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Follicular fluid (FF) is rich in extracellular vesicles (EVs). EVs carries a variety of miRNA involved in regulating follicular development, the function of cells in follicles, primordial follicular formation, follicular recruitment and selection, follicular atresia, oocyte communication, granulosa cells (GCs) function and luteinization and other biological processes of follicular development. Previous studies in our laboratory have shown that bovine follicular fluid (bFF) high density-small extracellular vesicles (HD-sEVs)-miRNA was enriched in autophagy-related pathways. However, the mechanism of bFF EVs carrying miRNA regulating GCs autophagy is not clear. Thus, this study carried out a series of studies on the previous HD-sEVs sequencing data and miR-128-3p contained in bFF HD-sEVs. A total of 38 differentially expressed genes were detected by RNA-Seq after overexpression of miR-128-3p in bovine GCs (bGCs). Through cell transfection, Western blot (WB) and Immunofluorescence (IF), it was proved that overexpression of miR-128-3p could promote the expression of LC3 (microtubule-associated protein I light chain 3), inhibit p62, promote the number of autophagosome, promote the formation of autophagy lysosome and autophagy flow, and activate bGCs autophagy. MiR-128-3p inhibitor significantly inhibited the expression of LC3 and monodansylcadaverine (MDC) in bGCs, and promoted the expression of autophagy substrate p62, indicating that HD-sEVs-miR-128-3p could activate bGCs autophagy. In addition, through double luciferase assay, bioinformatics analysis, WB and RT-qPCR, it was concluded that bFF HD-sEVs-miR-128-3p could target TFEB (transcription factor EB) and FoxO4 (Forkhead box O4) and activate GCs autophagy., Competing Interests: Declaration of competing interest The authors have no disclosure of conflicts of interest related to this investigation., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Dielectric barrier discharge (DBD) plasma regulates the levels of reactive oxygen species (ROS), which are critical for sperm quality. MicroRNAs (miRNAs) are non-coding single-stranded RNA molecules encoded by endogenous genes, which regulate post-transcriptional gene expression in animals. At present, it is unknown whether DBD plasma can regulate sperm ROS levels through miRNAs. To further understand the regulatory mechanism of DBD plasma on sperm ROS levels, miRNAs in fresh boar spermatozoa were detected using Illumina deep sequencing technology. We found that 25 known miRNAs and 50 novel miRNAs were significantly upregulated, and 14 known miRNAs and 74 novel miRNAs were significantly downregulated in DBD plasma-treated spermatozoa. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that target genes of differentially expressed miRNAs were involved in many activities and pathways associated with antioxidants. We verified that DBD plasma significantly increased boar sperm quality and reduced ROS levels. These results suggest that DBD plasma can improve sperm quality by regulating ROS levels via miRNAs. Our findings provide a potential strategy to improve sperm quality through miRNA-targeted regulation of ROS, which helps to increase male reproduction and protect cryopreserved semen in clinical practice., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The function of exosomal miRNAs (miRs) in retinal degeneration is largely unclear. We were aimed to investigate the functions of exosomes as well as their miRs derived from retinal pigment epithelial (RPE) cells following exposure to oxidative stress (OS). After the OS by lipopolysaccharide and rotenone on RPE cells, interleukin-1 beta (IL-1β), Interleukin-6 (IL-6), Tumor Necrosis Factor-alpha (TNF-α) were upregulated, along with the decreased mitochondrial membrane potential and upregulated oxidative damage marker 8-OH-dG in RPE cells. RPE-derived exosomes were then isolated, identified, injected into the subretinal space in mice. After subretinal injection, RPE-exosomes after OS not only induced higher ROS level and apoptotic retinal cells, but also elevated IL-1β, IL-6 alongside TNF-α expressions among retina/RPE/choroidal complex. Next, miRs inside the exosomes were sequenced by the next generation sequencing (NGS) technology. NGS revealed that certain miRs were abundant in exosomes, while others were selectively kept by RPE cells. Further, downregulated miRs, like miR-125b-5p, miR-125a-5p, alongside miR-128-3p, and upregulated miR, such as miR-7-5p were validated byRT-qPCR. Finally, Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to find the possible target genes of those selective exosomal miRs. Our results proved that the RPE-derived exosomes after OS selectively express certain miRs, providing novel insights into the pathogenesis of age-related macular degeneration (AMD) in future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Atherosclerosis is considered as a chronic inflammatory disease. MircoRNA-126-5p (miR-126-5p) may be pathophysiological relevant with the apoptotic processes in the endothelial cells in the arterial wall. Here, this study determined the role of circulating atherosclerosis-regulatory miR-126-5p in atherosclerotic mice and explored the possible mechanism in human aortic endothelial cells (HAECs). Atherosclerotic mice model was established, oxidative stress-induced apoptosis of HAECs was analyzed, and nuclear factor kappa B (NF-κB)/PI3K/AKT/mTOR signaling pathway was investigated both in vitro and in vivo. This study showed that miR-126-5p mice had less coronary atherosclerotic plaque and lower blood lipid than control mice after being induced by high cholesterol diet. Apoptosis of endothelial cells was inhibited and NF-κB/PI3K/AKT/mTOR signal pathway was downregulated in miR-126-5p mice compared to control. MiR-126-5p increased proliferation and inhibited apoptosis of HAECs induced by oxidative stress. In vitro assay showed that miR-126-5p regulated apoptosis of HAECs via downregulation of NF-κB-mediated PI3K/AKT/mTOR signaling pathway. In conclusion, these data indicated that transfection of miR-126-5p rescued apoptosis of HAECs and limited atherosclerosis, introducing a potential therapeutic approach for atherosclerosis., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Subarachnoid hemorrhage (SAH), a common devastating cerebrovascular accident, is a great threat to human health and life. Exploration of the potential therapeutic target of SAH is urgently needed. Previous studies showed that long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) promotes cell apoptosis in various diseases, while its role in SAH remains unclear. In our study, we established a mouse model of SAH and used the oxyhemoglobin (OxyHb) to induce neuronal injury in vitro. Interestingly, MALAT1 was found upregulated in brain tissues of SAH mice and OxyHb-stimulated neurons. In addition, knockdown of MALAT1 attenuated apoptosis and decreased reactive oxygen species (ROS) production in OxyHb-stimulated neurons. Mechanistically, we demonstrated that MALAT1 bound with miR-499-5p. Furthermore, our findings indicated that miR-499-5p bound to SOX6 3' untranslated region (UTR) and negatively regulated SOX6 mRNA and protein levels. Rescue assays suggested that SOX6 overexpression counteracted the effects of MALAT1 knockdown on neurocyte apoptosis, and ROS production in OxyHb-stimulated neurons. The in vivo assays indicated that knockdown of MALAT1 improved brain injury of SAH mice. Our study demonstrates that silencing of MALAT1 alleviates neurocyte apoptosis and reduces ROS production through the miR-499-5p/SOX6 axis after SAH injury., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Endothelial defects significantly contribute to cardiovascular pathology in the premature aging disease Hutchinson-Gilford progeria syndrome (HGPS). Using an endothelium-specific progeria mouse model, we identify a novel, endothelium-specific microRNA (miR) signature linked to the p53-senescence pathway and a senescence-associated secretory phenotype (SASP). Progerin-expressing endothelial cells exert profound cell-non-autonomous effects initiating senescence in non-endothelial cell populations and causing immune cell infiltrates around blood vessels. Comparative miR expression analyses revealed unique upregulation of senescence-associated miR34a-5p in endothelial cells with strong accumulation at atheroprone aortic arch regions but also, in whole cardiac- and lung tissues as well as in the circulation of progeria mice. Mechanistically, miR34a-5p knockdown reduced not only p53 levels but also late-stage senescence regulator p16 with no effect on p21 levels, while p53 knockdown reduced miR34a-5p and partially rescued p21-mediated cell cycle inhibition with a moderate effect on SASP. These data demonstrate that miR34a-5p reinforces two separate senescence regulating branches in progerin-expressing endothelial cells, the p53- and p16-associated pathways, which synergistically maintain a senescence phenotype that contributes to cardiovascular pathology. Thus, the key function of circulatory miR34a-5p in endothelial dysfunction-linked cardiovascular pathology offers novel routes for diagnosis, prognosis and treatment for cardiovascular aging in HGPS and potentially geriatric patients.

Objective: MicroRNAs (miRNAs) are essential biomarkers during development of human diseases. We aimed to explore the role of hypoxia-induced bone marrow mesenchymal stem cells (BMSCs)-derived exosomal miR-98-5p in myocardial ischemia-reperfusion injury (MI/RI)., Methods: BMSCs were isolated, cultured, stimulated by hypoxia and transfected with adenovirus expressing miR-98-5p. The exosomes were extracted from BMSCs and named as BMSC-exos. The rat MI/RI models were established by ligation of left anterior descending artery and were respectively injected. Then, hemodynamic indices, myocardial enzymes, oxidative stress factors, inflammatory factors, macrophage infiltration and infarct size in these rats were determined. Expression of miR-98-5p, toll-like receptor 4 (TLR4) and the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) signaling pathway-related proteins was assessed. The target relation between miR-98-5p and TLR4 was confirmed by bioinformatic method and dual luciferase report gene assay., Results: MiR-98-5p was downregulated, TLR4 was upregulated and the PI3K/Akt signaling pathway was inactivated in MI/RI rat myocardial tissues. Exosomal miR-98-5p from hypoxic BMSCs promoted cardiac function and suppressed myocardial enzyme levels, oxidative stress, inflammation response, macrophage infiltration and infarct size in I/R myocardial tissues. Moreover, TRL4 was targeted by miR-98-5p and miR-98-5p activated PI3K/Akt signaling pathway., Conclusion: Hypoxia-induced BMSC-exos elevated miR-98-5p to protect against MI/RI. This study may be helpful for treatment of MI/RI., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Objective: This study aimed to probe the function of microRNA-181b-5p (miR-181b-5p)/protein inhibitor of activated STAT1 (PIAS1)/protein arginine methyltransferase 1 (PRMT1) axis in the progression of alcoholic fatty liver disease (AFLD)., Methods: A rat model of AFLD was established and treated with altered miR-181b-5p, PIAS1 or PRMT1 expression constructs to identify their effects on liver function, serum inflammation, liver tissue oxidative stress, hepatocyte apoptosis and pathological changes of liver tissue in rats using a series of assays. miR-181b-5p, PIAS1 and PRMT1 levels were detected, and the targeting relationship between miR-181b-5p and PIAS1 was confirmed., Results: MiR-181b-5p and PRMT1 were elevated while PIAS1 was reduced in AFLD rat liver tissues, miR-181b-5p inhibition, PIAS1 overexpression or PRMT1 inhibition improved liver function, attenuated inflammation, oxidative stress, pathological changes and hepatocyte apoptosis in AFLD rat liver tissues. The impacts of miR-181b-5p inhibition on AFLD rats were reversed by PIAS1 silencing. PIAS1 was confirmed as a target gene of miR-181b-5p, and miR-181b-5p regulated PRMT1 expression through binding to PIAS1., Conclusion: Inhibiting miR-181b-5p can promote the expression of PIAS1, thereby inhibiting PRMT1 and ultimately improving AFLD., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Purpose: Previously, we demonstrated that miR-183/96/182 cluster (miR-183C) knockout mice exhibit decreased severity of Pseudomonas aeruginosa (PA)-induced keratitis. This study tests the hypothesis that prophylactic knockdown of miR-183C ameliorates PA keratitis indicative of a therapeutic potential., Methods: Eight-week-old miR-183C wild-type and C57BL/6J inbred mice were used. Locked nucleic acid-modified anti-miR-183C or negative control oligoribonucleotides with scrambled sequences (NC ORNs) were injected subconjunctivally 1 day before and then topically applied once daily for 5 days post-infection (dpi) (strain 19660). Corneal disease was graded at 1, 3, and 5 dpi. Corneas were harvested for RT-PCR, ELISA, immunofluorescence (IF), myeloperoxidase and plate count assays, and flow cytometry. Corneal nerve density was evaluated in flatmounted corneas by IF staining with anti-β-III tubulin antibody., Results: Anti-miR-183C downregulated miR-183C in the cornea. It resulted in an increase in IL-1β at 1 dpi, which was decreased at 5 dpi; fewer polymorphonuclear leukocytes (PMNs) at 5 dpi; lower viable bacterial plate count at both 1 and 5 dpi; increased percentages of MHCII+ macrophages (Mϕ) and dendritic cells (DCs), consistent with enhanced activation/maturation; and decreased severity of PA keratitis. Anti-miR-183C treatment in the cornea of naïve mice resulted in a transient reduction of corneal nerve density, which was fully recovered one week after the last anti-miR application. miR-183C targets repulsive axon-guidance receptor molecule Neuropilin 1, which may mediate the effect of anti-miR-183C on corneal nerve regression., Conclusions: Prophylactic miR-183C knockdown is protective against PA keratitis through its regulation of innate immunity, corneal innervation, and neuroimmune interactions.

The dysregulation of circular RNA (circRNA) has been monitored in osteoarthritis (OA) cartilage, hinting that circRNA deregulation modulates OA progression. We thus aimed to unveil the role of circRNA spastic paraplegia 11 (circ_SPG11) in OA conditions. The upregulation of circ_SPG11 was observed in OA cartilage and IL-1β-treated chondrocytes. Knockdown of circ_SPG11 restored IL-1β-depleted cell proliferation and alleviated IL-1β-induced cell apoptosis and ECM degradation. Circ_SPG11 bound to miR-665 and negatively regulated miR-665 expression. Inhibition of miR-665 reversed the inhibitory effect on IL-1β-induced chondrocyte injury caused by circ_SPG11 knockdown. GREM1 was a target of miR-665, and circ_SPG11 knockdown depleted GREM1 expression by enriching miR-665. Overexpression of GREM1 also reversed the inhibitory effect on IL-1β-induced chondrocyte injury caused by miR-665 enrichment. Circ_SPG11 might promote IL-1β-induced chondrocyte apoptosis and ECM degradation via increasing GREM1 expression by decoying miR-665., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Purpose: This work explores the abnormal expression of long noncoding RNAs (lncRNAs), microRNAs (miRNAs) and messenger RNAs (mRNAs) in diabetic corneal epithelial cells (CECs) and constructs an associated competitive endogenous RNA (ceRNA) network. Moreover, we revealed that Rik may exert advantageous effects on diabetic corneal epithelial wound closure by sponging miR-181a-5p., Methods: We obtained the profiles of differentially expressed lncRNAs (DELs) of CECs of type 1 diabetic versus control corneas by microarray and summarized the differentially expressed miRNAs (DEmiRs) and differentially expressed genes (DEGs) data by published literature. Subsequently, the ceRNA network was constructed using bioinformatics analyses. The levels of lncRNA ENSMUST00000153610/3632454L22Rik (Rik) and miR-181a-5p were verified. The localization of Rik was identified with fluorescence in situ hybridization (FISH), and dual-luciferase assays proved the targeted relationship between Rik and miR-181a-5p. Furthermore, we validated the functional impact of Rik in vitro., Results: Overall, 111 upregulated and 117 downregulated DELs were detected in diabetic versus control CECs. The level of Rik located in both the cytoplasm and the nucleus was clearly downregulated, whereas miR-181a-5p was upregulated in vitro and in vivo in the diabetic group versus the control group. Rik can act as a ceRNA to bind to miR-181a-5p, thus promoting diabetic corneal epithelial wound healing in vitro., Conclusions: This work investigated the expression profile of DELs and constructed ceRNA networks of diabetic CECs for the first time. Furthermore, we revealed that Rik may positively impact diabetic corneal epithelial wound healing by sponging miR-181a-5p, providing a novel potential therapeutic target of diabetic keratopathy (DK).

Background: Congenital anomalies are increasingly becoming a global pediatric health concern, which requires immediate attention to its early diagnosis, preventive strategies, and efficient treatments. Guanine nucleotide binding protein, alpha inhibiting activity polypeptide 3 (Gnai3) gene mutation has been demonstrated to cause congenital small jaw deformity, but the functions of Gnai3 in the disease-specific microRNA (miRNA) upregulations and their downstream signaling pathways during osteogenesis have not yet been reported. Our previous studies found that the expression of Mir24-2-5p was significantly downregulated in the serum of young people with overgrowing mandibular, and bioinformatics analysis suggested possible binding sites of Mir24-2-5p in the Gnai3 3'UTR region. Therefore, this study was designed to investigate the mechanism of Mir24-2-5p-mediated regulation of Gnai3 gene expression and explore the possibility of potential treatment strategies for bone defects. Methods: Synthetic miRNA mimics and inhibitors were transduced into osteoblast precursor cells to regulate Mir24-2-5p expression. Dual-luciferase reporter assay was utilized to identify the direct binding of Gnai3 and its regulator Mir24-2-5p. Gnai3 levels in osteoblast precursor cells were downregulated by shRNA (shGnai3). Agomir, Morpholino Oligo (MO), and mRNA were microinjected into zebrafish embryos to control mir24-2-5p and gnai3 expression. Relevant expression levels were determined by the qRT-PCR and Western blotting. CCK-8 assay, flow cytometry, and transwell migration assays were performed to assess cell proliferation, apoptosis, and migration. ALP, ARS and Von Kossa staining were performed to observe osteogenic differentiation. Alcian blue staining and calcein immersions were performed to evaluate the embryonic development and calcification of zebrafish. Results: The expression of Mir24-2-5p was reduced throughout the mineralization process of osteoblast precursor cells. miRNA inhibitors and mimics were transfected into osteoblast precursor cells. Cell proliferation, migration, osteogenic differentiation, and mineralization processes were measured, which showed a reverse correlation with the expression of Mir24-2-5p. Dual-luciferase reporter gene detection assay confirmed the direct interaction between Mir24-2-5p and Gnai3 mRNA. Moreover, in osteoblast precursor cells treated with Mir24-2-5p inhibitor, the expression of Gnai3 gene was increased, suggesting that Mir24-2-5p negatively targeted Gnai3. Silencing of Gnai3 inhibited osteoblast precursor cells proliferation, migration, osteogenic differentiation, and mineralization. Promoting effects of osteoblast precursor cells proliferation, migration, osteogenic differentiation, and mineralization by low expression of Mir24-2-5p was partially rescued upon silencing of Gnai3. In vivo , mir24-2-5p Agomir microinjection into zebrafish embryo resulted in shorter body length, smaller and retruded mandible, decreased cartilage development, and vertebral calcification, which was partially rescued by microinjecting gnai3 mRNA. Notably, quite similar phenotypic outcomes were observed in gnai3 MO embryos, which were also partially rescued by mir24-2-5p MO. Besides, the expression of phospho-JNK (p-JNK) and p-p38 were increased upon Mir24-2-5p inhibitor treatment and decreased upon shGnai3-mediated Gnai3 downregulation in osteoblast precursor cells. Osteogenic differentiation and mineralization abilities of shGnai3-treated osteoblast precursor cells were promoted by p-JNK and p-p38 pathway activators, suggesting that Gnai3 might regulate the differentiation and mineralization processes in osteoblast precursor cells through the MAPK signaling pathway. Conclusions: In this study, we investigated the regulatory mechanism of Mir24-2-5p on Gnai3 expression regulation in osteoblast precursor cells and provided a new idea of improving the prevention and treatment strategies for congenital mandibular defects and mandibular protrusion., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Objective: Angiogenesis is involved in multiple biological processes, including atherosclerosis (AS) and cancer. Dickkopf1 (DKK1) plays many roles in both tumors and AS and has emerged as a potential biomarker of cancer progression and prognosis. Targeting DKK1 is a good choice for oncological treatments. Many anticancer therapies are associated with specific cardiovascular toxicity. However, the effects of DKK1 neutralizing therapy on AS are unclear. We focused on how DKK1 affected angiogenesis in AS and ox-LDL-induced human umbilical vein endothelial cells (HUVECs). Methods: ApoE-/- mice were fed a high-fat diet and then injected with DKK1i or DKK1 lentivirus to study the effects of DKK1. In vitro , promoter assays, protein analysis, database mining, dual-luciferase reporter assay (DLR), electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), and coimmunoprecipitation (co-IP) were used to study the mechanism of DKK1 biogenesis. Cell migration and angiogenesis assays were performed to investigate the function and regulatory mechanisms of DKK1. Results: DKK1 participated in angiogenesis both in the plaques of ApoE-/- mice by knockdown or overexpression of DKK1 and ox-LDL-induced HUVECs. DKK1 induced angiogenesis (increasing migration and capillary formation, inducing expression of VEGFR-2/VEGF-A/MMP) via the CKAP4/PI3K pathway, independent of Wnt/β-catenin. ox-LDL increased the expression and nuclear transfer of Ets-1 and c-jun, and induced the transcriptional activity of DKK1 in HUVECs. Ets-1, along with c-jun and CBP, could bind to the promoter of DKK1 and enhance DKK1 transcription. MiR33a-5p was downregulated in ox-LDL induced HUVECs and aortic artery of high-fat diet ApoE-/- mice. Ets-1 was a direct target of miR33a-5p. MiR33a-5p/Ets-1/ DKK1 axis contributed to angiogenesis. Conclusions: MiR33a-5p/Ets-1/DKK1 signaling participated in ox-LDL-induced angiogenesis of HUVECs via the CKAP4/PI3K pathway. These new findings provide a rationale and notable method for tumor therapy and cardiovascular protection., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Senile cataract is a common age-related disease in ophthalmology. Hsa_circ_0004058 has been reported to be down-regulated in the lens epithelial cells of senile cataract patients, suggesting that hsa_circ_0004058 is associated with senile cataract. However, the underlying mechanism is still unknown. This study attempted to determine the functional role of hsa_circ_0004058 in senile cataract. We treated human lens epithelial cells (SRA01/04) with H 2 O 2 as senile cataract model, and found that cell viability and autophagy of SRA01/04 cells were severely decreased by H 2 O 2 treatment. Hsa_circ_0004058 was notably down-regulated in H 2 O 2 -treated SRA01/04 cells. Moreover, hsa_circ_0004058 overexpression reduced apoptotic cells and the expression of Cleaved-caspase-3 and Bax, and enhanced Bcl-2 expression in H 2 O 2 -treated SRA01/04 cells. However, hsa_circ_0004058 silencing caused the opposite results. Hsa_circ_0004058 up-regulation accelerated the expression of autophagy-related proteins LC3-II/LC3-I and Beclin-1 in H 2 O 2 -treated SRA01/04 cells, which was partly abolished by 3-Methyladenine (autophagy inhibitor). Additionally, hsa_circ_0004058 functioned as a competing endogenous RNA to competitive binding miR-186, and thus accelerated the expression of its down-stream target, ATG7. Hsa_circ_0004058 promoted autophagy of SRA01/04 cells by regulating miR-186/ATG7 axis. In conclusion, these data demonstrates that hsa_circ_0004058 inhibits apoptosis of SRA01/04 cells by promoting autophagy, which attributes to regulate miR-186/ATG7 axis. Thus, hsa_circ_0004058 may be a potential target for senile cataract treatment., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Objective: To study the possible effects of miR-26b-5p on fibroblast-like synovial cells in rheumatoid arthritis (RA-FLS) through targeting enhancer of zeste homolog 2 (EZH2)., Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect miR-26b-5p and EZH2 expressions in synovial tissues of RA patients and healthy controls. Dual luciferase reporter assay was adopted to verify the targeting relationship between miR-26b-5p and EZH2. RA-FLS was divided into Blank, mimics NC, mimics, NC siRNA, EZH2 siRNA and inhibitors + EZH2 siRNA groups, followed by the assessment of proliferation, apoptosis, migration and invasion. The expression of genes and proteins in RA-FLS was tested by qRT-PCR and western blotting, respectively., Results: MiR-26b-5p expression was lower, while EZH2 expression was higher in synovial tissue of RA patients than healthy controls; and miR-26b-5p was negatively correlated with the EZH2 in synovial tissue of RA patients, which were both related with disease activities. MiR-26b-5p can target EZH2 in RA-FLS. In vitro, miR-26b-5p mimics down-regulated EZH2 expression in RA-FLS. Compared with EZH2 siRNA group, the miR-26b-5p expression in inhibitors + EZH2 siRNA group was reduced, but EZH2 expression was increased. EZH2 siRNA inhibited the proliferation, invasion and migration of RA-FLS, promoted cell apoptosis, and inhibited the expression of TNF-α, IL-1β, IL-6, IL-17, MMP-2, MMP-9, which were reversed by miR-26b-5p inhibitor., Conclusion: MiR-26b-5p may affect the biological characteristics of RA-FLS via targeting EZH2, including proliferation, apoptosis, invasion and migration, as well as the secretion of cytokines, thus playing a potential therapeutic role in RA., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

AMPA receptors (AMPARs) are the major excitatory neurotransmitter receptor in the brain, and their expression at synapses is a critical determinant of synaptic transmission and therefore brain function. Synaptic plasticity involves increases or decreases in synaptic strength, caused by changes in the number or subunit-specific subtype of AMPARs expressed at synapses, and resulting in modifications of functional connectivity of neuronal circuits, a process which is thought to underpin learning and the formation or loss of memories. Furthermore, numerous neurological disorders involve dysregulation of excitatory synaptic transmission or aberrant recruitment of plasticity processes. MicroRNAs (miRNAs) repress the translation of target genes by partial complementary base pairing with mRNAs, and are the core component of a mechanism widely used in a range of cell processes for regulating protein translation. MiRNA-dependent translational repression can occur locally in neuronal dendrites, close to synapses, and can also result in relatively rapid changes in protein expression. MiRNAs are therefore well-placed to regulate synaptic plasticity via the local control of AMPAR subunit synthesis, and can also result in synaptic dysfunction in the event of dysregulation in disease. Here, I will review the miRNAs that have been identified as playing a role in physiological or pathological changes in AMPAR subunit expression at synapses, focussing on miRNAs that target mRNAs encoding AMPAR subunits, and on miRNAs that target AMPAR accessory proteins involved in AMPAR trafficking and hence the regulation of AMPAR synaptic localisation. This article is part of the special Issue on 'Glutamate Receptors - AMPA receptors'., (Copyright © 2021. Published by Elsevier Ltd.)

Microglia and macrophages play important roles in ischemic brain injury. Changes in their M1/M2 polarization phenotypes significantly impact disease progression. The M2 microglia/macrophages are anti-inflammatory and have a protective effect against ischemic injury. The microRNA 24 (miR-24) promotes M2 macrophage polarization and suppresses inflammation. We tested the hypothesis that miR-24 is protective in ischemic brain injury by regulating microglia polarization. We treated rats with miR-24 inhibitor or mimic and subsequently subjected the rats to middle cerebral artery occlusion (MCAO) to induce ischemic brain injury. Neurological deficit and infarct volume were analyzed. Microglia and macrophages were assessed by fluorescence-activated cell sorting. Microglia polarization was determined by genes specific for M1 and M2 both in vivo and in BV-2 cells. The effect of miR-24 target Clcn3 on microglia polarization was examined. We found that miR-24 inhibition aggravated MCAO induced damage, while miR-24 overexpression alleviated brain injury by suppressing microglia/macrophage infiltration. miR-24 suppressed M1 and promoted M2 microglia polarization both in vivo and in vitro. Finally, we showed that miR-24 targeted Clcn3 to regulate microglia polarization. Our study indicates that miR-24 plays a neuroprotective role by promoting anti-proinflammatory microglia polarization during ischemic brain injury., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Materials and Methods: LECs were cultured and induced with TGF-β2 (10 ng/mL). SiRNA against MALAT1 (Si-MALAT1) was transfected into LECs to knockdown the expression of MALAT1. To overexpress or knockdown miR-204-5p, miR-204-5p mimics (miR-204-5p mimics) and anti-miR-204-5p (miR-204-5p inhibitor) were transfected into LECs. We used RNA FISH to identify the location of MALAT1. RNA levels of MALAT1 and miR-204-5p were analyzed by RT-qPCR. Additionally, target protein levels of Smad4, epithelial differentiation and mesenchymal markers were analyzed with Western blot. We employed EdU Labeling to measured cell proliferation and performed Transwell Assay to analyze the cell migration. Dual-luciferase reporter assays in LECs were conducted to verify whether miRNA-204-5p was negatively regulated by MALAT1 and Smad4 was a direct target of miR-204-5p., Results: The expression of MALAT1 was upregulated in PCO specimens. MALAT1 was overexpressed in TGF-β2 induced LECs, and the knockdown of MALAT1 could attenuate TGF-β2 induced EMT. Besides, the upregulation of MALAT1 was correlated with the downregulation of miR-204-5p and upregulation of Smad4. Importantly, MALAT1 was revealed to be located in the cytoplasm of LECs. Furthermore, luciferase reporter assays confirmed that MALAT1 could negatively regulate the expression of miR-204-5p and then regulate its direct target Smad4. Finally, the knockdown of MALAT1 could inhibit the EMT, proliferation, and migration of LECs; however, those can be reversed by anti-miR-204-5p., Conclusions: Our findings reveal that MALAT1 may regulate EMT, proliferation, and migration of LECs as a ceRNA by "sponging" miR-204-5p and targeting Smad4, and serve as a promising therapeutic target in preventing PCO.

Myocardial infarction (MI) is a great threat to patients all over the word. MicroRNAs (miRNAs) are a group of non-coding RNAs and can regulate initiation and progression of MI. The current research aimed to investigate the role of miR-467a-5p in MI. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was conducted to detective relative expression of miR-467a-5p in cardiac tissues and mouse cardiomyocytes (MCMs). Hematoxylin and eosin staining was used to reveal the histology of the myocardium. Echocardiography was utilized to reveal cardiac function of mice. Flow cytometer analysis was used to reveal cell apoptosis. Luciferase reporter assay was applied for determining the binding capacity between molecules. We discovered that the level of miR-467a-5p was up-regulated in MI mice and in MCMs induced by H 2 O 2 or hypoxia. Functionally, an elevation of left ventricular end-diastolic diameter and left ventricular end-systolic diameter, as well as a decrease of left ventricular ejection fraction and left ventricular fractional shortening were observed in MI mice. In addition, deficiency of miR-467a-5p improved MI in mice by increasing the contents of lactate dehydrogenase, creatine kinase and malondialdehyde and reducing the activity of superoxide dismutase in serum. Moreover, silencing of miR-467a-5p reversed hypoxia-induced apoptosis of MCMs. Mechanistically, zinc finger E-box binding homeobox 1 (ZEB1) was confirmed as the target of miR-467a-5p. Moreover, miR-467a-5p negatively regulated ZEB1 level in MI mice and MCMs. Finally, the promotive effect of miR-467a-5p inhibition on cell apoptosis was reversed by knockdown of ZEB1. All the experimental results demonstrate that miR-467a-5p aggravates MI by modulating ZEB1 expression in mice, which may provide a novel therapeutic strategy for MI., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Objective: This study aimed to determine the effects of emodin on the viability, proliferation and apoptosis of human pulmonary artery smooth muscle cells (PASMCs) under hypoxia and to explore the underling molecular mechanisms., Methods: PASMCs were cultured in a hypoxic environment (1% oxygen) and then treated with emodin. Cell viability, proliferation and apoptosis were evaluated using CCK-8 assay, EdU staining assay, western blot and Mito-tracker red CMXRos and Annexin V-FITC apoptosis detection assay. The microRNA (miRNA)/mRNA and protein expression levels were assessed by quantitative real-time PCR and western blotting, respectively. Based on transcriptomics and proteomics were used to identify potential signaling pathways. Luciferase reporter assay was utilized to examine the interaction between miR-244-5p and DEGS1., Results: Emodin at 40 and 160 µM concentration-dependently suppressed cell viability, proliferation and migration, but enhanced cell apoptosis of PASMCs under hypoxia. Transcriptomic and proteomic analysis revealed that emodin could attenuate the activity of PI3K/Akt signaling in PASMCs under hypoxia. In addition, delta 4-desaturase, sphingolipid 1 (DEGS1) was found to be a direct target of miR-244-5p. Emodin could significantly up-regulated miR-244-5p expression and down-regulated DEGS1 expression in PASMCs under hypoxia. Furthermore, emodin-mediated effects on cell viability, migration, apoptosis and PI3K/Akt signaling activity of PASMCs under hypoxia were significantly attenuated by miR-244-5p knockdown., Conclusions: Our results indicated that emodin suppressed cell viability, proliferation and migration, promoted cell apoptosis of PASMCs under hypoxia via modulating miR-244-5p-mediated DEGS1/PI3K/Akt signaling pathway. MiR-244-5p/DEGS1 axis was initially investigated in this current study, which is expected to further the understanding of the etiology of pulmonary arterial hypertension., (© 2021. The Author(s).)

microRNAs (miRNAs) are ~21-22 nucleotide (nt) RNAs that mediate broad post-transcriptional regulatory networks. However, genetic analyses have shown that the phenotypic consequences of deleting individual miRNAs are generally far less overt compared to their misexpression. This suggests that miRNA deregulation may have broader phenotypic impacts during disease situations. We explored this concept in the Drosophila eye, by screening for miRNAs whose misexpression could modify the activity of pro-apoptotic factors. Via unbiased and comprehensive in vivo phenotypic assays, we identify an unexpectedly large set of miRNA hits that can suppress the action of pro-apoptotic genes hid and grim. We utilize secondary assays to validate that a subset of these miRNAs can inhibit irradiation-induced cell death. Since cancer cells might seek to evade apoptosis pathways, we modeled this situation by asking whether activation of anti-apoptotic miRNAs could serve as "second hits". Indeed, while clones of the lethal giant larvae (lgl) tumor suppressor are normally eliminated during larval development, we find that diverse anti-apoptotic miRNAs mediate the survival of lgl mutant clones in third instar larvae. Notably, while certain anti-apoptotic miRNAs can target apoptotic factors, most of our screen hits lack obvious targets in the core apoptosis machinery. These data highlight how a genetic approach can reveal distinct and powerful activities of miRNAs in vivo, including unexpected functional synergies during disease or cancer-relevant settings., (Copyright © 2021 Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) are recognized as an essential component of the RNA family, exerting multiple and intricate biological functions, particularly in the process of tumorigenesis, proliferation, and metastatic progression. MiRNAs are altered in gastric cancer (GC), showing activity as both tumor suppressors and oncogenes, although their true roles have not been fully understood. This review will focus upon the recent advances of miRNA studies related to the regulatory mechanisms of gastric tumor cell proliferation, apoptosis, and cell cycle. We hope to provide an in-depth insight into the mechanistic role of miRNAs in GC development and progression. In particular, we summarize the latest studies relevant to miRNAs' impact upon the epithelial-mesenchymal transition, tumor microenvironment, and chemoresistance in GC cells. We expect to elucidate the molecular mechanisms involving miRNAs for better understanding the etiology of GC, and facilitating the development of new treatment regimens for the treatment of GC.

Background : Glaucoma is an optic neuropathic disease and contributed to the irreversible blindness caused by the slow death of retinal ganglion cells (RGCs). Long non-coding RNA (lncRNA) metastasis associated lung adenocarcinoma transcript 1 (MALAT1) was reported to be aberrantly expressed in diverse diseases, including glaucoma. However, the mechanism of MALAT1 in glaucoma was still undefined. Methods : The levels of MALAT1, microRNA-149-5p (miR-149-5p) in RGCs cultured under elevated pressure were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The putative target of MALAT1 was predicted by starBase v2.0 online database, and dual luciferase reporter assay, RNA immunoprecipitation (RIP) assay and RNA pull-down assay were performed to verify this interaction. The cell viability of RGCs was measured by Cell Counting Kit-8 (CCK-8) assay. The apoptotic rate was evaluated via flow cytometry. The protein levels of apoptosis-related proteins (Bax, B-cell lymphoma 2 (Bcl-2)) and Cleaved caspase 3 were assessed by Western blot. Results : The level of MALAT1 was significantly down-regulated, and the level of miR-149-5p was distinctly up-regulated in RGCs under pressure in a dose-dependent manner. Functionally, MALAT1 overexpression or miR-149-5p inhibitor alleviated the inhibitory effect on cell viability and the promoted effect on apoptotic rate of RGCs in EIOP. The interaction between MALAT1 and miR-149-5p was predicted by starBase v2.0 online database, and dual luciferase reporter assay, RIP assay and RNA pull-down assay validated the interaction. Combined with the loss and gain experiment results, miR-149-5p was negatively interacted with MALAT1. Furthermore, miR-149-5p mimics mitigated the promoted impact on cell viability and the suppressive impact on apoptotic rate by targeting MALAT1. Conclusion : MALAT1 promoted cell proliferation and inhibited cell apoptosis of RGCs via targeting miR-149-5p in glaucoma in vitro , which might shed light on the mechanism of glaucoma pathogenesis.

Yanbian yellow cattle breeding is limited by slow growth. We previously found that the miRNA miR-93 was differentially expressed between the blood exosomes of Yanbian yellow cattle and Han Yan cattle, which differ in growth characteristics. In this experiment, we evaluated the effects of miR-93 on growth hormone (GH) secretion by pituitary cells of Yanbian yellow cattle using qPCR, Western blot, Targetscan and RNA hybrid analysis software and Dual-Luciferase reporter gene system. The results showed that miR-93 targeted 3' UTR of GHRHR(growth hormone releasing hormone receptor); GH mRNA and protein levels in pituitary cells of Yanbian yellow cattle were significantly lower in the miR-93-mi group than in the NC control group ( p  < 0.01), while GH mRNA and protein levels were higher in the miR-93-in group than in the iNC control group, but the difference was not significant ( p  > 0.05); GHRHR mRNA and protein levels were significantly lower in the miR-93-mi group than in the NC control group ( p  < 0.01), while GHRHR protein levels were significantly higher in the miR-93-in group than in the iNC control group ( p  < 0.05), but there was no significant difference about GHRHR mRNA level between two groups ( p  > 0.05). These results prove that miR-93 regulates GH secretion in pituitary cells via GHRHR.

Background: Posterior capsular opacification (PCO) is the major vision-disrupting complication arising after cataract surgery. Circular RNAs (circRNAs) are biological active RNAs which were involved in various physiological functions. So far, the role of circRNA caspase recruitment domain family member 6 (circ-CARD6) in PCO is still unclear., Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to detect the expression of circ-CARD6, microRNA 31 (miR-31) and fibroblast growth factor 7 (FGF7) message RNA (mRNA). Western blot was used to analyze the protein expression. Transmission electron microscopy (TEM) was employed to capture the exosome image. The proliferation and metastasis were analyzed by cell counting kit-8 (CCK8), transwell and wound healing assays. The potential binding sequences between miR-31 and circ-CARD6 or FGF7 were respectively predicted by Circinteractome and Targetscan online tool, and verified by dual-luciferase reporter and RNA binding protein immunoprecipitation (RIP) assays., Results: Exosome-transmitted circ-CARD6 was highly expressed in PCO tissues and TGF-β2-treated SRA01/04 cells. Circ-CARD6 deletion repressed the proliferation, metastasis, EMT process and MAPK pathway, which was reversed by anti-miR-31 in TGF-β2-treated SRA01/04 cells. Meanwhile, circ-CARD6 sponged miR-31 which directly targeted FGF7 in TGF-β2-treated SRA01/04 cells. FGF7 overexpression allayed miR-31 overexpression-induced suppression in proliferation, metastasis, EMT process and MAPK pathway. Besides, circ-CARD6 regulated FGF7 expression by sponging miR-31., Conclusion: Circ-CARD6 promoted PCO development via miR-31/FGF7 axis. This finding might contribute to the development of the targeted therapy for PCO., (Copyright © 2021. Published by Elsevier Ltd.)

Diabetic retinopathy (DR), characterized by intraretinal vessel formation, is a major complication in diabetes. Neovascularization is an important characteristic of DR, but its formation mechanism remains unclear. In this research, Malat1, miR-205-5p, and VEGF-A levels in high glucose (HG) treat-human retinal microvascular endothelial cells (hRMECs) was detected with qRT-PCR. CCK-8 assay, transwell assay, and tube formation assay was applied to access hRMEC viability, migration, and angiogenesis. Expression level of endothelial-mesenchymal transition (EndMT) markers (VE-cadherin, FSP1, and α-SMA) was detected by western blotting assay. Interaction among Malat1, miR-205-5p, and VEGF-A was confirmed by dual-luciferase reporter assay. Furthermore, in vivo DR mouse model was induced, and the effect of Malat1 on DR and EndMT markers was confirmed through hematoxylin-eosin (HE) staining and western blotting. As a result, Malat1 and VEGF-A was upregulated while miR-205-5p was suppressed under HG conditions. Malat1 could sponge miR-205-5p to regulate VEGF-A expression. Malat1 knockdown inhibited hRMEC proliferation, migration, and tube formation by targeting miR-205-5p under HG conditions. Furthermore, inhibition of Malat1 prevented the HG-induced EndMT process. In summary, Malat1 knockdown diminished hRMEC dysfunctions by regulating miR-205-5p/VEGF-A, providing a useful insight for exploring new therapeutic target for DR., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Coupling between osteogenesis and angiogenesis determines bone morphology. A decrease in the osteogenic ability of bone marrow mesenchymal stem cells (BMSCs) is one of the underlying causes of senile osteoporosis (OP). Here, we investigated the involvement of circular RNAs (circRNAs) in the osteogenic differentiation of BMSCs and the pathogenesis of senile OP. We sequenced RNA and found decreases expression of hsa&#95;circ&#95;0006215 in BMSCs from patients with OP. We further assessed the role of hsa&#95;circ&#95;0006215 in the osteogenic differentiation of BMSCs using lentivirus-mediated hsa&#95;circ&#95;0006215 overexpression and knockdown. Overexpression of hsa&#95;circ&#95;0006215 promoted the osteogenic differentiation of BMSCs. Luciferase reporter and RNA pull-down assays revealed that hsa&#95;circ&#95;0006215 bound to miRNA-942-5p and thus regulated RUNX2 and vascular endothelial growth factor (VEGF) expression in BMSCs. We assessed osteogenesis and vascular coupling in co-cultured cells, and the role of hsa&#95;circ&#95;0006215 in bone formation in vivo using a cortical bone defect model. We found that hsa&#95;circ&#95;0006215 promoted bone defect repair. Overall, our results showed that hsa&#95;circ&#95;0006215 has an important function in osteogenesis and could be a novel target for treating senile OP.

This study finds significant differences in hepatic fatty acid composition between four groups of Atlantic salmon (Salmo salar) consisting of offspring from families selected for high and low capacities to express the delta 6 desaturase isomer b and fed diets with 10% or 75% fish oil. The results demonstrated that hepatic lipid metabolism was affected by experimental conditions (diet/family). The fatty acid composition in the four groups mirrored the differences in dietary composition, but it was also associated with the family groups. Small RNA sequencing followed by RT-qPCR identified 12 differentially expressed microRNAs (DE miRNAs), with expression associated with family groups (miR-146 family members, miR-200b, miR-214, miR-221, miR-125, miR-135, miR-137, miR&#95;nov&#95;1), diets (miR-203, miR-462) or both conditions. All the conserved DE miRNAs have been reported as associated with lipid metabolism in other vertebrates. In silico predictions revealed 37 lipid metabolism pathway genes, including desaturases, transcription factors and key enzymes in the synthesis pathways as putative targets (e.g., srebp-1 and 2, Δ6fad&#95;b and c, hmdh, elovl4 and 5b, cdc42). RT-qPCR analysis of selected target genes showed expression changes that were associated with diet and with family groups (d5fad, d6fad&#95;a, srebp-1). There was a reciprocal difference in the abundance of ssa-miR-203a-3p and srebp-1 in one group comparison, whereas other predicted targets did not reveal any evidence of being negatively regulated by degradation. More experimental studies are needed to validate and fully understand the predicted interactions and how the DE miRNAs may participate in the regulation of hepatic lipid metabolism., (© 2020 The Authors. Journal of Fish Biology published by John Wiley & Sons Ltd on behalf of The Fisheries Society of the British Isles.)

MicroRNAs (miRNAs) are a class of conserved non-coding RNAs that are widely regarded as important regulators in a variety of biological processes. Increasing evidence has revealed that skeletal muscle fiber-type conversion is regulated by miRNAs, but the molecular mechanism is still not fully understood. In this study, we confirmed the role of miR-22-3p on skeletal muscle fiber-type conversion and investigated its potential mechanism in C2C12 myotubes. Here, we found that the miR-22-3p mimics inhibited the expressions of myosin heavy chain I (MyHC I), MyHC IIa and promoted the expression of MyHC IIb, while miR-22-3p inhibitor got inverse results. miR-22-3p mimics also downregulated phosphorylated AMPK, SIRT1 and PGC-1ɑ protein levels, which control the expression of oxidative fiber-related genes. Furthermore, Compound C (AMPK inhibitor) eliminated the effect of miR-22-3p inhibitor on MyHC I, MyHC IIa and MyHC IIb expressions. However, AICAR (AMPK activator) also abolished the effect of miR-22-3p mimics on MyHC I, MyHC IIa and MyHC IIb expressions. Collectively, our results suggest that miR-22-3p regulates skeletal muscle fiber-type conversion through inhibiting AMPK/SIRT1/PGC-1ɑ signaling pathway.

The pathogenesis of congenital cataract (CC), a major disease associated with blindness in infants, is complex and diverse. Aquaporin 5 (AQP5) represents an essential membrane water channel. In the present study, whole exome sequencing revealed a novel heterozygous missense mutation of AQP5 (c.152 T > C, p. L51P) in the four generations of the autosomal dominant CC (adCC) family. By constructing a mouse model of AQP5 knockout (KO) using the CRISPR/Cas9 technology, we observed that the lens of AQP5-KO mice showed mild opacity at approximately six months of age. miR-124-3p.1 expression was identified to be downregulated in the lens of AQP5-KO mice as evidenced by qRT-PCR analysis. A dual luciferase reporter assay confirmed that vimentin was a target gene of miR-124-3p.1. Organ-cultured AQP5-KO mouse lenses were showed increased opacity compared to those of WT mice, and vimentin expression was upregulated as determined by RT-PCR, western blotting, and immunofluorescence staining. After miR-124-3p.1 agomir was added, the lens opacity in WT mice and AQP5-KO mice decreased, accompanied by the downregulation of vimentin. AQP5-L51P increased vimentin expression of in human lens epithelial cells. Therefore, a missense mutation in AQP5 (c.152 T > C, p. L51P) was associated with adCC, and AQP5 could participate in the maintenance of lens transparency by regulating vimentin expression via miR-124-3p.1., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

MicroRNAs(miRNAs) regulate and control gene expression at the post-transcriptional level by base pairing with its target gene 3'UTR, resulting in degradation of the target mRNA or inhibition of its translation. The previous high-throughput sequencing results indicated that miR-27a was involved in coat color regulation. However, the mechanism of action is not still illuminated. In this research, using western blotting and real-time quantitative polymerase chain reaction(qRT-PCR), the expression of miR-27a, WNT3A and KITLG were examined in the skin of Cashmere goats with white and brown, and human embryonic kidney 293 T cells (HEK-293T cells) which over-express miR-27a. Targeting relationship between miR-27a and WNT3A or KITLG was examined by the luciferase reporter gene system. The qRT-PCR detection showed that miR-27a was more highly expressed in white Cashmere goats skin than that in brown Cashmere goats skin. Furthermore, WNT3A and KITLG mRNA and protein expression were down-regulated by miR-27a in vitro and in vivo . A dual-luciferase reporter gene indicated that miR-27a negatively correlates with WNT3A or KITLG. In a word, our research demonstrated that the expression of miR-27a was evidently differential in the white and brown Cashmere goats skin and WNT3A or KITLG was negatively regulated by miR-27a.

A fraction of the transcriptome is translated into proteins. The rest is classified as non-protein coding RNA (Ribonucleic Acid) but has gained increased attention as functional and regulatory group of transcripts. The gene regulatory role of non-coding RNAs (ncRNAs) has now been widely accepted in diverse biological processes in both physiology and disease. MicroRNAs fall into this latter group and are widely known for their diverse post-transcriptional regulatory role. MicroRNA sequences are embedded in the long ncRNAs, known as primary microRNAs, are processed into precursor microRNAs and are typically transported out of the nucleus for maturation and loading into a protein complex forming RNA-induced silencing complex (RISC) that either drives the degradation of messenger RNA (mRNA) or blocks its translation. A new phenomenon is emerging where microRNAs have active roles within the nucleus. The presence of RISC components including microRNAs in the nucleus supports this notion. They may integrate with chromatin modifiers, microprocessing machinery and mRNA stabilizing transcripts to play a multifunctional role in the nucleus. Although a limited number of studies appreciate this novel activity of microRNAs relevant to the cardiovascular system, they provide proof-of-concept that requires consideration while targeting miRNAs with therapeutic potential., (Copyright © 2020 Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) are small, non-coding RNAs that post-transcriptionally regulate gene expression by binding to specific mRNA targets and promoting their degradation and/or translational inhibition. miRNAs regulate both physiological and pathological liver functions. Altered expression of miRNAs is associated with liver metabolism dysregulation, liver injury, liver fibrosis and tumour development, making miRNAs attractive therapeutic strategies for the diagnosis and treatment of liver diseases. Here, we review recent advances regarding the regulation and function of miRNAs in liver diseases with a major focus on miRNAs that are specifically expressed or enriched in hepatocytes (miR-122, miR-194/192), neutrophils (miR-223), hepatic stellate cells (miR-29), immune cells (miR-155) and in circulation (miR-21). The functions and target genes of these miRNAs are emphasised in alcohol-associated liver disease, non-alcoholic fatty liver disease, drug-induced liver injury, viral hepatitis and hepatocellular carcinoma, as well liver fibrosis and liver failure. We touch on the roles of miRNAs in intercellular communication between hepatocytes and other types of cells via extracellular vesicles in the pathogenesis of liver diseases. We provide perspective on the application of miRNAs as biomarkers for early diagnosis, prognosis and assessment of liver diseases and discuss the challenges in miRNA-based therapy for liver diseases. Further investigation of miRNAs in the liver will help us better understand the pathogeneses of liver diseases and may identify biomarkers and therapeutic targets for liver diseases in the future., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

The study aims to investigate the effects of long noncoding RNA (lncRNA) transmitted nuclear factor-κB interacting lncRNA (NKILA)-containing astrocyte-derived small extracellular vesicles (EVs) on traumatic brain injury (TBI). TBI was modeled in vitro by exposing human neurons to mechanical injury and in vivo by controlled cortical impact in a mouse model. The gain- and loss-function approaches were conducted in injured neurons to explore the role of NKILA, microRNA-195 (miR-195) and nucleotide-binding leucine-rich repeat containing family member X1 (NLRX1) in neuronal injury. EVs extracted from NKILA-overexpressing astrocytes were used to treat injured neurons. It was revealed that NKILA was downregulated in injured neurons. Astrocyte co-culture participated in the upregulation of NKILA in injured neurons. Additionally, NKILA could competitively bind to miR-195 that directly targeted NLRX1. Next, the upregulation of NLRX1 or NKILA relived neuronal injury by promoting neuronal proliferation but inhibiting apoptosis. Astrocyte-derived EVs transferred NKILA into neurons, which led to the downregulation of miR-195, upregulation of NLRX1, increased cell proliferation, and decreased cell apoptosis. The in vivo experiments validated that NKILA-containing EVs promoted brain recovery following TBI. Collectively, astrocyte-derived EVs carrying NKILA was found to alleviate neuronal injury in TBI by competitively binding to miR-195 and upregulating NLRX1.

Purpose: Apoptosis of the retinal ganglion cells (RGCs) can cause irreversible damage to visual function after retinal ischemia reperfusion injury (RIR). Using a lncRNA chip assay, we selected lncRNA Ttc-209 and characterized its role in RGCs during ischemia reperfusion (I/R)-induced apoptosis., Methods: We created an ischemic model of RGCs by applying Hank's balanced salt solution containing 10 µM antimycin A and 2 µM calcium ionophore for 2 hours. RIR was induced in mice by elevating the intraocular pressure to 120 mm Hg for 1 hour by cannulation of the cornea; this was followed by reperfusion. Real-time quantitative PCR was used to detect the expression levels of long noncoding RNA (lncRNA), microRNA (miRNA), and target gene mRNA. Western blotting, flow cytometry, immunofluorescent staining, and TUNEL assays were performed to detect cell apoptosis. Dual-luciferase reporter assays and FISH were used to identify endogenous competitive RNA (ceRNA) mechanisms that link lncRNAs, miRNAs, and target genes. We also used scotopic electroretinography examinations to evaluate visual function in treated mice., Results: lncRNA Ttc3-209 was significantly upregulated after I/R injury and played a proapoptotic role in RGCs during I/R-induced apoptosis. Mechanistically, lncRNA Ttc3-209 is a ceRNA that competitively binds to miR-484 and upregulates the translation of its target (Wnt8a mRNA), thus promoting apoptosis in RGCs., Conclusions: Reducing the expression of lncRNA Ttc3-209 had a protective effect against apoptosis in RGCs. This may provide a new therapeutic option for the prevention of RGC apoptosis in response to RIR injury.

Vitreous alterations occur from early stages and continue through the normal aging, with gradual lamellae formation and the appearance of liquefied spaces, which eventually leads to complications, such as retinal tear, retinal detachment, and intravitreal hemorrhage. The aim of the present study was to investigate the expression of let-7 miRNA family in the vitreous and retina in newborn (1-3- day-old), young adult (2-month-old), and aging (12-month-old) rats, as well as their role as regulators of vitreous components. MicroRNAs are small, non-coding RNAs that post-transcriptionally regulate gene expression. Our results showed detection of all investigated let-7 isoforms (let-7a, let-7b, let-7c, let-7d, let-7e, let-7f and let-7i) in the retina and vitreous. Although most let-7 members were significantly upregulated in the vitreous during development, only let-7b, let-7c, and let-7e followed this same expression pattern in the retina. Let-7b and -7c increased in aging vitreous as well, and were expressed in vitro by Müller glial cells and their extracellular vesicles. Moreover, let-7 targeted hyaluronan synthase 2 (Has2) mRNA, a synthesizing enzyme of hyaluronan. These observations indicate that let-7 function is important during retina and vitreous development, and that isoforms of let-7 increased with aging, potentially modulating hyaluronan content., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Objectives: Parkinson's disease (PD) is a neurodegenerative problem correlated with neuronal damage. Long noncoding RNAs (lncRNAs) are implicated in neuronal damage in PD development. This research aims to analyze the function and mechanism of LINC00943 in 1-methyl-4-phenylpyridinium (MPP + )-caused neuronal injury., Methods: MPP + -challenged SK-N-SH cells served as a PD-like model of neuronal damage. LINC00943, microRNA-15b-5p (miR-15b-5p) and RAB3A interacting protein (RAB3IP) abundances were examined via quantitative reverse transcription polymerase chain reaction or western blot. MPP + -caused neuronal damage was assessed via cell viability, apoptosis, inflammatory injury and oxidative injury. The association between miR-15b-5p and LINC00943 or RAB3IP was determined via dual-luciferase reporter analysis and RNA immunoprecipitation., Results: LINC00943 abundance was up-regulated in MPP + -challenged SK-N-SH cells. LINC00943 silence alleviated MPP + -caused decrease of cell viability and elevation of apoptosis, inflammatory injury and oxidative injury. miR-15b-5p was inhibited via LINC00943, and miR-15b-5p inhibition reversed knockdown of LINC00943-mediated suppression of MPP + -induced neuronal damage. RAB3IP was targeted via miR-15b-5p, and LINC00943 could regulate RAB3IP via miR-15b-5p. miR-15b-5p addition mitigated MPP + -induced neuronal damage through decreasing RAB3IP., Conclusion: LINC00943 inhibition alleviated MPP + -induced neuronal injury via miR-15b-5p/RAB3IP axis, indicating a potential target for treatment of PD.

Purpose: Ciliary epithelium (CE) of adult mammalian eyes contains quiescent retinal progenitor/stem cells that generate neurospheres in vitro and differentiate into retinal neurons. This ability doesn't evolve efficiently probably because of regulatory mechanisms, such as microRNAs (miRNAs) that control pluripotent, progenitor, and differentiation genes. Here we investigate the presence of Let-7 miRNAs and its regulator and target, Lin28 and Hmga2, in CE cells from neurospheres, newborns, and adult tissues., Methods: Newborn and adult rats CE cells were dissected into pigmented and nonpigmented epithelium (PE and NPE). Newborn PE cells were cultured with growth factors to form neurospheres and we analyzed Let-7, Lin28a, and Hmga2 expression. During the neurospheres formation, we added chemically modified single-stranded oligonucleotides designed to bind and inhibit or mimic endogenous mature Let-7b and Let-7c. After seven days in culture, we analyzed neurospheres size, number and expression of Let-7, Lin28, and Hmga2., Results: Let-7 miRNAs were expressed at low rates in newborn CE cells with significant increase in adult tissues, with higher levels on NPE cells, that does not present the stem cells reprogramming ability. The Lin28a and Hmga2 protein and transcripts were more expressed in newborns than adults cells, opposed to Let-7. Neurospheres presented higher Lin28 and Hmga2 expression than newborn and adult, but similar Let-7 than newborns. Let-7b inhibitor upregulated Hmga2 expression, whereas Let-7c mimics upregulated Lin28 and downregulated Hmga2., Conclusions: This study shows the dynamic of Lin28-Let-7-Hmga regulatory axis in CE cells. These components may develop different roles during neurospheres formation and postnatal CE cells.

Ischemia/reperfusion injury (IRI) initiates from oxidative stress caused by lack of blood supply and subsequent reperfusion. It is often associated with sterile inflammation, cell death and microvascular dysfunction, which ultimately results in myocardial, cerebral and hepatic IRIs. Reportedly, deregulation of Nrf2 pathway plays a significant role in the oxidative stress-induced IRIs. Further, microRNAs (miRNAs/miRs) are proved to regulate the expression and activation of Nrf2 by targeting either the 3'-UTR or the upstream regulators of Nrf2. Additionally, compounds (crocin, ZnSO 4 and ginsenoside Rg1) that modulate the levels of the Nrf2-regulating miRNAs were found to exhibit a protective effect against IRIs of different organs. Therefore, the current review briefs the impact of ischemia reperfusion (I/R) pathogenesis in various organs, role of miRNAs in the regulation of Nrf2 and the I/R protective effect of compounds that alter their expression., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Sperm-mediated gene transfer (SMGT) has a potential application in the generation of transgenic animals. Capillary electroporation consists of the application of electrical pulses, resulting in an increased transfection rate. Little is known about the impacts of the transfection of exogenous DNA on sperm epigenetics. MicroRNAs are epigenetic factors that are related to sperm motility. MiRNA-122-5p regulates genes that influence motility, and consequently, the fertilizing potential of sperm. Therefore, we aimed at identifying whether epigenetic factors such as microRNAs could be altered after DNA transfection, using the capillary electroporation technique. In this study, bull sperm was electroporated using voltages of 600 V, 1500 V, and 0 V (control group), with or without exogenous DNA. Parameters of sperm quality were analyzed using CASA and flow cytometry, and expression of the miRNA-122-5p was analyzed using RT-qPCR. It was observed that electroporation increased the internalization of exogenous DNA (P < 0.05), but did not impair the mitochondrial activity (P > 0.05). It reduced sperm motility (P < 0.05). The expression of miRNA-122-5p was upregulated in sperm electroporated at 1500 V, and the presence of exogenous DNA did not affect its expression. Thus, we can conclude that electroporation influences the expression of miRNA-122-5p from bull sperm cells., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor-in-Chief. Concern was raised about the reliability of the Western blot results in Figs. 1C and 4B+J, which appear to have the same eyebrow shaped phenotype as many other publications tabulated here (https://docs.google.com/spreadsheets/d/149EjFXVxpwkBXYJOnOHb6RhAqT4a2llhj9LM60MBffM/edit#gid=0). The journal requested the corresponding author comment on these concerns and provide the raw data. However, the authors were not responsive to the request for comment. Since original data could not be provided, the overall validity of the results could not be confirmed. Therefore, the Editor-in-Chief decided to retract the article., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Purpose: Thyroid eye disease (TED) is a condition that causes the tissue behind the eye to become inflamed and can result in excessive fatty tissue accumulation in the orbit. Two subpopulations of fibroblasts reside in the orbit: those that highly express Thy1 (Thy1+) and those with little or no Thy1 (Thy1-). Thy1- orbital fibroblasts (OFs) are more prone to lipid accumulation than Thy1+ OFs. The purpose of this study was to investigate the mechanisms whereby Thy1- OFs more readily accumulate lipid., Methods: We screened Thy1+ and Thy1- OFs for differences in microRNA (miRNA) expression. The effects of increasing miR-130a levels in OFs was investigated by measuring lipid accumulation and visualizing lipid deposits. To determine if adenosine monophosphate-activated protein kinase (AMPK) is important for lipid accumulation, we performed small interfering RNA (siRNA)-mediated knockdown of AMPKβ1. We measured AMPK expression and activity using immunoblotting for AMPK and AMPK target proteins., Results: We determined that miR-130a was upregulated in Thy1- OFs and that miR-130a targets two subunits of AMPK. Increasing miR-130a levels enhanced lipid accumulation and reduced expression of AMPKα and AMPKβ in OFs. Depletion of AMPK also increased lipid accumulation. Activation of AMPK using AICAR attenuated lipid accumulation and increased phosphorylation of acetyl-CoA carboxylase (ACC) in OFs., Conclusions: These data suggest that when Thy1- OFs accumulate in TED, miR-130a levels increase, leading to a decrease in AMPK activity. Decreased AMPK activity promotes lipid accumulation in TED OFs, leading to excessive fatty tissue accumulation in the orbit.

Slow transit constipation (STC) is a gastrointestinal disorder characterized by abnormal prolonged colonic transit time, which affects the life quality of many people. The decrease number of interstitial cells of Cajal (ICCs) is involved in the pathogenesis of STC. However, the molecular mechanism of loss of ICCs in STC remains unclear, making it difficult to develop new agents for the disease. In this study, we investigated the mechanism of decreasing ICCs in the pathogenesis of STC. We constructed the STC model rats by using atropine and diphenoxylate. A series of methods were used including immunofluorescence and immunochemistry staining, western blot, qRT-PCR, exosomes extraction and exosomes labeling. The results indicate that ICCs decreased in the STC rats accompanied with the macrophages activation. Further studies suggested that macrophages decreased the cell viability of ICCs by secretion exosomes containing miR-34c-5p. miR-34c5p targeted the 3Ꞌ -UTR of stem cell factor(SCF) mRNA and regulated the expression of SCF negatively. In conclusion, we demonstrated a novel regulatory mechanism of ICCs cell viability in STC. We found that exosome miR-34c-5p mediate macrophage-ICCs cross-talk. M1 macrophages derived exosomes miR-34c-5p decreased ICCs cell viability by directly targeting SCF.

Objective: Parkinson's disease (PD) is a frequent degenerative disease of the nervous system with undefined pathogenesis. This study explored the protective effect of microRNA (miR)-218-5p on dopaminergic neuron injury in substantia nigra (SN) of rats with PD through the regulation of LIM and SH3 domain protein 1 (LASP1)., Methods: The PD rat model was established by fixed point injection of 6-hydroxydopamine into the rats. The PD rats were injected with miR-218-5p overexpressed recombinant adeno-associated virus (rAAV) or LASP1 silenced rAAV to explore their roles in dopaminergic neurons in SN of rats with PD. The changes in pathological structure of SN were observed and the expression of tyrosine hydroxylase (TH) and deacetylvindoline acetyltransferase (DAT), the dopaminergic neuron apoptosis and oxidative stress factor in the SN were detected. The expression of miR-218-5p, LASP1, Bcl-2 and Bax in SN was detected. The targeting relationship between miR-218-5p and LASP1 was confirmed., Results: Declined miR-218-5p and overexpressed LASP1 existed in the brain SN of PD rats. Up-regulated miR-218-5p or inhibited LASP1 improved the pathological damage of dopaminergic neurons and increased the number of TH and DAT positive cells in brain SN of PD rats. Furthermore, elevated miR-218-5p or depressed LASP1 inhibited the apoptosis, and oxidative stress of dopaminergic neurons in brain SN of PD rats. In addition, increased miR-218-5p repressed the expression of LASP1 in the brain SN of PD rats. LASP1 was proven to be a direct target of miR-218-5p., Conclusion: The study highlights that up-regulated miR-218-5p could improve the damage of dopaminergic neurons in PD rats, which was related to the inhibition of LASP1., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Ischemic stroke is an urgent public health concern and one of the major causes of deaths and disabilities over the world. MicroRNA (miRNA) has become a key mediator of cerebral ischemia-reperfusion (I/R) injuries. However, whether miR-190 is involved in cerebral I/R-induced neuronal damage remains unknown. This study was to investigate the role of miR-190 in the brain I/R injury. We divided the rats into sham, I/R, control, and miR-190-mim (miR-190 mimics) groups. Quantitative real-time polymerase chain reaction (qRT-PCR), Nissl staining, flow cytometry, and western blot were conducted to examine the expression of miR-190 and cell apoptosis in different groups. The results showed that the expression of miR-190 was greatly decreased in rats suffering with I/R. Overexpression of miR-190 significantly reduced the increased neurological scores, brain water contents, infarct volumes, and neuronal apoptosis in rats suffering with I/R. In addition, we found that the expression of RhoA and Rho kinase was greatly elevated in rats suffering with I/R. Bioinformatics analysis indicated that Rho was a target of miR-190. Moreover, overexpression of miR-190 significantly downregulated the increased mRNA and protein expression of Rho/Rho kinase and cell apoptosis, while inhibition of miR-190 further upregulated the increased mRNA and protein expression of Rho/Rho kinase and cell apoptosis in rats suffering with I/R. Furthermore, knockdown of Rho significantly downregulated the increased mRNA and protein expression of Rho/Rho kinase and cell apoptosis, while these effects were inhibited by miR-190 inhibitors in rats suffering with I/R. These results indicate that miR-190 confers protection against brain I/R damage by modulating Rho/Rho-kinase signaling.

Diabetic retinopathy (DR) is the leading cause of visual impairment and acquired blindness among adults worldwide. Retinal microvascular pericyte deficiency is one of the earliest pathological changes associated with DR, and long noncoding RNA myocardial infarction-associated transcript (MIAT) has been implicated as a crucial regulator of microvascular dysfunction in DR. Pyroptosis is a caspase-1-dependent proinflammatory form of cell death, and in the present study, we investigated the potential pyroptosis of primary human retinal pericytes (HRPCs) and the mechanism by which MIAT is involved in this process. We applied advanced glycation end product modified bovine serum albumin (AGE-BSA) to simulate the DR environment. The results suggested that AGE-BSA induced the active cleavage of caspase-1 and gasdermin D, the release of IL-1β, IL-18 and LDH, and reduced cell viability, which was prevented by the inhibition of caspase-1, indicating the occurrence of caspase-1-mediated pyroptosis in HRPCs. Immunofluorescence images revealed the phenotypic characteristics of pyroptosis, including pyknosis, swelling and hyperpermeability in plasmolemma. MIAT and CASP1 expression were substantially increased, while that of miR-342-3p was decreased in AGE-BSA-treated HRPCs. MIAT knockdown inhibited pyroptosis in HRPCs, which was reinforced by cotreatment with miR-342-3p mimic but relieved by cotreatment with miR-342-3p inhibitor. Furthermore, HRPC pyroptosis was inhibited by treatment with the miR-342-3p mimic alone but enhanced by the miR-342-3p inhibitor. Luciferase reporter assay results demonstrated binding between MIAT and miR-342-3p, as well as between miR-342-3p and CASP1. MIAT antagonized the effect of miR-342-3p on the depression of its target CASP1 and promoted AGE-BSA-induced pericyte pyroptosis. These findings may promote a better understanding of retinal pericyte depletion pathogenesis and the development of new therapeutic strategies for the treatment of diabetic retinopathy., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Chronic Chagas disease cardiomyopathy (CCC), an especially aggressive inflammatory dilated cardiomyopathy caused by lifelong infection with the protozoan Trypanosoma cruzi, is a major cause of cardiomyopathy in Latin America. Although chronic myocarditis may play a major pathogenetic role, little is known about the molecular mechanisms responsible for its severity. The aim of this study is to study the genes and microRNAs expression in tissues and their connections in regards to the pathobiological processes. To do so, we integrated for the first time global microRNA and mRNA expression profiling from myocardial tissue of CCC patients employing pathways and network analyses. We observed an enrichment in biological processes and pathways associated with the immune response and metabolism. IFNγ, TNF and NFkB were the top upstream regulators. The intersections between differentially expressed microRNAs and differentially expressed target mRNAs showed an enrichment in biological processes such as Inflammation, inflammation, Th1/IFN-γ-inducible genes, fibrosis, hypertrophy, and mitochondrial/oxidative stress/antioxidant response. MicroRNAs also played a role in the regulation of gene expression involved in the key cardiomyopathy-related processes fibrosis, hypertrophy, myocarditis and arrhythmia. Significantly, a discrete number of differentially expressed microRNAs targeted a high number of differentially expressed mRNAs (>20) in multiple processes. Our results suggest that miRNAs orchestrate expression of multiple genes in the major pathophysiological processes in CCC heart tissue. This may have a bearing on pathogenesis, biomarkers and therapy., Competing Interests: The authors have declared that no competing interests exist.

MicroRNAs (miRNAs) are endogenous non-coding RNAs in eukaryotic cells that modulate apoptosis of ovarian granulosa cells (GCs), which is an important cause of mammalian follicular atresia. In the present study, associations were evaluated between miR-21-5p and the extent of Smad7 protein production in regulation of ovarian granulosa cell (pGC) apoptosis. There was detection of miR-21-5p and Smad7 primarily in the cytoplasm and nucleus of pGCs, respectively. When there was an enhanced abundance of miR-21-5p and decreased abundance of Smad7 there were similar effects in pGCs, including inducing proliferation, inhibiting apoptosis, increasing the number of cells in S and G2/M phases, increasing serum estradiol, and decreasing serum progesterone concentrations. Furthermore, the Smad7 mRNA transcript was identified as a target for miR-21-5p actions, with enhanced abundances of miR-21-5p being associated with a lesser abundance of Smad7 mRNA transcript and protein in pGCs. Overall, results from the present study indicate that miR-21-5p has actions on the Smad7 mRNA transcript during the process of ovarian granulosa cell apoptosis in pigs., (Copyright © 2020. Published by Elsevier B.V.)

MicroRNAs are important coordinators of circadian regulation that mediate the fine-tuning of gene expression. Although many studies have shown the effects of individual miRNAs on the circadian clock, the global functional miRNA-mRNA interaction network involved in the circadian system remains poorly understood. Here, we used CLEAR (Covalent Ligation of Endogenous Argonaute-bound RNAs)-CLIP (Cross-Linking and Immuno-Precipitation) to explore the regulatory functions of miRNAs in the circadian system by comparing the miRNA-mRNA interactions between Drosophila wild-type strain W 1118 and a mutant of the key circadian transcriptional regulator Clock (Clk jrk ). This experimental approach unambiguously identified tens of thousands of miRNA-mRNA interactions in both the head and body. The miRNA-mRNA interactome showed dramatic changes in the Clk jrk flies. Particularly, among ~300 miRNA-mRNA circadian relevant interactions, multiple interactions involving core clock genes pdp1, tim, and vri displayed distinct changes as a result of the Clk mutation. Based on the CLEAR-CLIP analysis, we found a novel regulation of the circadian rhythm and sleep by the miR-375-timeless interaction. The results indicated that Clk disruption abolished normal rhythmic expression of miR-375 and the functional regulation occurred in the l-LNv neurons, where miR-375 modulated the circadian rhythm and sleep via targeting timeless. This work provides the first global view of miRNA regulation in the circadian rhythm., (© 2020 Federation of American Societies for Experimental Biology.)