Endogenous Small RNAs Research Articles

MiR-223 as a Regulator and Therapeutic Target in Liver Diseases.

MicroRNAs (miRNAs) are endogenous non-coding single-stranded small molecule RNAs consisting of 20–24 nucleotides that are highly conserved in species evolution. Expression of miRNAs is strictly tissue-specific, and it is chronological in fungi and plants, as well as in animals. MiR-223 has been shown to play a key role in innate immunity, and dysregulation of its expression contributes to the pathogenesis of multiple inflammatory diseases, and cancers. In this article the biosynthesis and functions of miR-223 in innate immunity are reviewed, and the role of miR-223 in liver physiopathology and therapeutic prospects are highlighted.

Identification and Characterization of MicroRNAs Involving in Initial Sex Differentiation of Chlamys farreri Gonads.

Simple SummarySex formation of gonads encompasses two ancient and highly conserved biological processes, sex determination and sex differentiation. The processes are strictly regulated by a complex of gene networks. There is increasing evidence that miRNAs play key roles in many biological processes. however, information is limited in their contribution to sex differentiation in animals. In the present study, we identified the novel miRNAs involved in sex-related genes regulation and explored the miRNA–mRNA networks underlying the posttranscriptional regulation during the initial sex differentiation in Zhikong scallop, Chlamys farreri. Our findings provide an important basis for studying the sex differentiation mechanisms, as well as developing sex control techniques in bivalves.Research on expressional regulation of genes at the initial sex differentiation of gonads will help to elucidate the mechanisms of sex determination and differentiation in animals. However, information on initial sex differentiation of gonads is limited in bivalves. MicroRNAs (miRNAs) are a class of endogenous small noncoding RNAs that can regulate the target gene expression at the posttranscription level by degrading the mRNA or repressing the mRNA translation. In the present study, we investigated the small RNAs transcriptome using the testes and ovaries of Zhikong scallop Chlamys farreri juveniles with a shell height of 5.0 mm, a critical stage of initial sex differentiation of gonads. A total of 75 known mature miRNAs and 103 novel miRNAs were identified. By comparing the expression of miRNAs between the ovary and testis, 11 miRNAs were determined to be differentially expressed. GO annotations and KEGG analyses indicated that many putative target genes that matched to these differentially expressed miRNAs participated in the regulation of sex differentiation. Furthermore, two selected miRNAs, cfa-novel_miR65 and cfa-miR-87a-3p_1, were confirmed to downregulate expressions of Foxl2 (a female-critical gene) and Klf4 (a male-critical gene), respectively, using a dual-luciferase reporter analysis. Our findings provided new insights into the initial sex differentiation of gonads regulated by miRNAs in bivalves.

Fragments of rDNA Genes Scattered over the Human Genome Are Targets of Small RNAs.

Small noncoding RNAs of different origins and classes play several roles in the regulation of gene expression. Here, we show that diverged and rearranged fragments of rDNA units are scattered throughout the human genome and that endogenous small noncoding RNAs are processed by the Microprocessor complex from specific regions of ribosomal RNAs shaping hairpins. These small RNAs correspond to particular sites inside the fragments of rDNA that mostly reside in intergenic regions or the introns of about 1500 genes. The targets of these small ribosomal RNAs (srRNAs) are characterized by a set of epigenetic marks, binding sites of Pol II, RAD21, CBP, and P300, DNase I hypersensitive sites, and by enrichment or depletion of active histone marks. In HEK293T cells, genes that are targeted by srRNAs (srRNA target genes) are involved in differentiation and development. srRNA target genes are enriched with more actively transcribed genes. Our data suggest that remnants of rDNA sequences and srRNAs may be involved in the upregulation or downregulation of a specific set of genes in human cells. These results have implications for diverse fields, including epigenetics and gene therapy.

Overexpression of microRNA-381-3p ameliorates hypoxia/ischemia-induced neuronal damage and microglial inflammation via regulating the C-C chemokine receptor type 2 /nuclear transcription factor-kappa B axis

ABSTRACT microRNAs, as small endogenous RNAs, influence umpteen sophisticated cellular biological functions regarding neurodegenerative and cerebrovascular diseases. Here, we interrogated miR-381-3p’s influence on BV2 activation and neurotoxicity in ischemic and hypoxic environment. Oxygen-glucose deprivation (OGD) was adopted to induce microglial activation and HT-22 neuron damage. Quantitative polymerase chain reaction (qRT-PCR) was taken to check miR-381-3p expression in OGD-elicited BV2 cells and HT-22 neurons. It transpired that miR-381-3p expression was lowered in BV2 cells and HT-22 cells elicited by OGD. miR-381-3p up-regulation remarkably hampered inflammatory mediator expression in BV2 cells induced by OGD and weakened HT22 neuron apoptosis. In vivo, miR-381-3p expression was abated in HI rats’ ischemic lesions, and miR-381-3p up-regulation could ameliorate inflammation and neuron apoptosis in their brain. C-C chemokine receptor type 2 (CCR2) was identified as the downstream target of miR-381-3p, and miR-381-3p suppressed the CCR2/NF-κB pathway to mitigate microglial activation and neurotoxicity. Therefore, we believed that miR-381-3p overexpression exerts anti-inflammation and anti-apoptosis in ischemic brain injury by targeting CCR2

A miRNA-Disease Association Identification Method Based on Reliable Negative Sample Selection and Improved Single-Hidden Layer Feedforward Neural Network

miRNAs are a category of important endogenous non-coding small RNAs and are ubiquitous in eukaryotes. They are widely involved in the regulatory process of post-transcriptional gene expression and play a critical part in the development of human diseases. By utilizing recent advancements in big data technology, using bioinformatics methods to identify causative miRNA becomes a hot spot. In this paper, a method called RNSSLFN is proposed to identify the miRNA-disease associations by reliable negative sample selection and an improved single-hidden layer feedforward neural network (SLFN). It involves, firstly, obtaining integrated similarity for miRNAs and diseases; next, selecting reliable negative samples from unknown miRNA-disease associations via distinguishing up-regulated or down-regulated miRNAs; then, introducing an improved SLFN to solve the prediction task. The experimental results on the latest data sets HMDD v3.2 and the framework of 5-fold cross-validation (CV) show that the average AUC and AUPR of RNSSLFN achieve 0.9316 and 0.9065 m, respectively, which are superior to the other three state-of-the-art methods. Furthermore, in the case studies of 10 common cancers, more than 70% of the top 30 predicted miRNA-disease association pairs are verified in the databases, which further confirms the reliability and effectiveness of the RNSSLFN model. Generally, RNSSLFN in predicting miRNA-disease associations has prodigious potential and extensive foreground.

Identification, expression analysis, and potential roles of microRNAs in the regulation of polysaccharide biosynthesis in Polygonatum cyrtonema Hua

MicroRNAs (miRNAs) are a group of endogenous small non-coding RNAs with important roles in plant growth, development, and metabolic processes. Polygonatum cyrtonema Hua (P. cyrtonema) is an important Chinese traditional medicinal herb with broad pharmacological functions, and polysaccharides are the main biological substance accumulated in the P. cyrtonema rhizome. However, regulation of the process of polysaccharide biosynthesis in P. cyrtonema remains largely unknown.To elucidate the miRNAs and their targets involved in polysaccharide biosynthesis in P. cyrtonema, four small RNA libraries were constructed from flower, leaf, rhizome, and root tissues and sequenced. A total of 69 conserved and 5 novel miRNAs were identified, of which 6 miRNAs (miR156a-5p, miR156f-5p, miR395a-5, miR396a-3p, miR396g-3p, and miR397-5p-1) were down-regulated and 7 miRNAs (miR160, miR160h-1, miR160e-5p, miR319b-1, miR319-1, miR319c-5p-3, and miR319c-1) were up-regulated in rhizomes compared with flower, leaf, and root tissues. Bioinformatics analysis showed that the predicted targets of these miRNAs were mostly transcription factors and functional genes enriched in metabolic and secondary metabolite biosynthetic pathways, and 7 genes and their paired miRNAs were identified in carbohydrate metabolism. qRT-PCR expression analysis demonstrated that 6 miRNAs and their targets involved in carbohydrate metabolism were existed a negative correlation in. P. cyrtonema tissues. MiR396a-3p and one of its target genes, abfA, were possibly involved in polysaccharide biosynthesis pathway. This is the first report on the identification of conserved and novel miRNAs and their potential targets in P. cyrtonema, thus providing molecular evidence for the role of miRNAs in the regulation of polysaccharide biosynthesis in P. cyrtonema.

Analysis of the miRNA expression profile involved in the tomato spotted wilt orthotospovirus–pepper interaction

MicroRNAs (miRNAs) are a class of about 22 nucleotide endogenous non-coding small RNAs that play important roles in plant development and defense responses to biotic and abiotic stress. Tomato spotted wilt orthotospovirus (TSWV) is among the most devastating plant viruses worldwide, causing leaf spotting and wilting symptoms in many plants. In this study, high-throughput sequencing technology was used to identify miRNAs in peppers that were regulated by TSWV infection, and bioinformatics techniques were used to analyze the function of these miRNAs and their target genes. The sequencing results showed that, in the pepper plants infected by TSWV, a total of 101 miRNAs were differentially expressed, and several differentially expressed miRNAs were verified by the qRT-PCR method. Based on the GO function and KEGG pathway enrichment analysis, the functions of these miRNAs and their target genes were predicted. These target genes play a regulatory role in pathways such as plant-pathogen interactions and plant hormone signal transduction. Our results provide information about pepper miRNAs and will help to further understand the mechanism of TSWV–host interactions.

An Efficient Genetic Transformation and CRISPR/Cas9-Based Genome Editing System for Moso Bamboo (Phyllostachys edulis).

Moso bamboo (Phyllostachys edulis) is the most important monopodial bamboo species worldwide. Without a genetic transformation system, it is difficult to verify the functions of genes controlling important traits and conduct molecular breeding in moso bamboo. Here, we established a plant regeneration system from immature embryos. Calli were induced on MS medium added 4–6 mg⋅L–1 2,4-dichlorophenoxyacetic acid (2,4-D) with high efficiency (>60%). A plant growth regulator combination of 0.5 mg⋅L–1 1-naphthylacetic acid (NAA), 2.0 mg⋅L–1 6-benzylaminopurine (BAP), and 3.0 mg⋅L–1 zeatin (ZT) was suitable for shoot differentiation, and the shoot induction frequency was increased to 43% after 0.5 mg⋅L–1 abscisic acid (ABA) pretreatment. An effective antibiotic screening concentration was determined by hygromycin sensitivity test. We further optimized the Agrobacterium concentration and added vacuum infiltration for infection, which improves the transient expression efficiency. A genetic transformation system was established for the first time in moso bamboo, with the transformation efficiency of approximately 5%. To optimize genome editing, two endogenous U3 small nuclear RNA (snRNA) promoters were isolated and used to drive small guide RNA (sgRNA) expression. The results showed that the PeU3.1 promoter exhibited higher efficiency, and it was used for subsequent genome editing. Finally, homozygous pds1pds2 mutants were obtained by an efficient CRISPR/Cas9 genome-editing system. These technical systems will be conducive to gene functional validation and accelerate the molecular breeding process of moso bamboo.

Virus and endogenous viral element-derived small non-coding RNAs and their roles in insect–virus interaction

RNA interference pathways mediated by different types of small non-coding RNAs (siRNAs, miRNAs and piRNAs) are conserved biological responses to exotic stresses, including viral infection. Aside from the well-established siRNA pathway, the miRNA pathway and the piRNA pathway process viral sequences, exogenously or endogenously, into miRNAs and piRNAs, respectively. During the host-virus interaction, viral sequences, including both coding and non-coding sequences, can be integrated as endogenous viral elements (EVEs) and thereby become present within the germline of a non-viral organism. In recent years, significant progress has been made in characterizing the biogenesis and function of viruses and EVEs associated with snRNAs. Overall, the siRNA pathway acts as the primarily antiviral defense against a wide range of exogenous viruses; the miRNA pathways associated with viruses or EVEs function in antiviral response and host gene regulation; EVE derived piRNAs with a ping-pong signature have the potential to limit cognate viral infection.

MiR-340-5p Mediates Cardiomyocyte Oxidative Stress in Diabetes-Induced Cardiac Dysfunction by Targeting Mcl-1.

Diabetic cardiomyopathy (DCM) is initially characterized by early diastolic dysfunction, left ventricular remodeling, hypertrophy, and myocardial fibrosis, and it is eventually characterized by clinical heart failure. MicroRNAs (miRNAs), endogenous small noncoding RNAs, play significant roles in diabetes mellitus (DM). However, it is still largely unknown about the mechanism that links miRNAs and the development of DCM. Here, we aimed to elucidate the mechanism underlying the potential role of microRNA-340-5p in DCM in db/db mouse, which is a commonly used model of type 2 DM and diabetic complications that lead to heart failure. We first demonstrated that miR-340-5p expression was dramatically increased in heart tissues of mice and cardiomyocytes under diabetic conditions. Overexpression of miR-340-5p exacerbated DCM, which was reflected by extensive myocardial fibrosis and more serious dysfunction in db/db mice as represented by increased apoptotic cardiomyocytes, elevated ROS production, and impaired mitochondrial function. Inhibition of miR-340-5p by a tough decoy (TUD) vector was beneficial for preventing ROS production and apoptosis, thus rescuing diabetic cardiomyopathy. We identified myeloid cell leukemia 1 (Mcl-1) as a major target gene for miR-340-5p and showed that the inhibition of Mcl-1 was responsible for increased functional loss of mitochondria, oxidative stress, and cardiomyocyte apoptosis, thereby caused cardiac dysfunction in diabetic mice. In conclusion, our results showed that miR-340-5p plays a crucial role in the development of DCM and can be targeted for therapeutic intervention.

Characterization of miRNAs in Embryonic, Larval, and Adult Lumpfish Provides a Reference miRNAome for Cyclopterus lumpus.

Simple SummaryLumpfish (Cyclopterus lumpus) is an emergent aquaculture species, and its miRNA repertoire is still unknown. miRNAs are critical post-transcriptional modulators of teleost gene expression. Therefore, a lumpfish reference miRNAome was characterized by small RNA sequencing and miRDeep analysis of samples from different organs and developmental stages. The resulting miRNAome, an essential reference for future expression analyses, consists of 443 unique mature miRNAs from 391 conserved and eight novel miRNA genes. Enrichment of specific miRNAs in particular organs and developmental stages indicates that some conserved lumpfish miRNAs regulate organ and developmental stage-specific functions reported in other teleosts.MicroRNAs (miRNAs) are endogenous small RNA molecules involved in the post-transcriptional regulation of protein expression by binding to the mRNA of target genes. They are key regulators in teleost development, maintenance of tissue-specific functions, and immune responses. Lumpfish (Cyclopterus lumpus) is becoming an emergent aquaculture species as it has been utilized as a cleaner fish to biocontrol sea lice (e.g., Lepeophtheirus salmonis) infestation in the Atlantic Salmon (Salmo salar) aquaculture. The lumpfish miRNAs repertoire is unknown. This study identified and characterized miRNA encoding genes in lumpfish from three developmental stages (adult, embryos, and larvae). A total of 16 samples from six different adult lumpfish organs (spleen, liver, head kidney, brain, muscle, and gill), embryos, and larvae were individually small RNA sequenced. Altogether, 391 conserved miRNA precursor sequences (discovered in the majority of teleost fish species reported in miRbase), eight novel miRNA precursor sequences (so far only discovered in lumpfish), and 443 unique mature miRNAs were identified. Transcriptomics analysis suggested organ-specific and age-specific expression of miRNAs (e.g., miR-122-1-5p specific of the liver). Most of the miRNAs found in lumpfish are conserved in teleost and higher vertebrates, suggesting an essential and common role across teleost and higher vertebrates. This study is the first miRNA characterization of lumpfish that provides the reference miRNAome for future functional studies.

Small RNAs Participate in Plant-Virus Interaction and Their Application in Plant Viral Defense.

Small RNAs are significant regulators of gene expression, which play multiple roles in plant development, growth, reproductive and stress response. It is generally believed that the regulation of plants’ endogenous genes by small RNAs has evolved from a cellular defense mechanism for RNA viruses and transposons. Most small RNAs have well-established roles in the defense response, such as viral response. During viral infection, plant endogenous small RNAs can direct virus resistance by regulating the gene expression in the host defense pathway, while the small RNAs derived from viruses are the core of the conserved and effective RNAi resistance mechanism. As a counter strategy, viruses evolve suppressors of the RNAi pathway to disrupt host plant silencing against viruses. Currently, several studies have been published elucidating the mechanisms by which small RNAs regulate viral defense in different crops. This paper reviews the distinct pathways of small RNAs biogenesis and the molecular mechanisms of small RNAs mediating antiviral immunity in plants, as well as summarizes the coping strategies used by viruses to override this immune response. Finally, we discuss the current development state of the new applications in virus defense based on small RNA silencing.

Understanding the role of miRNAs in the pathogenesis of brain arteriovenous malformations

Brain arteriovenous malformations (AVMs) are abnormal vessels that are prone to rupture, causing life-threatening intracerebral hemorrhage (ICH). Understanding the molecular basis of pathogenesis, timely diagnosis, and treatment of brain AVMs are some of the urgent problems in neurosurgery. MicroRNAs (miRNAs) are small endogenous RNAs that regulate gene-expression posttranscriptionally. MiRNAs are involved in almost all biological processes, including cell proliferation, apoptosis, and cell differentiation. Recent studies have shown that miRNAs can be involved in brain AVMs formation and rupture. There are also extracellular forms of miRNAs. Circulating miRNAs have been detected in the blood circulation and other body fluids. Owing to their stability and resistance to endogenous RNase activity, circulating miRNAs have been proposed as diagnostic and prognostic biomarkers for various diseases, such as tumors, cardiovascular and autoimmune diseases. In this review, we summarized the role of some miRNAs in brain AVMs pathogenesis and discussed their potential clinical application as non-invasive biomarkers.

MicroRNAs: An opportunity to overcome significant challenges in malaria detection and control

Organ damage and pathological disease states lead to the rapid release of microRNAs (miRNAs), a class of endogenous small non-coding RNAs, into the blood circulation. Because secreted miRNAs can be detected in biologic fluids such as plasma, they are currently being explored as promising non-invasive biomarkers of infectious and non-infectious diseases. Malaria remains a major global health challenge but still the potential of miRNAs has not been explored extensively in the context of malaria compared to other diseases. Here, we highlight important miRNAs found during different phases of the malaria life cycle in the anopheline vector and the human host. We have also put forward our opinion on how malaria parasite-stage-specific miRNAs can be incorporated into new diagnostic and prognostic tools to detect carrier mosquitoes and infected patients. In addition, we have emphasised the potential of miRNAs to be used as new therapeutics to treat severe malaria patients, an unresearched area of malaria control.

How to Design U1 snRNA Molecules for Splicing Rescue.

Mutations affecting constitutive splice donor sites (5'ss) are among the most frequent genetic defects that disrupt the normal splicing process. Pre-mRNA splicing requires the correct identification of a number of cis-acting elements in an ordered fashion. By disrupting the complementarity of the 5'ss with the endogenous small nuclear RNA U1 (U1 snRNA), the key component of the spliceosomal U1 ribonucleoprotein, 5'ss mutations may result in exon skipping, intron retention or activation of cryptic splice sites. Engineered modification of the U1 snRNA seemed to be a logical method to overcome the effect of those mutations. In fact, over the last years, a number of in vitro studies on the use of those modified U1 snRNAs to correct a variety of splicing defects have demonstrated the feasibility of this approach. Furthermore, recent reports on its applicability in vivo are adding up to the principle that engineered modification of U1 snRNAs represents a valuable approach and prompting further studies to demonstrate the clinical translatability of this strategy.Here, we outline the design and generation of U1 snRNAs with different degrees of complementarity to mutated 5'ss. Using the HGSNAT gene as an example, we describe the methods for a proper evaluation of their efficacy in vitro, taking advantage of our experience to share a number of tips on how to design U1 snRNA molecules for splicing rescue.

Extending and Running the Mosquito Small RNA Genomics Resource Pipeline.

The Mosquito Small RNA Genomics (MSRG) resource is a repository of analyses on the small RNA transcriptomes of mosquito cell cultures and somatic and gonadal tissues. This resource allows for comparing the regulation dynamics of small RNAs generated from transposons and viruses across mosquito species. This chapter covers the procedures to set up the MSRG resource pipeline as a new installation by detailing the necessary collection of genome reference and annotation files and lists of microRNAs (miRNAs) hairpin sequences, transposon repeats consensus sequences, and virus genome sequences. Proper execution of the MSRG resource pipeline yields outputs amenable to biologists to further analyze with desktop and spreadsheet software to gain insights into the balance between arthropod endogenous small RNA populations and the proportions of virus-derived small RNAs that include Piwi-interacting RNAs (piRNAs) and endogenous small interfering RNAs (siRNAs).

Significance of miRNA in enhancement of flavonoid biosynthesis.

Flavonoid metabolism shows very strong plasticity in plant development and coping with the changing environment. Flavonoid biosynthesis is regulated by many metabolic pathways, including transcriptional regulation, post-transcriptional control, post-translational regulationand epigenetic regulation. miRNA is a form of endogenous noncoding single-strand small molecule RNA that primarily regulates the expression of target genes horizontally after transcription through splicing and translational suppression. It also plays an important role in regulating plant growth and development, secondary metabolism and biotic and abiotic stress. miRNA can regulate the formation of flavonoids by acting on structural genes or indirectly by using an MBW transcription complex comprising MYB-bHLH-WD40. This study summarizes the biosynthesis and mechanisms of miRNA, and provides a summary of the mechanisms of miRNAs involved in production of flavonoids, in order to elucidate the biosynthesis pathway and complex regulatory network of plant flavonoids. We aim to provide new insights into improving the content of flavonoid active ingredients in plants.

Small RNA sequencing reveals various microRNAs involved in piperine biosynthesis in black pepper (Piper nigrum L.)

BackgroundBlack pepper (Piper nigrum L.), an important and long-cultivated spice crop, is native to South India and grown in the tropics. Piperine is the main pungent and bioactive alkaloid in the berries of black pepper, but the molecular mechanism for piperine biosynthesis has not been determined. MicroRNAs (miRNAs), which are classical endogenous noncoding small RNAs, play important roles in regulating secondary metabolism in many species, but less is known regarding black pepper or piperine biosynthesis.ResultsTo dissect the functions of miRNAs in secondary metabolism especially in piperine biosynthesis, 110 known miRNAs, 18 novel miRNAs and 1007 individual targets were identified from different tissues of black pepper by small RNA sequencing. qRT-PCR and 5′-RLM-RACE experiments were conducted to validate the reliability of the sequencing data and predicted targets. We found 3 miRNAs along with their targets including miR166-4CL, miR396-PER and miR397-CCR modules that are involved in piperine biosynthesis.ConclusionMiRNA regulation of secondary metabolism is a common phenomenon in plants. Our study revealed new miRNAs that regulate piperine biosynthesis, which are special alkaloids in the piper genus, and they might be useful for future piperine genetic improvement of black pepper.

Building Endogenous Gene Connections through RNA Self-Assembly Controlled CRISPR/Cas9 Function.

Construction of synthetic circuits that can artificially establish endogenous gene connections is essential to introduce new phenotypes for cellular behaviors. Given the diversity of endogenous genes, it lacks a general and easy-to-design toolbox to manipulate the genetic network. Here we present a type of self-assembly-induced RNA circuit that can directly build regulatory connections between endogenous genes. Inspired from the natural assembling process of guide RNA in the CRISPR/Cas9 complex, this design employs an independent trigger RNA strand to induce the formation of a ternary guide RNA assembly for functional control of CRISPR/Cas9. With this general principle, expressional regulations of endogenous genes can be controlled by totally independent endogenous small RNAs and mRNAs in E. coli via activatable CRISPR/Cas9 function. Moreover, the cellular phenotype of E. coli is successfully programmed with introduction of new gene connections. In addition, the functionality of this design is also verified in the mammalian system. This self-assembly-based RNA circuit exhibits a great flexibility and simplicity of design and provides a unique approach to build endogenous gene connections, which paves a broad way toward manipulation of cellular genetic networks.

High miR-3609 expression is associated with better prognosis in TNBC based on mining using systematic integrated public sequencing data.

MicroRNAs (miRNAs/miRs) are small endogenous RNAs that regulate gene expression post-transcriptionally. Abnormal miR-3609 expression is associated with the occurrence of pancreatic cancer, glioma and other diseases, such as polycystic ovary syndrome. However, the prognostic potential of miR-3609 has been reported in breast cancer. Thus, the present study aimed to investigate the differential expression and prognostic value of miR-3609 in patients with breast cancer from the UALCAN, cBioportal and Kaplan-Meier Plotter databases, respectively. Furthermore, the co-expression genes of miR-3609 in breast cancer were investigated using data from the LinkedOmics database, and functional enrichment analysis was performed using the LinkInterpreter module in LinkedOmics. The co-expression gene network was constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins database, and the cytoHubba plug-in was used to identify the hub genes, which were visualized using Cytoscape software. The prognoses of the hub genes were performed using the Kaplan-Meier Plotter database. The Cell Counting Kit-8 and cell cycle assays were performed to confirm the functions of miR-3609 mimics transfection in MDA-MB-231 cells. Survival analysis using the Kaplan-Meier Plotter database demonstrated that high miR-3609 expression in triple-negative breast cancer (TNBC) was associated with a better prognosis. Furthermore, the experimental results indicated that high miR-3609 expression inhibited the proliferation of TNBC cells and induced cell cycle arrest of TNBC cells in the G0/G1 phase. Taken together, the results of the present study suggest that miR-3609 plays a vital role in mediating cell cycle arrest and inhibiting the proliferation of TNBC cells.