted of 2 lncRNAs, 15 mRNAs, and 4 miRNAs. This network represents an appropriate target for design of anti-cancer modalities and documentation of novel markers for breast cancer.

Constructing Integrative ceRNA Networks and Finding Prognostic Biomarkers in Renal Cell Carcinoma.

Inspired by a newly discovered gene regulation mechanism known as competing endogenous RNA (ceRNA) interactions, several computational methods have been proposed to generate ceRNA networks. However, most of these methods have focused on deriving restricted types of ceRNA interactions such as lncRNA-miRNA-mRNA interactions. Competition for miRNA-binding occurs not only between lncRNAs and mRNAs but also between lncRNAs or between mRNAs. Furthermore, a large number of pseudogenes also act as ceRNAs, thereby regulate other genes. In this study, we developed a general method for constructing integrative networks of all possible interactions of ceRNAs in renal cell carcinoma (RCC). From the ceRNA networks we derived potential prognostic biomarkers, each of which is a triplet of two ceRNAs and miRNA (i.e., ceRNA-miRNA-ceRNA). Interestingly, some prognostic ceRNA triplets do not include mRNA at all, and consist of two non-coding RNAs and miRNA, which have been rarely known so far. Comparison of the prognostic ceRNA triplets to known prognostic genes in RCC showed that the triplets have a better predictive power of survival rates than the known prognostic genes. Our approach will help us construct integrative networks of ceRNAs of all types and find new potential prognostic biomarkers in cancer.

The intracellular parasite Anncaliia algerae induces a massive miRNA down-regulation in human cells.

Anncaliia algerae belongs to microsporidia, a group of obligate intracellular parasites related to fungi. These parasites are largely spread in water and food-webs and can infect a wide variety of hosts ranging from invertebrates to vertebrates including humans. In humans, microsporidian infections are mainly opportunistic as immunocompetent hosts can clear parasites naturally. Recent studies however have reported persistent microsporidian infections and have highlighted them as a risk factor in colon cancer. This may be a direct result of cell infection or may be an indirect effect of the infectious microenvironment and the host's response. In both cases, this raises the question of the effects of microsporidian infection at the host and host-cell levels. We aimed to address the question of human host intracellular response to microsporidian infection through a transcriptomic kinetic study of human foreskin fibroblasts (HFF) infected with A.algerae, a human infecting microsporidia with an exceptionally wide host range. We focused solely on host response studying both coding and small non-coding miRNA expression. Our study revealed a generalized down-regulation of cell miRNAs throughout infection with up to 547 different miRNAs downregulated at some timepoints and also transcriptomic dysregulations that could facilitate parasite development with immune and lipid metabolism genes modulation. We also hypothesize possible small nucleic acid expropriation explaining the miRNA downregulation. This work contributes to a better understanding of the dialogue that can occur between an intracellular parasite and its host at the cellular level, and can guide future studies on microsporidian infection biology to unravel the mode of action of these minimalist parasites at the tissue or host levels.We have also generated a kinetic and comprehensive transcriptomic data set of an infectious process that can help support comparative studies in the broader field of parasitology. Lastly, these results may warrant for caution regarding microsporidian exposure and persistent infections.

Molecular mechanisms that regulate the heat stress response in sea urchins (Strongylocentrotus intermedius) by comparative heat tolerance performance and whole-transcriptome RNA sequencing

In the context of climate change and extreme high temperature, the commercially important sea urchin Strongylocentrotus intermedius suffers high mortality during summer in Northern China. How sea urchins respond to high temperatures is of great concern to academia and industry. How to understand the heat tolerance of sea urchin from the whole transcriptome level. In this study, the heat-resistant S. intermedius bred by our team and its control group were used as the research objects, then we applied whole-transcriptome RNA sequencing to detect differentially expressed mRNAs, microRNAs, long noncoding RNAs that respond to heat stress in the heat-resistant and control S. intermedius. A competitive endogenous RNA (ceRNA) regulatory network was constructed with predicted pairs of differentially expressed mRNAs and noncoding RNAs and revealed the molecular regulatory mechanisms in S. intermedius responding to heat stress. A functional analysis suggested that the ceRNAs were involved in basal metabolism, calcium ion transport, endoplasmic reticulum stress, and apoptosis. This is the whole-transcriptomic analysis of S. intermedius under heat stress to propose ceRNA networks that will provide a basis for studying the potential functions of long noncoding RNAs and miRNAs in the heat stress response in S. intermedius and provide a theoretical basis for the study of the molecular mechanism of sea urchins in response to environmental changes.

Whole transcriptome analysis reveals dysregulation of molecular networks in schizophrenia.

To characterize the regulatory relationships between different types of transcripts and the altered molecular networks in schizophrenia (SCZ), we performed a whole transcriptome study by quantifying mRNAs, long noncoding RNAs (lncRNAs), miRNAs, and circular RNAs (circRNAs) in the same individuals simultaneously. A total of 807 dysregulated genes showed differential expression in SCZ cases compared with controls. Network-based analysis revealed dysregulation of molecular networks in SCZ. Finally, integration of the transcriptome data with published data identified promising SCZ candidate genes. Our study reveals that dysregulated molecular networks and regulatory relationships between different types of transcript may have a role in SCZ.

Urinary MicroRNA Analysis Indicates an Epigenetic Regulation of Chronic Kidney Disease of Unknown Etiology in Sri Lanka.

Chronic kidney disease of unknown etiology (CKDu) is reported among male paddy farmers in the dry zone of Sri Lanka. The exact cause of this disease remains undetermined. Genetic susceptibility is identified as a major risk factor for CKDu Objectives: In this study, small urinary RNAs were characterized in CKDu patients, healthy endemic and non-endemic controls. Differently expressed urinary miRNAs and their associated pathways were identified in the study population. Healthy and diseased male volunteers (n = 9) were recruited from Girandurukotte (endemic) and Mawanella (non-endemic) districts. Urinary small RNAs were purified and sequenced using Illumina MiSeqTM. The sequence trace files were assembled and analyzed. Differentially ex-pressed miRNAs among these three groups were identified and pathway analysis was conducted. The urine samples contained 130,623 sequence reads identified as non-coding RNAs, PIWI-interacting RNAs (piRNA), and miRNAs. Approximately four percent of the total small RNA reads represented miRNA, and 29% represented piRNA. A total of 409 miRNA species were ex-pressed in urine. Interestingly, both diseased and endemic controls population showed significantly low expression of miRNA and piRNA. Regardless of the health status, the endemic population ex-pressed significantly low levels of miR-10a, miR-21, miR-148a, and miR-30a which have been linked with several environmental toxins Conclusion: Significant downregulation of miRNA and piRNA expression in both diseased and healthy endemic samples indicates an epigenetic regulation of CKDu involving genetic and environmental interaction. Further studies of specific miRNA species are required to develop a miRNA panel to identify individuals susceptible to CKDu.

Targeting long non-coding RNA MALAT1 reverses cancerous phenotypes of breast cancer cells through microRNA-561-3p/TOP2A axis

Non-coding RNAs, including Inc-RNA and miRNA, have been reported to regulate gene expression and are associated with cancer progression. MicroRNA-561-3p (miR-561-3p), as a tumor suppressor, has been reported to play a role in preventing cancer cell progression, and MALAT1 (Lnc-RNA) have also been demonstrated to promote malignancy in various cancers, such as breast cancer (BC). In this study, we aimed to determine the correlation between miR-561-3p and MALAT1 and their roles in breast cancer progression. The expression of MALAT1, mir-561-3p, and topoisomerase alpha 2 (TOP2A) as a target of miR-561-3p was determined in BC clinical samples and cell lines via qRT-PCR. The binding site between MALAT1, miR-561-3p, and TOP2A was investigated by performing the dual luciferase reporter assay. MALAT1 was knocked down by siRNA, and cell proliferation, apoptotic assays, and cell cycle arrest were evaluated. MALAT1 and TOP2A were significantly upregulated, while mir-561-3p expression was downregulated in BC samples and cell lines. MALAT1 knockdown significantly increased miR-561-3p expression, which was meaningfully inverted by co-transfection with the miR 561-3p inhibitor. Furthermore, the knockdown of MALAT1 by siRNA inhibited proliferation, induced apoptosis, and arrested the cell cycle at the G1 phase in BC cells. Notably, the mechanistic investigation revealed that MALAT1 predominantly acted as a competing endogenous RNA in BC by regulating the miR-561-3p/TOP2A axis. Based on our results, MALAT1 upregulation in BC may function as a tumor promoter in BC via directly sponging miRNA 561-3p, and MALAT1 knockdown serves a vital antitumor role in BC cell progression through the miR-561-3p/TOP2A axis.

Construction of ceRNA network based on RNA-seq for identifying prognostic lncRNA biomarkers in Perthes disease.

Introduction: Legg-Calvé-Perthes disease or Perthes disease is a condition that occurs in children aged 2 to 15years, and is characterized by osteonecrosis of the femoral head, which results in physical limitations. Despite ongoing research, the pathogenesis and molecular mechanisms underlying the development of Perthes disease remain unclear. In order to obtain further insights, the expression patterns of long non-coding RNAs (lncRNAs), miRNAs, and mRNAs in a rabbit model of Perthes disease were analyzed in this study by transcriptome sequencing. Methods and results: The results of RNA-seq analyses revealed that 77 lncRNAs, 239 miRNAs, and 1027 mRNAs were differentially expressed in the rabbit model. This finding suggested that multiple genetic pathways are involved in the development of Perthes disease. A weighted gene co-expression network analysis (WGCNA) network was subsequently constructed using the differentially expressed mRNAs (DEmRNAs), and network analysis revealed that the genes associated with angiogenesis and platelet activation were downregulated, which was consistent with the findings of Perthes disease. A competing endogenous RNA (ceRNA) network was additionally constructed using 29 differentially expressed lncRNAs (including HIF3A and LOC103350994), 28 differentially expressed miRNAs (including ocu-miR-574-5p and ocu-miR-324-3p), and 76 DEmRNAs (including ALOX12 and PTGER2). Disscusion: The results obtained herein provide novel perspectives regarding the pathogenesis and molecular mechanisms underlying the development of Perthes disease. The findings of this study can pave the way for the development of effective therapeutic strategies for Perthes disease in future.

The long non-coding RNA, miRNA and mRNA landscapes ofcementoblasts during cementogenesis.

Stimulation of cementogenesis is essential to cementum regeneration and root restoration. Long non-coding RNAs (lncRNAs) participate in the regulatory networks of periodontal regeneration processes. We identified and analysed differentially expressed lncRNAs, miRNAs and mRNAs associated with cementogenic differentiation of cementoblasts. OCCM-30 immortalized mouse cementoblast cells were induced in cementogenic medium for 7 and 14 days. Total RNA was extracted and subjected to RNA sequencing to screen for differentially expressed lncRNAs, miRNAs and mRNAs. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed to determine the expression levels of RNAs. Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were used to clarify the potential functions of differentially expressed genes in biological processes and pathways. lncRNA-miRNA-mRNA networks were constructed based on correlation and algorithmic analyses. In all, 461 lncRNAs, 89 miRNAs and 2157 mRNAs showed differential expression in OCCM-30 cells after cementoblast differentiation. At day 7, upregulation of 248 lncRNAs, 30 miRNAs and 905 mRNAs was observed, along with downregulation of 127 lncRNAs, 34 miRNAs and 960 mRNAs. At day 14, 197 lncRNAs, 13 miRNAs and 847 mRNAs were upregulated, while 74 lncRNAs, 12 miRNAs and 760 mRNAs were downregulated. The results of qRT-PCR showed that four candidate lncRNAs, H19, Gdap10, Foxo6os and Ipw, were significantly upregulated after 7 and 14 days of cementogenic induction. The lncRNA-miRNA-mRNA network illustrated a possible competitive endogenous RNA regulatory mechanism. GO analysis showed that consistently differentially expressed mRNAs were involved in blood vessel morphogenesis, cell-substrate adhesion, cell adhesion, ossification and extracellular matrix organization. KEGG analysis indicated that extracellular matrix-receptor interaction, focal adhesion, and the PI3K-Akt, Rap1, mitogen-activated protein kinase, and Ras signalling pathways varied significantly during cementogenesis. The expressions of lncRNA, miRNA and mRNA were significantly altered in cementoblasts after cementogenesis. This study highlighted the effect of lncRNAs in the process of cementogenesis and revealed their potential for the discovery of novel biomarkers and therapeutic targets for cementum regeneration.