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By employing an aptamer as the bridge and combining catalytic hairpin assembly with the Au aggregation amplification effect, a lateral flow assay (LFA) is designed for simultaneous detection of liver cancer-associated miRNA and exosomes. The LFA can differentiate between liver cancer patients and healthy individuals with simple operation and high accuracy.

Background: Circular RNAs are highly enriched in the synapses of the mammalian brain and play important roles in neurological function by acting as molecular sponges of microRNAs. circAnk3 is derived from the 11th intron of the ankyrin-3 gene, Ank3, a strong genetic risk factor for neuropsychiatric disorders; however, the function of circAnk3 remains elusive. In this study, we investigated the function of circAnk3 and its downstream regulatory network for target genes in the hippocampus of mice., Methods: The DNA sequence from which circAnk3 is generated was modified using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9) technology, and neurobehavioral tests (anxiety and depression-like behaviors, social behaviors) were performed in circAnk3 +/- mice. A series of molecular and biochemical assays were used to investigate the function of circAnk3 as a microRNA sponge and its downstream regulatory network for target genes., Results: circAnk3 +/- mice exhibited both anxiety-like behaviors and social deficits. circAnk3 was predominantly located in the cytoplasm of neuronal cells and functioned as a miR-7080-3p sponge to regulate the expression of Iqgap1. Inhibition of miR-7080-3p or restoration of Iqgap1 in the hippocampus ameliorated the behavioral deficits of circAnk3 +/- mice. Furthermore, circAnk3 deficiency decreased the expression of the NMDA receptor subunit GluN2a and impaired the structural plasticity of dendritic synapses in the hippocampus., Conclusions: Our results reveal an important role of the circAnk3/miR-7080-3p/IQGAP1 axis in maintaining the structural plasticity of hippocampal synapses. circAnk3 might offer new insights into the involvement of circular RNAs in neuropsychiatric disorders., (Copyright © 2023 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Background: The prevalence of laryngeal squamous cell carcinoma (LSCC) is increasing, and it poses a significant threat to human health; therefore, identifying specific targets for LSCC remains crucial., Methods: Bioinformatics analysis was used to compare the different expression genes expressed in LSCC. Immunohistochemical assay and western blotting were used to analysis protein expression. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)((4,5 Dimethyl thiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide)4,5 Dimethyl thiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) and 5-ethynyl 2'-deoxyuridine (Edu) assay. Flow cytometry was used to measure the cell cycle. Cell migration was measured by wound healing assay and transwell assay., Results: Our analysis revealed 36 upregulated and 65 downregulated differentially expressed genes (DEGs) when comparing LSCC tumors to adjacent tissues, with cornulin (CRNN) identified as a key hub gene connecting these DEGs. We observed a consistent downregulation of CRNN expression in LSCC cell lines and tissues and was associated with poor patient survival and the tumor microenvironment. CRNN overexpression was found to significantly inhibit cell growth, cell cycle progression, migration and invasion, while CRNN knockdown had the opposite effects. Additionally, in vivo experiments demonstrated that CRNN overexpression suppressed tumor growth in nude mice., Conclusions: CRNN functions as a potential tumor suppressor and regulates important aspects of LSCC, providing valuable insights into the role of CRNN in LSCC pathogenesis and potential for targeted therapeutic interventions., Competing Interests: The authors declare no conflict of interest., (© 2024 The Author(s). Published by IMR Press.)

Liver hepatocellular carcinoma (LIHC) is among the most lethal human cancers. Studies have shown that Homer scaffold protein 3 (HOMER3) plays important roles in various diseases and cancers, but its biological function and molecular mechanism in LIHC have never been investigated. Our study discovered the aberrantly high expression of HOMER3 and its promising diagnostic and prognostic significance in LIHC. Functionally, HOMER3 knockdown inhibited the proliferative and migrative abilities of LIHC cells and tumor growth in vivo. Mechanically, HOMER3 mediated the aggressiveness of LIHC cells via GPNMB. Meanwhile, miR-361 directly targeted GPNMB and attenuated LIHC progression by suppressing GPNMB expression. The regulatory effect of HOMER3 during LIHC progression was exerted through the miR-361/GPNMB axis. Furthermore, EZH2 supplementation or miR-361 depletion effectively abated the tumor-suppressive effect of HOMER3 knockdown on LIHC progression. In conclusion, HOMER3 mediated LIHC progression through the EZH2/miR-361/GPNMB axis., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Background: MicroRNA-1 (miR1), encoded by the genes miR1-1 and miR1-2 , is the most abundant microRNA in the heart and plays a critical role in heart development and physiology. Dysregulation of miR1 has been associated with various heart diseases, where a significant reduction (>75%) in miR1 expression has been observed in patient hearts with atrial fibrillation or acute myocardial infarction. However, it remains uncertain whether miR1-deficiency acts as a primary etiological factor of cardiac remodeling., Methods: miR1-1 or miR1-2 knockout mice were crossbred to produce 75%-miR1-knockdown (75%KD; miR1-1 +/- :miR1-2 -/- or miR1-1 -/- :miR1-2 +/- ) mice. Cardiac pathology of 75%KD cardiomyocytes/hearts was investigated by ECG, patch clamping, optical mapping, transcriptomic, and proteomic assays., Results: In adult 75%KD hearts, the overall miR1 expression was reduced to ≈25% of the normal wild-type level. These adult 75%KD hearts displayed decreased ejection fraction and fractional shortening, prolonged QRS and QT intervals, and high susceptibility to arrhythmias. Adult 75%KD cardiomyocytes exhibited prolonged action potentials with impaired repolarization and excitation-contraction coupling. Comparatively, 75%KD cardiomyocytes showcased reduced Na + current and transient outward potassium current, coupled with elevated L-type Ca 2+ current, as opposed to wild-type cells. RNA sequencing and proteomics assays indicated negative regulation of cardiac muscle contraction and ion channel activities, along with a positive enrichment of smooth muscle contraction genes in 75%KD cardiomyocytes/hearts. miR1 deficiency led to dysregulation of a wide gene network, with miR1's RNA interference-direct targets influencing many indirectly regulated genes. Furthermore, after 6 weeks of bi-weekly intravenous tail-vein injection of miR1 mimics, the ejection fraction and fractional shortening of 75%KD hearts showed significant improvement but remained susceptible to arrhythmias., Conclusions: miR1 deficiency acts as a primary etiological factor in inducing cardiac remodeling via disrupting heart regulatory homeostasis. Achieving stable and appropriate microRNA expression levels in the heart is critical for effective microRNA-based therapy in cardiovascular diseases., Competing Interests: Disclosures None.

As a commonly used antibacterial agent in daily consumer products, triclosan (TCS) has attracted significant attention due to its potential environmental risks. In this study, we investigated the toxic effects of TCS exposure (1.4 μM) on heart development in zebrafish embryos. Our findings revealed that TCS exposure caused significant cardiac dysfunction, characterized by pericardial edema, malformations in the heart structure, and a slow heart rate. Additionally, TCS exposure induced oxidative damage and abnormal apoptosis in heart cells through the up-regulation of β-adrenergic receptor (β-AR) signaling pathway genes (adrb1, adrb2a, arrb2b), similar to the effects induced by β-AR agonists. Notably, the adverse effects of TCS exposure were alleviated by β-AR antagonists. Using high-throughput transcriptome miRNA sequencing and targeted miRNA screening, we focused on miR-133b, which targets adrb1 and was down-regulated by TCS exposure, as a potential contributor to TCS-induced cardiotoxicity. Inhibition of miR-133b produced similar toxic effects as TCS exposure, while overexpression of miR-133b down-regulated the β-AR signaling pathway and rescued heart defects caused by TCS. In summary, our findings provide new insights into the mechanisms underlying the cardiotoxic effects of TCS. We suggest that targeting the β-AR pathway and miR-133b may be effective strategies for pharmacotherapy in cardiotoxicity induced by environmental pollutants such as TCS., 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 © 2023 Elsevier Ltd. All rights reserved.)

Background: Long non-coding RNAs (lncRNAs) have been brought great attention for their crucial roles in diverse biological processes. However, systematic identification of lncRNAs associated with specialized rice pest, brown planthopper (BPH), defense in rice remains unexplored., Results: In this study, a genome-wide high throughput sequencing analysis was performed using leaf sheaths of susceptible rice Taichung Native 1 (TN1) and resistant rice IR36 and R476 with and without BPH feeding. A total of 2283 lncRNAs were identified, of which 649 lncRNAs were differentially expressed. During BPH infestation, 84 (120 in total), 52 (70 in total) and 63 (94 in total) of differentially expressed lncRNAs were found only in TN1, IR36 and R476, respectively. Through analyzing their cis-, trans-, and target mimic-activities, not only the lncRNAs targeting resistance genes (NBS-LRR and RLKs) and transcription factors, but also the lncRNAs acting as the targets of the well-studied stress-related miRNAs (miR2118, miR528, and miR1320) in each variety were identified. Before the BPH feeding, 238 and 312 lncRNAs were found to be differentially expressed in TN1 vs. IR36 and TN1 vs. R476, respectively. Among their putative targets, the plant-pathogen interaction pathway was significantly enriched. It is speculated that the resistant rice was in a priming state by the regulation of lncRNAs. Furthermore, the lncRNAs extensively involved in response to BPH feeding were identified by Weighted Gene Co-expression Network Analysis (WGCNA), and the possible regulation networks of the key lncRNAs were constructed. These lncRNAs regulate different pathways that contribute to the basal defense and specific resistance of rice to the BPH., Conclusion: In summary, we identified the specific lncRNAs targeting the well-studied stress-related miRNAs, resistance genes, and transcription factors in each variety during BPH infestation. Additionally, the possible regulating network of the lncRNAs extensively responding to BPH feeding revealed by WGCNA were constructed. These findings will provide further understanding of the regulatory roles of lncRNAs in BPH defense, and lay a foundation for functional research on the candidate lncRNAs., (© 2023. The Author(s).)

Diabetic nephropathy (DN) is one of the most common and intractable microvascular complications of diabetes worldwide, serving as the main cause of terminal renal disease. Due to the lack of early specific symptoms and diagnostic markers, DN severely threatens the sufferer's life. MicroRNA-192 (miR-192) was early identified in human renal cortical tissue and stored and excreted in urine as microvesicles. MiR-192 was found to be involved in the development of DN. For the first time, the present review summarized all the current evidence on the topic of the roles of miR-192 in DN. Finally, 28 studies (ten clinical trials and eighteen experimental studies) were eligible for thorough reviewing. Most of the clinical trials (7/10, 70%) indicated miR-192 might be a protective factor for DN development and progression, while the majority of experimental studies (14/18, 78%) suggested miR-192 might be a pathogenic factor for DN. Mechanistically, miR-192 interacts with various direct targeted proteins (i.e., ZEB1, ZEB2, SIP1, GLP1R, and Egr1) and signaling cascades (i.e., SMAD/TGF-β and PTEN/PI3K/AKT), together contribute to the pathogenesis of DN through epithelial-to-mesenchymal transition (EMT), extracellular matrix deposition, and fibrosis formation. The current review highlights the dual role of miR-192 in the development of DN. Low serum miR-192 expression could be applied for the early prediction of DN (the early stage of DN), while the high miR-192 level in renal tissues and urine may imply the progression of DN (the late stage of DN). Further investigations are still warranted to illustrate this inconsistent phenomenon, which may facilitate promoting the therapeutic applications of miR-192 in predicting and treating DN., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wan, Liao, Lai, Zhang, Cui and Chen.)

Age-related macular degeneration (AMD) is one of the leading causes of irreversible blindness in the elderly population. Neovascular AMD is the late stage, characterized by choroidal neovascularization (CNV). Non-coding RNAs have been implicated in CNV; however, the role of circular RNAs (circRNAs) has not yet been elucidated. Herein, we comprehensively investigated circRNA profiles in laser-induced CNV mouse models and patient specimens. A novel circRNA, circRNA Uxs1, was identified, and its function in CNV regulation was investigated in the present study. CircRNA Uxs1 was consistently upregulated in CNV patient specimens and CNV mouse models. Knockdown of circRNA Uxs1 interrupted the tube formation, migration, and proliferation of endothelial cells in vitro. Silencing circRNA Uxs1 in vivo alleviated neovascularization formation, as shown by the decreased size of laser spots. Mechanistically, circRNA Uxs1 functioned by binding to miR-335-5p, which further upregulated the expression of placental growth factor (PGF) gene and activated the mammalian target of rapamycin/p70 S6 Kinase (mTOR/p70 S6k) pathway. By subretinal injections of adeno-associated virus (AAV), we demonstrated the anti-angiogenic function of circRNA Uxs1 knockdown in vivo. In conclusion, circRNA Uxs1 promoted CNV by sponging miR-335-5p, which stimulated PGF expression and subsequently activated the mTOR/p70 S6k pathway. Therefore, circRNA Uxs1 may serve as a promising therapeutic target for CNV., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Glioma is a tumor in the brain and spinal cord originating in the glial cells that surround the nerve cells. Among several microRNAs reported, miRNA-363 is associated with human glioma. Based on miRNA-363 levels, the development and progression of glioma can be monitored. The current study used an interdigitated electrode sensor to monitor microRNA-363 levels, which indeed reflects the severity of glioma. The interdigitated electrode was generated using a photolithography technique followed by surface chemical modification carried out to insert miRNA-363 complementary oligo as the probe complexed with gold nanoparticles. The proposed sensor works based on the dipole moment between two electrodes, and when molecular immobilization or interaction occurs, the response by the signal output changes. The changes in the target microRNA-363 sequence were standardized to identify glioma. The limit of detection of miRNA-363 was 10 fM with an R 2 value of 0.996 on the linear coefficient regression ranges between 1 fM and 100 pM. Furthermore, unrelated sequences failed to increase the response of the current with the complementary probe, indicating specific miRNA-363 detection on the interdigitated electrode. This study demonstrates the platform to be used for determining the presence of microRNA-363 in glioma and as the basis for other biomarker analyses., (© 2022 International Union of Biochemistry and Molecular Biology, Inc.)

Salinity stress is a serious limitation to tomato growth and development. The aim of this study was to investigate the effects of Sly-miR164a on tomato growth and fruit nutritional quality under salt stress. The results showed that the root length, fresh weight, plant height, stem diameter and ABA content of miR164a#STTM (knockdown of Sly-miR164a) lines were higher than those of WT and miR164a#OE (overexpression of Sly-miR164a) lines under salt stress. Compared with WT, miR164a#STTM tomato lines exhibited lower ROS accumulation under salt stress. In addition, the fruits of miR164a#STTM tomato lines had higher soluble solids, lycopene, ascorbic acid (ASA) and carotenoid content compared with WT. The study indicated that tomato plants were more sensitive to salt when Sly-miR164a was overexpressed, while knockdown of Sly-miR164a enhanced plant salt tolerance and improved fruit nutritional value., Competing Interests: The authors declare no conflict of interest.

Background: Sepsis is one of the most lethal diseases worldwide. Pyroptosis is a unique form of cell death, and the mechanism of interaction with sepsis is not yet clear. The aim of this study was to uncover pyroptosis genes associated with sepsis and to provide early therapeutic targets for the treatment of sepsis., Methods: Based on the GSE134347 dataset, sepsis-related genes were mined by differential expression analysis and weighted gene coexpression network analysis (WGCNA). Subsequently, the sepsis-related genes were analysed for enrichment, and a protein‒protein interaction (PPI) network was constructed. We performed unsupervised consensus clustering of sepsis patients based on 33 pyroptosis-related genes (PRGs) provided by prior reviews. We finally obtained the PRGs mostly associated with sepsis by machine learning prediction models combined with prior reviews. The GSE32707 dataset served as an external validation dataset to validate the model and PRGs via receiver operating characteristic (ROC) curves. The NetworkAnalyst online tool was utilized to create a ceRNA network of lncRNAs and miRNAs around PRGs mostly associated with sepsis., Results: A total of 170 genes associated with sepsis and 13 hub genes were acquired by WGCNA and PPI network analysis. The results of the enrichment analysis implied that these genes were mainly involved in the regulation of the inflammatory response and the positive regulation of bacterial and fungal defence responses. The prolactin signalling pathway and IL-17 signalling pathway were the primary enrichment pathways. Thirty-three PRGs can effectively classify septic patients into two subtypes, implying that there is a reciprocal relationship between sepsis and pyroptosis. Eventually, NLRC4 was considered the PRG most strongly associated with sepsis. The validation results of the prediction model and NLRC4 based on ROC curves were 0.74 and 0.67, respectively, both of which showed better predictive values. Meanwhile, the ceRNA network consisting of 6 lncRNAs and 2 miRNAs was constructed around NLRC4., Conclusion: NLRC4, as the PRG mostly associated with sepsis, could be considered a potential target for treatment. The 6 lncRNAs and 2 miRNAs centred on NLRC4 could serve as a further research direction to uncover the deeper pathogenesis of sepsis., (© 2023. The Author(s).)

Introduction: The aims of this study were to investigate the molecular alterations of cuproptosis-related genes and to construct the cuproptosis-related circular RNA (circRNA)-microRNA (miRNA)-mRNA networks in neovascular age-related macular degeneration (nAMD)., Methods: The transcriptional profiles of laser-induced choroid neovascularization (CNV) mouse models and nAMD patient samples were obtained from sequencing and from the GEO database (GSE146887), respectively. The expression levels of ten cuproptosis-related genes (FDX1, DLAT, LIAS, DLD, PDHB, MTF1, CDKN2A, GLS, LIPT1, and PDHA1) were extracted and verified in both mouse CNV models and patient peripheral blood mononuclear cells (PBMCs) samples. The cuproptosis-related circRNA-miRNA-mRNA network was further constructed based on miRNet database, the dataset GSE131646 of small RNA expression profile, and the dataset GSE140178 of circRNA expression profile in mouse CNV models., Results: The significant upregulation of Cdkn2a and Mtf1 and the downregulation of other 5 cuproptosis-related genes were verified in the mouse CNV model, but only CDKN2A significantly upregulated in PBMCs of patients with nAMD. Four miRNAs were detected in the intersection between miRNet prediction and sequencing data: miR-129-5p, miR-129-2-3p, miR-182-5p, and miR-615-3p. There were 9 circRNAs at the intersection of hsa-miR-182-5p and hsa-miR-615-3p predictions, one circRNA predicted by hsa-miR-129-5p and GSE140178 (hsa-circASH1L), and one circRNA predicted by hsa-miR-182-5p and hsa-miR-615-3p (hsa-circNPEPPS)., Conclusion: This study suggested the repression of cuproptosis in nAMD pathologies and constructed a cuproptosis-related network of 8 cuproptosis-related genes, 4 miRNAs, and 11 circRNAs., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

Lung squamous cell carcinoma (LSCC) is a prevalent and progressive subtype of lung cancer. This study aimed to substantiate the regulatory effect of the PAK2/SOX2/DEK axis on the LSCC development. LSCC tissues (n = 83) and adjacent normal tissues were collected and SOX2 expression was determined by qRT-PCR and Western blotting. Correlation between SOX2 expression and the prognosis of LSCC patients was then explored utilizing Kaplan-Meier analysis. Co-immunoprecipitation and glutathione-S-transferase pull-down assays were conducted to validate the binding of SOX2 to DEK. Gain- and loss- of function assays were then performed on LSCC cells, with CCK-8 and Transwell assays applied to detect the malignant behaviors of cells. A mouse xenograft model of LSCC was further established for in vivo validation. The expression levels of SOX2, PAK2 and DEK were up-regulated in LSCC tissues and cells. SOX2 overexpression was correlated with poor prognosis of LSCC patients. Knockdown of SOX2 weakened the viability and the migratory and invasive potential of LSCC cells. Further, PAK2 directly interacted with SOX2. PAK2 overexpression accelerated the malignant phenotypes of LSCC cells through interplay with SOX2. Moreover, SOX2 activated the expression of DEK, and silencing DEK attenuated the malignant behaviors of LSCC cells. In conclusion, PAK2 could bind to the transcription factor SOX2 and thus activate the expression of DEK, thereby driving the malignant phenotypes of LSCC cells both in vivo and in vitro., (© 2022. The Author(s), under exclusive licence to United States and Canadian Academy of Pathology.)

Background: Metastasis is still the major cause of endometrial cancer (EC)-related death. Because of their biological function and regenerative properties, exosomes have been applied to therapeutic regimens. SERPINA5 expression is downregulated in several tumors and linked to tumor cell migration and invasion. However, the expression and biological functions of SERPINA5 in EC remain unclear., Methods: The levels of SERPINA5 in plasma exosomes were determined with ELISAs. SERPINA5 expression in EC and its relationship with survival outcomes were analyzed using the TCGA database and clinical EC tissue samples. The effect of SERPINA5 overexpression or exosomal SERPINA5 on EC metastasis was examined by cell migration and invasion assays in vitro. Mechanistically, overexpression of SERPINA5 or high exosomal SERPINA5 levels mediated the regulation of the integrin β1/FAK signaling pathway in EC cell lines. The therapeutic effect of exosomal SERPINA5 was determined with xenograft models., Results: This study revealed that the level of exosomal SERPINA5 was increased in the circulating plasma of EC patients. In addition, the expression of SERPINA5 was decreased in EC patients with distant metastasis, and low expression of SERPINA5 indicated worse survival. In addition, SERPINA5 was elevated in normal tissues adjacent to EC tumors. Moreover, overexpression of SERPINA5 inhibited metastatic potential of EC cell lines in vitro. Furthermore, SERPINA5 loaded on secreted exosomes reduced the metastatic ability of EC cells. Notably, overexpression of SERPINA5 or high exosomal SERPINA5 levels suppressed EC metastatic potential by suppressing integrin β1/FAK signaling pathway activation. Finally, exosomal SERPINA5 impeded tumor growth and metastasis in xenograft models., Conclusions: Our findings revealed that a low level of SERPINA5 expression indicated poor survival outcomes in EC and that exogenous SERPINA5 loading of exosomes may be a novel therapeutic strategy for metastatic EC., (© 2022. Springer Nature Switzerland AG.)

(R,S)-ketamine is known to elicit persistent prophylactic effects in rodent models of depression. However, the precise molecular mechanisms underlying its action remain elusive. Using RNA-sequencing analysis, we searched for novel molecular target(s) that contribute to the prophylactic effects of (R)-ketamine, a more potent enantiomer of (R,S)-ketamine in chronic restraint stress (CRS) model. Pretreatment with (R)-ketamine (10 mg/kg, 1 day before CRS) significantly ameliorated body weight loss, increased immobility time of forced swimming test, and decreased sucrose preference of sucrose preference test in CRS-exposed mice. RNA-sequencing analysis of prefrontal cortex (PFC) revealed that several miRNAs such as miR-132-5p might contribute to sustained prophylactic effects of (R)-ketamine. Methyl CpG binding protein 2 (MeCP2) is known to regulate brain-derived neurotrophic factor (BDNF) expression. Quantitative RT-PCR confirmed that (R)-ketamine significantly attenuated altered expression of miR-132-5p and its regulated genes (Bdnf, Mecp2, Tgfb1, Tgfbr2) in the PFC of CRS-exposed mice. Furthermore, (R)-ketamine significantly attenuated altered expression of BDNF, MeCP2, TGF-β1 (transforming growth factor β1), and synaptic proteins (PSD-95, and GluA1) in the PFC of CRS-exposed mice. Administration of agomiR-132-5p decreased the expression of Bdnf and Tgfb1 in the PFC, resulting in depression-like behaviors. In contrast, administration of antagomiR-132-5p blocked the increased expression of miR-132-5p and decreased expression of Bdnf in the PFC of CRS-exposed mice, resulting in antidepressant-like effects. In conclusion, our data show a novel role of miR-132-5p in the PFC underlying depression-like phenotypes in CRS model and the sustained prophylactic effects of (R)-ketamine., (© 2022. The Author(s).)

Recently, the effects of competing endogenous RNA (ceRNA) on molecular biological mechanism of cancer have aroused great interest. In this study, long noncoding RNA-microRNA-messenger RNA (lncRNA-miRNA-mRNA) ceRNA network was screened and constructed based on the Cancer Genome Atlas (TCGA) database, and its efficacy in predicting the prognosis of breast cancer patients was evaluated. The RNA-sequencing, miRNA-sequencing, and corresponding clinical information were downloaded from the TCGA database, and differentially expressed genes were screened after data searching. The similarity between two groups of genes was analyzed by weighted correlation network analysis (WGCNA). Next, the interaction among lncRNA, miRNA, and mRNA was predicted followed construction of the lncRNA-miRNA-mRNA ceRNA network. Finally, univariate and multivariate Cox regression analysis was used to screen prognostic factors to construct prognostic risk model. Receiver operating characteristic (ROC) curve was used to evaluate the efficacy of this model in predicting the prognosis of breast cancer patients. In total 5056 differentially expressed lncRNAs, 712 differentially expressed miRNAs, and 9878 differentially expressed mRNAs were identified in breast cancer tissues. WGCNA predicted that 823 lncRNAs and 1813 mRNAs were closely related to breast cancer. The lncRNA-miRNA-mRNA ceRNA network involved in breast cancer was constructed based on 27 lncRNA, 14 miRNAs, and 4 mRNAs. ZC3H12B, HRH1, TMEM132C, and PAG were the possible independent risk factors for the prognosis of breast cancer patients with the area under the signal characteristic curve under ROC curve of 0.609. This study suggested that the prognosis risk model based on ZC3H12B, HRH1, TMEM132C, and PAG1 accurately predicted the prognosis of breast cancer patients., (© 2022 Wiley Periodicals LLC.)

This work was to investigate mechanism by which mir-22 targeting nod-like receptor protein 3 (NLRP3) inflammasome affected activity of human skin malignant melanoma (MM) A375 cells. Twenty-four mice were rolled into a control group (Group X) and an experimental group (Group Y) randomly. Without treatment in Group X, Group Y established MM model. After cell transfection, the mice were divided into group A (blank group), group B (negative group), group C (miR-22 mimics group), group D (miR-22 inhibitor group), and group E (miR-22 inhibitor+siNLRP3 group). The results were summarized as follows. The level of miR-22 mRNA in Group Y was obviously lower than that in Group X, and levels of NLRP3 and caspase-1 mRNA and NLRP3 and caspase-1 protein in Group Y were greatly higher than those in Group X ( P < 0.05). The mRNA levels of miR-22 mRNA in group C were much higher in contrast to those in group A, and the mRNA levels of NLRP3 and caspase-1 were lower. The contrast results in group D and group A were the opposite, P < 0.05. The levels of NLRP3 and caspase-1 proteins in group C were greatly elevated, and those in group D were decreased compared with those in group A ( P < 0.05). Therefore, miR-22 may target and inhibit the activation of the NLRP3 inflammasome to reduce the activity of cutaneous malignant melanoma A375 cells., Competing Interests: The authors declare that they have no competing interest., (Copyright © 2022 Akebaier Sulaiman et al.)

Background: Breast cancer (BC) is the leading cause of cancer-related mortality in females and the most common malignancy with high morbidity worldwide. It is imperative to develop new biomarkers and therapeutic targets for early diagnosis and effective treatment in BC., Methods: We revealed the oncogene function of cadherin 1 (CDH1) via bioinformatic analysis in BC. Moreover, miRNA database was utilized to predict miRNAs upstream of CDH1. Expression of CDH1-related miRNAs in BC and their values in BC stemness and prognosis were analyzed through TCGA-BRCA datasets. In addition, Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) were performed to explore the potential functions and signaling pathways of CDH1 in combination with CDH1-related miRNAs in BC progression. Finally, the differential expressions of soluble E-cadherin (sE-cad), which is formed by the secretion of CDH1-encoded E-cadherin into serum, analyzed by enzyme-linked immunosorbent assay (ELISA). Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the expression level of CDH1-related miRNAs in serum samples., Results: The mRNA and protein expressions of CDH1 were elevated in BC tissues compared with normal counterparts. Moreover, CDH1 overexpression was positively correlated with BC stage, metastatic, stemness characteristics, and poor prognosis among patients. In predictive analysis, miR-340, miR-185, and miR-20a target CDH1 and are highly expressed in BC. miR-20a overexpression alone was strongly associated with high stemness characteristics and poor prognosis of BC. Additionally, GO, KEGG, and hallmark effect gene set analysis demonstrated that CDH1 in combination with overexpression of miR-340, miR-185, or miR-20a participated in multiple biological processes and underly signaling pathways involving in tumorigenesis and development of BC. Finally, we provide experimental evidence that the combined determination of serum sE-cad and miR-20a in BC has highly diagnostic efficiency., Conclusions: This study provides evidence for CDH1 as an oncogene in BC and suggests that miR-20a may regulate the stemness characteristics of BC to exert a pro-oncogenic effect by regulating CDH1. Moreover, sE-cad and miR-20a in serum can both be used as valid noninvasive markers for BC diagnosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Xie, Chen, Wan, Li, Pei, Luo, Liu and Ye.)

Cholangiocarcinoma (CCA) is a severe malignancy originating from the bile duct and the second most common primary liver cancer. NF-kappa B interacting lncRNA (NKILA) is a functional lncRNA, which play important role in human cancers. However, the role and underlying mechanism of NKILA in CCA remains largely unknown. Here, our study demonstrated that NKILA was significantly upregulated in CCA tissues and cells. Overexpression of NKILA is associated with advanced TNM stage, lymph node and distant metastasis, and also indicated poor prognosis in CCA patients. Functionally, NKILA facilitated CCA growth and metastasis in vitro and in vivo. The 5-methylcytosine (m 5 C) methyltransferase NSUN2 interacts with NKILA, increasing its m 5 C level and promoting its interaction with YBX1. Moreover, NKILA physically interacted with and suppressed miR-582-3p, which was regulated by METTL3-mediated N 6 -methyladenosine (m 6 A) modification. Finally, we showed that YAP1 was a target of NKILA via miR-582-3p and NKILA functioned partially via YAP1 in CCA. Taken together, our findings indicate a novel regulatory mechanism of NKILA for promoting CCA progression and that NKILA may be a promising target for CCA treatment., (© 2022 John Wiley & Sons A/S . Published by John Wiley & Sons Ltd.)

The aberrant expression or genomic mutations of microRNA are associated with several human diseases. This study analyzes the relationship between genetic variations of miRNA and schizophrenia or bipolar disorder. We performed case-control studies for ten SNPs in a total sample of 1584 subjects. All these ten SNPs were on or near mature microRNAs. We identified the association between bipolar disorder and the T/C polymorphism at rs895819. To illustrate the function of miR-27a, we constructed several miR-27a knockout (KO) cell lines, determined candidates of miR-27a, and then verified NCAM1 as a target gene of miR-27a. Further studies revealed that the T/C polymorphism on miR-27a led to the differential expression of mature and precursor miR-27a without affecting the expression of primary miR-27a. Furthermore, the C mutation on pre-miR-27a suppresses cell migration and dopamine expression levels. Our study highlights the importance of miR-27a and its polymorphism at rs895819 in bipolar disorder., (© 2022. The Author(s).)

One of the most prevalent forms of endocrine malignancies is thyroid cancer. Herein, we explored the mechanisms whereby miR-1246 is involved in thyroid cancer. Phosphoinositide 3-kinase adapter protein 1 (PIK3AP1) was identified as a potential miR-1246 target, with the online Gene Expression Omnibus (GEO) database. The binding between miR-1246 and PIK3AP1 and the dynamic role of these two molecules in downstream PI3K/AKT signaling were evaluated. Analysis of GEO data demonstrated significant miR-1246 downregulation in thyroid cancer, and we confirmed that overexpression of miR-1246 can inhibit migratory, invasive, and proliferative activity in vitro and tumor growth in vivo. Subsequent studies indicated that miR-1246 overexpression decreased the protein level of PIK3AP1 and the phosphorylation of PI3K and AKT, which were reversed by PIK3AP1 overexpression. At the same time, overexpression of PIK3AP1 also reversed the miR-1246 mimics-induced inhibition proliferative, migratory, and invasive activity, while promoting increases in apoptotic death, confirming that miR-1246 function was negatively correlated with that of PIK3AP1. Subsequently, we found that the miR-1246 mimics-induced inhibition of PI3K/AKT phosphorylation was reversed by the PI3K/AKT activator IGF-1. miR-1246 mimics inhibited proliferative, migratory, and invasive activity while promoting increases in apoptotic death, which were reversed by IGF-1. Furthermore, miR-1246 agomir can inhibit tumor growth in vivo. We confirmed that miR-1246 affects the signaling pathway of PI3K/AKT via targeting PIK3AP1 and inhibits the development of thyroid cancer. Thus, miR-1246 is a new therapeutic target for thyroid cancer., (© 2021. The Author(s).)

Objective: Ulcerative colitis (UC) is a chronic colitis with unknown etiology. Circular RNA (circRNA) has shown regulatory effect in many diseases, but the role of circRNA in UC is barely known. This study uncovers the function and regulatory mechanism of circRNA HECTD1 (circHECTD1) in UC., Methods: Colonic mucosal tissues of 60 patients with active UC and 30 healthy controls were collected for H&E staining. Lipopolysaccharide (LPS) and dextran sulfate sodium (DSS) were used to induce inflammation and UC in Caco-2 cells and C57BL/6 mice where modification of circHECTD1, miR-182-5p and/or human antigen R (HuR) took place. The Caco-2 cells and the colon tissues of DSS-treated mice were collected for analysis of the expression levels of inflammatory cytokines, NLRP3 inflammasome, and autophagy-related proteins. The interactions among circHECTD1, miR-182-5p, and HuR were verified., Results: The colonic mucosal tissues of UC patients showed impaired autophagy and decreased expressions of circHECTD1 and HuR. Overexpression of circHECTD1 or HuR or inhibition of miR-182-5p suppressed inflammation and promoted autophagy of LPS-induced Caco-2 cells. The expression of HuR was promoted by circHECTD1 via miR-182-5p in Caco-2 cells. Overexpression of circHECTD1 reduced colonic injuries and inflammation by promoting autophagy in DSS-treated mice., Conclusion: Overexpression of circHECTD1 alleviates UC by promoting HuR-dependent autophagy via miR-182-5p. This study highlights the therapeutic potential of circHECTD1 for UC and adds to the knowledge of circRNA in the pathogenesis of UC., (© 2021 Crohn’s & Colitis Foundation. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Dopamine agonists (DAs) are preferred for the treatment of prolactinomas and are usually very effective. Nonetheless, 20-30% of bromocriptine- and approximately 10% of cabergoline-treated individuals exhibit resistance to DAs. In addition, the mechanism underlying this phenomenon remains elusive. In this study, we summarize the major findings regarding the role of microRNAs (miRNAs) in the pathogenesis of DA-resistant prolactinoma (DARP). Currently available evidence suggests that miRNAs are usually dysregulated in DARP and that, although controversial, the dysregulated miRNAs target the transforming growth factor (TGF)-β, dopamine 2 receptor (D2R), or estradiol (E2)/estrogen receptor (ER) signaling pathways to mediate the therapeutic effect of DAs. These findings provide new incentives for research on innovative strategies for predicting patients' responsiveness to dopamine therapies and for developing treatment approaches. Unfortunately, recent studies tended to focus exclusively on the differential miRNA expression profiles between DARP and dopamine-sensitive prolactinoma, and no definitive consensus has been reached regarding the role of these miRNAs in the modulation mechanism. Therefore, current and future efforts should be directed toward the exploration of the mechanism underlying the dysregulation of miRNAs as well as of the target proteins that are affected by the dysregulated miRNAs. Furthermore, the modulation of the expression of dysregulated miRNAs, which target the D2R, TGF-β, or E2/ER signaling pathways, might be a promising alternative to treat patients with DARP and improve their prognosis., (© 2021 The Author(s) Published by S. Karger AG, Basel.)

Overexpression of C-X-C motif chemokine receptor 4 (CXCR4) and intercellular cell adhesion molecule-1 (ICAM-1) may promote homing of mesenchymal stem cells (MSC). In this study, we treated ulcerative colitis animals with MSC preconditioned with or without H19 and compared the therapeutic effect of MSC and MSC-H19. We evaluated the regulatory relationship of H19 vs. miR-141/miR-139 and miR-141/miR-139 vs. ICAM-1/CXCR4. We established an ulcerative colitis mouse model to assess the effect of MSC and MSC-H19. H19 was found to bind to miR-141 and miR-139. The activity of H19 was strongly decreased in cells c-transfected with miR-141/miR-139 and WT H19. ICAM-1 was confirmed to be targeted by miR-141 and CXCR4 was targeted by miR-139. The H19 expression showed a negative regulatory relationship with the miR-141 and miR-139 expression but a positive regulatory relationship with the ICAM-1 and CXCR4 expression. In summary, the overexpression of H19 in MSC downregulated miR-139 and miR-141, thus increasing the activity of their targets ICAM-1 and CXCR4, respectively, to exhibit therapeutic effects in ulcerative colitis., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2021 Ming-li Zhao et al.)

The function and regulation of lipid metabolic genes are essential for plant male reproduction. However, expression regulation of lipid metabolic genic male sterility (GMS) genes by noncoding RNAs is largely unclear. Here, we systematically predicted the microRNA regulators of 34 maize white brown complex members in ATP-binding cassette transporter G subfamily (WBC/ABCG) genes using transcriptome analysis. Results indicate that the ZmABCG26 transcript was predicted to be targeted by zma-miR164h-5p, and their expression levels were negatively correlated in maize B73 and Oh43 genetic backgrounds based on both transcriptome data and qRT-PCR experiments. CRISPR/Cas9-induced gene mutagenesis was performed on ZmABCG26 and another lipid metabolic gene, ZmFAR1 . DNA sequencing, phenotypic, and cytological observations demonstrated that both ZmABCG26 and ZmFAR1 are GMS genes in maize. Notably, ZmABCG26 proteins are localized in the endoplasmic reticulum (ER), chloroplast/plastid, and plasma membrane. Furthermore, ZmFAR1 shows catalytic activities to three CoA substrates in vitro with the activity order of C12:0-CoA > C16:0-CoA > C18:0-CoA, and its four key amino acid sites were critical to its catalytic activities. Lipidomics analysis revealed decreased cutin amounts and increased wax contents in anthers of both zmabcg26 and zmfar1 GMS mutants. A more detailed analysis exhibited differential changes in 54 monomer contents between wild type and mutants, as well as between zmabcg26 and zmfar1 . These findings will promote a deeper understanding of miRNA-regulated lipid metabolic genes and the functional diversity of lipid metabolic genes, contributing to lipid biosynthesis in maize anthers. Additionally, cosegregating molecular markers for ZmABCG26 and ZmFAR1 were developed to facilitate the breeding of male sterile lines.

Although microRNA-155 (miR-155) is considered a pro-inflammatory mediator, cumulative evidence indicates that it also has anti-inflammatory effects in macrophages and dendritic cells. In this study, we identified the dramatic expression changes of more than half of potential miR-155-targeted genes upon lipopolysaccharide (LPS) stimulation; 223 genes were down-regulated and 85 genes were up-regulated, including suppressor of cytokine signaling 1 ( SOCS1 ) and transforming growth factor-β-activated kinase 1-binding protein 2 ( TAB2 ), two well-known genes involved in miR-155-mediated regulation of the Toll-like receptor 4 (TLR4) signaling pathway. We also found that miR-155 acted as an anti-inflammatory mediator in the initial stage of LPS-induced inflammatory response mainly through repressing TAB2 protein translation, and as a pro-inflammatory mediator by down-regulating SOCS1 in the later stage. Meanwhile, overexpression of TAB2 3' untranslated region (UTR) in macrophages promoted the development of endotoxin tolerance by competing for binding with miR-155, which resulted in an elevated expression level of SOCS1 protein. These findings provide new insights for understanding the regulatory mechanisms in fine-tuning of LPS-induced innate immune response.

Existing studies have demonstrated that dysregulation of microRNAs (miRNAs or miRs) is involved in the initiation and progression of cancer. Many efforts have been devoted to identify microRNAs as potential biomarkers for cancer diagnosis, prognosis and therapeutic targets. With the rapid development of miRNA sequencing technology, a vast amount of miRNA expression data for multiple cancers has been collected. These invaluable data repositories provide new paradigms to explore the relationship between miRNAs and cancer. Thus, there is an urgent need to explore the complex cancer-related miRNA-gene patterns by integrating multi-omics data in a pan-cancer paradigm. In this study, we present a tensor sparse canonical correlation analysis (TSCCA) method for identifying cancer-related miRNA-gene modules across multiple cancers. TSCCA is able to overcome the drawbacks of existing solutions and capture both the cancer-shared and specific miRNA-gene co-expressed modules with better biological interpretations. We comprehensively evaluate the performance of TSCCA using a set of simulated data and matched miRNA/gene expression data across 33 cancer types from the TCGA database. We uncover several dysfunctional miRNA-gene modules with important biological functions and statistical significance. These modules can advance our understanding of miRNA regulatory mechanisms of cancer and provide insights into miRNA-based treatments for cancer., Competing Interests: The authors have declared that no competing interests exist.

Propofol is one of the most common intravenous anesthetics which may cause neuronal cell death in young mice. HOX transcript antisense RNA (HOTAIR) was abnormally expressed in neurodegenerative diseases. However, the effect of HOTAIR on propofol-induced pyroptosis of neurons and related mechanisms are still unknown. In this study, propofol treatment significantly reduced neuronal the viability of neurons, and promoted the expression of inflammation-related factors. Propofol treatment also promoted neuron death and neuronal pyroptosis. All the above effects might be related to the propofol-induced overexpression of HOTAIR. Interestingly, knockdown of HOTAIR by shRNA (sh-HOTAIR) significantly inhibited neuronal pyroptosis, but increased neuronal viability. Further analysis showed that HOTAIR and Nod-like receptor protein1 (NLRP1) were the targets of miR-455-3p, respectively. Notably, propofol treatment decreased the level of miR-455-3p, while increased the level of NLRP1. In addition, sh-HOTAIR increased the level of miR-455-3p, which further inhibited the expression of NLRP1 and the activation of NLRP1 inflammasome, thereby inhibiting neuronal pyroptosis. More importantly, NLRP1 overexpression decreased neuronal viability, and reactivated NLRP1 inflammasome, thus reversing the inhibitory effect of sh-HOTAIR on pyroptosis. Our findings indicated that HOTAIR inhibited propofol-induced pyroptosis of neurons by regulating miR-455-3p/NLRP1 axis, indicating that HOTAIR may be a potential therapeutic target for propofol-induced neurotoxicity.

Background: MicroRNAs (miRs) play critical roles in regulation of numerous biological events, including cardiac electrophysiology and arrhythmia, through a canonical RNA interference mechanism. It remains unknown whether endogenous miRs modulate physiologic homeostasis of the heart through noncanonical mechanisms., Methods: We focused on the predominant miR of the heart (miR1) and investigated whether miR1 could physically bind with ion channels in cardiomyocytes by electrophoretic mobility shift assay, in situ proximity ligation assay, RNA pull down, and RNA immunoprecipitation assays. The functional modulations of cellular electrophysiology were evaluated by inside-out and whole-cell patch clamp. Mutagenesis of miR1 and the ion channel was used to understand the underlying mechanism. The effect on the heart ex vivo was demonstrated through investigating arrhythmia-associated human single nucleotide polymorphisms with miR1-deficient mice., Results: We found that endogenous miR1 could physically bind with cardiac membrane proteins, including an inward-rectifier potassium channel Kir2.1. The miR1-Kir2.1 physical interaction was observed in mouse, guinea pig, canine, and human cardiomyocytes. miR1 quickly and significantly suppressed I K1 at sub-pmol/L concentration, which is close to endogenous miR expression level. Acute presence of miR1 depolarized resting membrane potential and prolonged final repolarization of the action potential in cardiomyocytes. We identified 3 miR1-binding residues on the C-terminus of Kir2.1. Mechanistically, miR1 binds to the pore-facing G-loop of Kir2.1 through the core sequence AAGAAG, which is outside its RNA interference seed region. This biophysical modulation is involved in the dysregulation of gain-of-function Kir2.1-M301K mutation in short QT or atrial fibrillation. We found that an arrhythmia-associated human single nucleotide polymorphism of miR1 (hSNP14A/G) specifically disrupts the biophysical modulation while retaining the RNA interference function. It is remarkable that miR1 but not hSNP14A/G relieved the hyperpolarized resting membrane potential in miR1-deficient cardiomyocytes, improved the conduction velocity, and eliminated the high inducibility of arrhythmia in miR1-deficient hearts ex vivo., Conclusions: Our study reveals a novel evolutionarily conserved biophysical action of endogenous miRs in modulating cardiac electrophysiology. Our discovery of miRs' biophysical modulation provides a more comprehensive understanding of ion channel dysregulation and may provide new insights into the pathogenesis of cardiac arrhythmias.

Amyloid β (Aβ) is a crucial component of drusen, the hallmark of the early stage of age-related macular degeneration (AMD), and can cause retinal pigment epithelium (RPE) cell damage through activation of the inflammatory response. MicroRNAs play a critical role in inflammation. However, the mechanism underlying the effect of microRNAs on the NLRP3 inflammasome induced by Aβ remains poorly understood. In the present study, we demonstrated that Aβ 1-40 -mediated RPE damage by inducing a decrease in endogenous miR-191-5p expression. This led to the upregulation of its target gene, C/EBPβ. C/EBPβ acts as a transcription factor for NLRP3, promotes its transcription, and upregulates the downstream inflammatory factors Caspase-1 and IL-1β. Correspondingly, overexpression of miR-191-5p alleviated RPE cell injury by suppressing inflammation. The present study elucidates a novel transcriptional regulatory mechanism of the NLRP3 inflammasome. Our findings suggest an anti-inflammatory effect of miR-191-5p in Aβ 1-40 -induced RPE impairment, shedding light on novel preventive or therapeutic approaches for AMD-associated RPE impairment., (© 2021 Federation of American Societies for Experimental Biology.)

Lately, long non-coding RNA (lncRNA) is recognized as a key regulator of gastric cancer (GC) which has aroused great interest in the fields of medicine, toxicology, and functional food. Studies related to LncRNA expression microarray data indicate that BX357664 is down-regulated in GC specimens. However, the expression pattern and molecular mechanism of BX357664 in GC have not been studied so far. The purpose of this study was to investigate the expression of lncRNA BX357664 in GC and its function in GC cell lines. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the level of BX357664 in 50 pairs of cancer tissues and adjacent non-cancer tissues collected from GC patients. It was found that BX357664 level was lowered in cancer specimens than adjacent non-cancer tissues and correlated with tumor size and TNM stage. Also, we used cell counting kit 8 (CCK8), cell clone formation assay and transwell assay, which affirmed that up-regulation of BX357664 inhibited the proliferation, migration, and invasion of GC cells, but promoted apoptosis. In the dual-luciferase report analysis, BX357664 acted as a miR-183-3p ceRNA to target and regulate the expression of PTEN and affect the PI3K/AKT pathway. These results indicate that BX357664 can inhibit the proliferation and metastasis of GC through the miR-183-3p/PTEN/PI3K/AKT pathway, which may serve as potential targets for the treatment of GC in the future., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

As a naturally occurring class of gene regulators, microRNAs (miRNAs) have attracted much attention as promising targets for therapeutic development. However, RNAs including miRNAs have long been considered undruggable, and most efforts have been devoted to using synthetic oligonucleotides to regulate miRNAs. Encouragingly, recent findings have revealed that miRNAs can also be drugged with small molecules that directly target miRNAs. In this review paper, we give a summary of recently emerged small-molecule inhibitors (SMIs) and small-molecule degraders (SMDs) for miRNAs. SMIs are small molecules that directly bind to miRNAs to inhibit their biogenesis, and SMDs are bifunctional small molecules that upon binding to miRNAs induce miRNA degradation. Strategies for discovering SMIs and developing SMDs were summarized. Applications of SMIs and SMDs in miRNA inhibition and cancer therapy were also introduced. Overall, SMIs and SMDs introduced here have high potency and specificity in miRNA inhibition. We envision that these small molecules will pave the way for developing novel therapeutics toward miRNAs that were previously considered undruggable.

Background: Interferon alfa (IFN-α) has been proved effective in treating chronic hepatitis B (CHB), owing to its ability to suppress hepatitis B surface antigen and hepatitis B virus (HBV) covalently closed circular DNA. However, the underlying mechanisms are unclear., Methods: We investigated the antiviral activities of exosomes from responders and nonresponders to pegylated IFN-α (PegIFN-α) as well as the supernatants of IFN-α-treated macrophages derived from THP-1 (the human leukemia monocyte cell line). Then the expression profiles of exosomal microRNAs (miRNAs) were analyzed using miRNA sequencing. The luciferase reporter assay was used to locate the binding position of HBV genomic sequence targeted by the identified miRNA., Results: Exosomes from PegIFN-α-treated patients, particularly responders, as well as the supernatants of IFN-α-treated macrophages exhibited anti-HBV activities, as manifested by the suppression of hepatitis B surface antigen, hepatitis B e antigen, HBV DNA, and covalently closed circular DNA levels in HBV-related cell lines. PegIFN-α treatment up-regulated exosomal hsa-miR-193a-5p, hsa-miR-25-5p, and hsa-miR-574-5p, which could partially inhibit HBV replication and transcription, and hsa-miR-574-5p reduced pregenomic RNA and polymerase messenger RNA levels by binding to the 2750-2757 position of the HBV genomic sequence., Conclusions: Exosomes can transfer IFN-α-related miRNAs from macrophages to HBV-infected hepatocytes, and they exhibit antiviral activities against HBV replication and expression., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Competing endogenous RNAs (ceRNAs) are transcripts with the ability to competitively titrate microRNAs (miRNAs) against miRNA repressing target genes to post-transcriptionally regulate the expression of corresponding miRNAs. It is a newly discovered gene regulation pattern between longer RNA and miRNA molecules. Recent research has gradually revealed the functional significance of ceRNAs in regulating normal development and stress response processes in plants and animals, as well as in cancer genesis and metastasis. Therefore, ceRNA identification is an important and necessary step to deepen our understanding of the regulation mechanisms of various biological processes. Here, we provide a pipeline used to computationally identify plant ceRNAs and reconstruct ceRNA regulatory networks based on RNA-seq and small RNA-seq data., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

The purpose of this study was to investigate the role of a human lens microRNA (miR-497-5p) in regulating epithelialmesenchymal transition (EMT) under the control of transforming growth factor beta (TGF-β). A microRNA array was used to evaluate the microRNA profiles of untreated and TGF-β-treated human lens epithelial cells in culture. This showed that TGF-β treatment led to the upregulation of 96 microRNAs and downregulation of 39 microRNAs. Thirteen microRNAs were predicted to be involved in the pathogenesis of posterior capsule opacification (PCO). Meanwhile, overexpression of miR-497-5p suppressed cell proliferation and EMT 48 h post-transfection, and inhibition of miR-497-5p accelerated cell proliferation and EMT. Treatment with TGF-β inhibited the expression of miR-497-5p, but not cell proliferation. miR-497-5p was also found to regulate the level of CCNE1 and FGF7, which are reported to be actively involved in EMT. CCNE1 and FGF7 were bona fide targets of miR-497-5p. The results suggest that miR-497-5p participates in the direct regulation of lens epithelial cell EMT and is regulated by TGF-β. miR-497-5p may be a novel target for PCO therapy.

The antioxidant activity of Spirulina platensis polysaccharide (SPP) was investigated in Caenorhabditis elegans. Nuclear magnetic resonance, Fourier-transform infrared, multi-angle laser light scattering, and GC-MS were used to identify the structural characteristics of SPP. It was composed of the →2)-α-L-Rhap-(1→, →4)-β-D-Manp-(1→, →6)-β-D-Glcp-(1→, →4)-β-Xylp-(1→, →3)-β-L-Araf-(1→, and →2)-β-L-Fucp-(1→, respectively. The modulation of gut microbiota in C. elegans was determined using 16S rRNA gene high-throughput sequencing. The malondialdehyde (MDA) content was significantly decreased, while the total superoxide dismutase (T-SOD) and reactive oxygen species (ROS) levels were improved after SPP supplementation. The cellular mitochondrial content and apoptosis were significantly down-regulated. The obvious increased levels of the DAF-16 and SKN-1 mRNAs were observed in the SPP-treated group, while the levels of miR-48 and miR-51 were significantly reduced. Moreover, SPP administration significantly increased the abundance of Flavobacterium, Achromobacter, Empedobacter, Anaerolinea, and Pseudoalteromonas of the intestinal flora. Based on these results, S. platensis polysaccharides may be used as a functional food to ameliorate diseases related to oxidative stress., Competing Interests: Declaration of competing interest No conflict of interest exits in the submission of this manuscript, and manuscript was approved by all authors for publication. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously, and not under consideration for publication elsewhere., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Idiopathic pulmonary fibrosis (IPF), a progressive and fatal lung disease, usually leads to an irreversible distortion of the pulmonary structure. The functional roles of bone marrow-derived mesenchymal stem cells (BMSC)-secreted extracellular vesicles (EVs) in fibroblasts have been implicated, yet their actions in the treatment of IPF are not fully understood. This study investigated the roles of BMSC-derived EVs expressing miR-29b-3p in fibroblasts in IPF treatment. EVs derived from BMSCs were successfully isolated and could be internalized by pulmonary fibroblasts, and Cell Counting Kit-8 (CCK-8) and Transwell assay results identified that EVs inhibited the activation of fibroblast in IPF. miR-29b-3p, frizzled 6 (FZD6), α-skeletal muscle actin (α-SMA), and Collagen I expressions were examined, which revealed that miR-29b-3p was poorly expressed and FZD6, α-SMA, and Collagen I were overexpressed in pulmonary tissues. Dual-luciferase reporter assay results demonstrated that miR-29b-3p could inversely target FZD6 expression. The gain- and loss-of-function assays were conducted to determine regulatory effects of FZD6 and miR-29b-3p on IPF. CCK-8 and Transwell assays results displayed that BMSCs-derived EVs overexpressing miR-29b-3p contributed to inhibited pulmonary interstitial fibroblast proliferation, migration, invasion, and differentiation. Furthermore, the effects of BMSCs-derived EVs overexpressing miR-29b-3p on IPF progression were assessed in vivo, which confirmed the repressive effects of BMSCs-derived EVs overexpressing miR-29b-3p on IPF progression. Collectively, BMSCs-derived EVs overexpressing miR-29b-3p relieve IPF through FZD6., (© 2020 Wiley Periodicals LLC.)

Myocardial ischemia/reperfusion (I/R) injury is a clinically fatal disease, caused by restoring myocardial blood supply after a period of ischemia or hypoxia. However, the underlying mechanism remains unclear. Recently, increasing evidence reveal that microRNAs (miRs) participate in myocardial I/R injury. This study aimed to investigate whether miR-128-1-5p contributed to cardiomyocyte apoptosis induced by myocardial I/R injury. Here, we showed that the expression of miR-128-1-5p was decreased in mice following myocardial I/R injury. Down-regulation of miR-128-1-5p was also showed in H9c2 cardiomyocytes after hypoxia/reoxygenation (H/R), and in neonatal rat cardiomyocytes (NRCMs) with H 2 O 2 treatment. Importantly, we found that overexpression of miR-128-1-5p ameliorates cardiomyocyte apoptosis both in H9c2 cardiomyocytes and NRCMs. Moreover, we also found that growth arrest DNA damage-inducible gene 45 gamma (Gadd45g) is identified as a direct target of miR-128-1-5p, which negatively regulated Gadd45g expression. Additionally, silencing of Gadd45g inhibits cardiomyocyte apoptosis in H9c2 cardiomyocytes and NRCMs. These results reveal a novel mechanism by which miR-128-1-5p regulates Gadd45g-mediated cardiomyocyte apoptosis in myocardial I/R injury., 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 © 2020 Elsevier Inc. All rights reserved.)

Pancreatic cancer (PC) is a lethal cancer in the digestive system. microRNAs (miRNAs) have been demonstrated to participate in PC progression. In this context, we, thus, aimed to explore the mechanism of miR-382 in epithelial mesenchymal transition (EMT) and lymph node metastasis in PC in relation to Anxa3 and the PI3K/Akt signaling pathway. Gene expression data sets GSE16515, GSE71989, and GSE32676 were screened out, with the findings showing the significance of miR-382 and annexin A3 (Anxa3) in PC. A total of 115 PC patients were selected for determination of miR-382 and Anxa3 expression with lowly expressed miR-382 and highly expressed Anxa3 found via RT-quantitative PCR and Western blot analysis. Additionally, negative correlation was found between miR-382 and Anxa3 in PC. Dual-luciferase reporter gene assay and in situ hybridization results confirmed that miR-382 negatively regulated Anxa3. miR-382 targeted Anxa3 and suppressed PC progression by blocking the PI3K/Akt signaling pathway. After a series of gain- and loss-of function approaches, upregulation of miR-382 or silencing of Anxa3 inhibited the EMT and lymph node metastasis, as evidenced by increased level of E-cadherin and decreased level of N-cadherin, vimentin, vascular endothelial growth factor(VEGFR)-3, VEGF-C, and VEGF-D. Overexpression of miR-382 or downregulation of Anxa3 was shown to inhibit colony formation, migration, and invasion abilities of PC cells. Further, tumor xenograft in nude mice in vivo also confirmed the inhibitory role of miR-382 and silenced Anxa3 in lymph node metastasis in PC. Thus, this study provides promising therapeutic targets for PC treatment. NEW & NOTEWORTHY This study focused on the mechanism of miR-382 in epithelial mesenchymal transition and lymph node metastasis in PC in relation to Anxa3 and the PI3K/Akt signaling pathway. We found the inhibitory role of miR-382 in PC in vitro and in vivo.

Increasing evidence shows that aberrant lncRNAs expression contributes to the progression of gastric cancer (GC). The role of the novel lncRNA OIP5-AS1 and its underlying mechanisms in the growth of GC is largely unknown. Here we demonstrate for the first time that OIP5-AS1 expression was up-regulated in GC tissues and cell lines, which significantly correlated with unfavorable clinical characteristics and shorter survival. The results of in vitro and in vivo gain- and loss-of-function experiments indicate that OIP5-AS1 promoted cell proliferation and colony formation while inhibiting apoptosis of GC cells. OIP5-AS1 functioned as an endogenous sponge for miR-367-3p in GC cells. Restoration of miR-367-3p expression abolished the biological effects of OIP5-AS1 on GC cells. Moreover, we show that HMGA2 was a downstream target of miR-367-3p and mediated the effects of OIP5-AS1 on GC cells. OIP5-AS1 regulated the activities of the PI3K/AKT and Wnt/β-catenin pathways through HMGA2. In conclusion, OIP5-AS1 functions as an oncogenic lncRNA that promotes the progression of GC and may serve as a therapeutic target for managing GC., (Copyright © 2019 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Drosha-dependent canonical microRNAs (miRNAs) play a crucial role in the biological functions and development of cancer. However, the effects of Drosha-independent non-canonical miRNAs remain poorly understood. In our previous work, we found a set of aberrant miRNAs, including some upregulated miRNAs, called Drosha-independent noncanonical miRNAs, in Drosha-knockdown gastric cancer (GC) cells. Surprisingly, Drosha-silenced GC cells still retained strong malignant properties (e.g., proliferation ability and cancer stem cell (CSC) characteristics), indicating that aberrantly upregulated non-canonical miRNAs may play an important role in the maintenance of the malignant properties in GC cells that express low Drosha levels. Here, we report that miR-6778-5p, a noncanonical miRNA, acts as a crucial regulator for maintenance of CSC stemness in Drosha-silenced GC cells. MiR-6778-5p belongs to the 5'-tail mirtron type of non-canonical miRNAs and is transcript splice-derived from intron 5 of SHMT1 (coding cytoplasmic serine hydroxymethyltransferase). It positively regulates expression of its host gene, SHMT1, via targeting YWHAE in Drosha-knockdown GC cells. Similar to its family member SHMT2, SHMT1 plays a crucial role in folate-dependent serine/glycine inter-conversion in one-carbon metabolism. In Drosha wild type GC cells, SHMT2 mediates a mitochondrial-carbon metabolic pathway, which is a major pathway of one-carbon metabolism in normal cells and most cancer cells. However, in Drosha-silenced or Drosha low-expressing GC cells, miR-6778-5p positively regulates SHMT1, instead of SHMT2, thus mediating a compensatory activation of cytoplasmic carbon metabolism that plays an essential role in the maintenance of CSCs in gastric cancer (GCSCs). Drosha wild type GCSCs with SHMT2 are sensitive to 5-fluorouracil; however, Drosha low-expressing GCSCs with SHMT1 are 5-FU-resistant. The loss of miR-6778-5p or SHMT1 notably mitigates GCSC sphere formation and increases sensitivity to 5-fluorouracil in Drosha-knockdown gastric cancer cells. Thus, our study reveals a novel function of Drosha-independent noncanonical miRNAs in maintaining the stemness of GCSCs., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: To investigate whether miR-449a can regulate the biological functions of hepatocellular carcinoma (HCC) cells by targeting special AT-rich sequence binding protein 1 (SATB1)., Methods: qRT-PCR and western blot were carried out to detect the expression of miR-449a and SATB1 in normal human hepatocyte cell line HL-7702 and in HCC cells SMMC-7721, Hep3B, HepG2, and Bel-7402. miR-449a-mimics, miR-negative control (miR-NC), specifically inhibited SATB1 RNA (si-SATB1), specifically overexpressed SATB1 RNA (sh-SATB1), and negative control RNA (Si-NC) were transfected into the Hep3B and Bel-7402 cells. MTT assay, Transwell assay and flow cytometry were conducted to detect cell proliferation, invasion, and apoptosis. Dual luciferase reporter assay was performed to determine the relationship between miR-449a and SATB1., Results: miR-449a was highly but SATB1 was poorly expressed in HCC cells. According to the cell experiments, the up-regulation of miR-449a expression could inhibit the proliferation and invasion of HCC cells, promote their apoptosis, and significantly reduce SATB1 expression. The inhibition of SATB1 expression could inhibit the proliferation and invasion and promote apoptosis. The dual luciferase reporter assay confirmed that there was a targeted regulatory relationship between miR-449a and SATB., Conclusion: miR-449a can inhibit the proliferation and invasion of HCC cells and promote their apoptosis through the targeted regulation of SATB1, so it is expected to become a potential therapeutic target for this disease in clinical practice.

Clear cell renal cell carcinoma (ccRCC) is the most common RCC subtype with high metastasis, poor prognosis and conventional chemotherapy resistance. Prostate cancer associated transcript 1 (PCAT1) is an important lncRNA that was reported to be involved in cell proliferation, migration and invasion of several types of cancer cells. However, its role in ccRCC is still undetermined. This study found that PCAT1 levels were elevated in ccRCC tumors as well as several ccRCC cells, and knockdown of PCAT1 with siRNA (si-PCAT1) alleviated cell proliferation, migration and invasion of Caki-2 and ACHN cells. With bioinformatics analysis, dual-luciferase reported assay, RNA pull-down assay and Spearman's correlation analysis, we demonstrated that PCAT1 acted as a sponge for miR-656 and miR-539. Moreover, we found dual competitive interaction of miR-656/539 with PCAT1 and yes-associated protein (YAP), resulting in the identification of PCAT1-miR-656/539-YAP axis in Caki-2 and ACHN cells. With CCK-8 assay and transwell assay, miR-656/539 inhibitor or YAP overexpression could alleviate the effects of si-PCAT1 on the proliferation, migration and invasion of Caki-2 and ACHN cells. Our data indicated that PCAT1 promotes proliferation, migration and invasion of ccRCC cells by upregulating YAP via sponging miR-656 and miR-539. Taken together, this study provided a novel therapeutic target for ccRCC treatment.

Idiopathic pulmonary fibrosis (IPF) is a condition that results in the progressive deterioration of lung function with poor prognosis. The current study is aimed at exploring how microRNA-448 (miR-448) targeting ABCC3 affects fibroblast proliferation, apoptosis, and collagen synthesis of mice with IPF via the Jun N-terminal kinase (JNK) signaling pathway. Bioinformatics and dual-luciferase polymerase chain reaction were used to predict the relationship of miR-448 and ABCC3. The expression of miR-448 and ABCC3 was detected in IPF tissues. Using IPF mouse models, lung fibroblasts for the experiments were treated with miR-448 mimic, miR-448 inhibitor, si-ABCC3, or SP600125 (inhibitor of JNK) to evaluate the cell proliferation and apoptosis in response to miR-448. Reverse transcription quantitative polymerase chain reaction and western blot analysis were used to identify the expression of miR-448, ABCC3, and the activation of the JNK signaling pathway. ABCC3 was targeted and downregulated by miR-448 based on bioinformatics prediction and dual-luciferase reporter gene assay. Additionally, miR-448 was found to be highly expressed in IPF lung tissues with low expression levels of ABCC3. In response to the treatment of miR-448 mimic or si-ABCC3, lung fibroblasts exhibited decreased cell proliferation and increased apoptotic rates, whereas the miR-448 inhibitor reversed the conditions. Notably, we also found that miR-448 mimic inhibited the JNK signaling pathway. In conclusion, by using miR-448 to target and downregulate ABCC3 to block the JNK signaling pathway in mice with IPF, we found an increase in fibroblast apoptosis, inhibited cell proliferation, and decreased collagen synthesis of fibroblasts., (© 2019 Wiley Periodicals, Inc.)

Background: The molecular mechanisms of abnormal palatogenesis were investigated in this study. A key regulator, miR-106a-5p, and its target pathway were analyzed., Objectives: This research is trying to clarify the underlying mechanism of the modulation of miRNA transcription during the formation of cleft palate by 7T and 9.4T NMR metabolomic platforms., Method: Differentially expressed miRNAs and mRNAs were analyzed by microarray analysis and verified by qRT-PCR. The protein expression in TGFβ signaling pathways were analyzed by Western Blotting. The relationship between miR-106a-5p and TGFβ were analyzed by luciferase reporter assay. Cell apoptosis were analyzed by flow cytometer. And finally, the metabonomics were analyzed by NMR and multivariate data analysis models (MVDA)., Results: The expression of miR-106a-5p increased in cleft palatal tissue and negatively correlated with the protein level of Tgfbr2. The luciferase assay further proved that the tgfbr2 was a direct target of miR-106a-5p. In another aspect, miR-106a-5p increased apoptosis level in palatal mesenchymal cells, possibly because its inhibition of TGFβ signaling pathway. Moreover, low cholesterol and choline levels with high citric acid and lipid levels were observed by 7T and 9.4T NMR metabonomic analysis, which inferred the disorder of cell membrane synthesis in cleft palate formation. Furthermore, transformation from choline to phosphatidylcholine regulated by miR-106a-5p was also disrupted, resulting in phosphatidic choline synthesis disorder and reduced cell membrane synthesis., Conclusions: The regulatory mechanism of cleft palate was studied at transcriptional and metabolomics levels, which may provide important information in understanding the primary cause of this abnormality., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Circular RNA FOXO3 (CircFOXO3, also termed as Hsa&#95;circ&#95;0006404) is derived from exon 2 of forkhead box O3 (FOXO3) gene, and abnormal expression is shown in different diseases. However, whether circFOXO3 plays important roles in tumorigenesis and progression of prostate cancer (PCa) remains unclear. In this study, we found that circFOXO3 was up-regulated in both PCa tissues and serum samples. Moreover, circFOXO3 was positively correlated with the Gleason score in PCa samples. CircFOXO3 was observed to be up-regulated in Gleason score > 6 PCa samples compared with Gleason score = 6 PCa samples. Knock-down circFOXO3 could remarkably inhibit PCa cell cycle, proliferation and promote cell apoptosis in vitro. Furthermore, we demonstrated circFOXO3 could act as miR-29a-3p sponge to up-regulate SLC25A15 expression by bioinformatics analysis, dual-luciferase reporter assays and biotinylated RNA pull-down assays. SLC25A15 could reverse the tumour suppressing roles of knock-down circFOXO3 in PCa. Of note, we found that miR-29a-3p was down-regulated; however, SLC25A15 was overexpressed in PCa samples compared with normal tissues. In conclusion, circFOXO3 acts as a miR-29a-3p sponge to exhibit oncogenic activity that affects the cell cycle and cell apoptosis in PCa through transcriptional up-regulation of SLC25A15. Our analysis suggests circFOXO3 could act as promising prostate cancer biomarkers., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Colorectal cancer (CRC) is a form of cancer developing from either the colon or rectum. Nowadays, research supports the functionality of exosome expressing microRNAs (miRNAs) as potential biomarker for various cancers including CRC. This study was performed with the intent of investigating the roles of both bone marrow-derived mesenchymal stem cells (BMSCs) and exosomal miR-16-5p in CRC by regulating integrin α2 (ITGA2). A microarray-based analysis was conducted to screen the CRC-associated differentially expressed genes (DEGs) as well as potential regulatory miRNAs. Next, the role of miR-16-5p in terms of its progression in association with CRC was determined. Subsequently, CRC cells were exposed to exosomes secreted by BMSCs transfected with miR-16-5p, isolated and cocultured with CRC cells in an attempt to identify the role of exosomes. Effects of BMSCs-derived exosomes overexpressing miR-16-5p on biological functions of CRC cells and tumorigenicity were all subsequently detected. Effects of miR-16-5p treated with CRC cells in regard to CRC in vivo were also measured. ITGA2 was overexpressed, while miR-16-5p was poorly expressed in CRC cells and miR-16-5p targeted ITGA2. The in vitro experiments revealed that the BMSCs-derived exosomes overexpressing miR-16-5p inhibited proliferation, migration, and invasion, while simultaneously stimulating the apoptosis of the CRC cells via downregulation of ITGA2. Furthermore, the results of in vivo experiments confirmed that the BMSCs-derived exosomes overexpressing miR-16-5p repressed the tumor growth of CRC. Collectively, BMSCs-derived exosomes overexpressing miR-16-5p restricted the progression of CRC by downregulating ITGA2., (© 2019 Wiley Periodicals, Inc.)

The "competing endogenous RNA (ceRNA) hypothesis" has recently been proposed for a new type of gene regulatory model in many organisms. Anther development is a crucial biological process in plant reproduction, and its gene regulatory network (GRN) has been gradually revealed during the past two decades. However, it is still unknown whether ceRNAs contribute to anther development and sexual reproduction in plants. We performed RNA and small RNA sequencing of anther tissues sampled at three developmental stages in two maize lines. A total of 28,233 stably transcribed loci, 61 known and 51 potentially novel microRNAs (miRNAs) were identified from the transcriptomes. Predicted ceRNAs and target genes were found to conserve in sequences of recognition sites where their corresponding miRNAs bound. We then reconstructed 79 ceRNA-miRNA-target gene regulatory networks consisting of 51 known miRNAs, 28 potentially novel miRNAs, 619 ceRNA-miRNA pairs, and 869 miRNA-target gene pairs. More than half of the regulation pairs showed significant negative correlations at transcriptional levels. Several well-studied miRNA-target gene pairs associated with plant flower development were located in some networks, including miR156 - SPL , miR159 - MYB , miR160 - ARF , miR164 - NAC , miR172 - AP2 , and miR319 - TCP pairs. Six target genes in the networks were found to be orthologs of functionally confirmed genes participating in anther development in plants. Our results provide an insight that the ceRNA-miRNA-target gene regulatory networks likely contribute to anther development in maize. Further functional studies on a number of ceRNAs, miRNAs, and target genes will facilitate our deep understanding on mechanisms of anther development and sexual plants reproduction.

Cancer-associated fibroblasts (CAFs) remain active even in the absence of cancer cells. However, the molecular mechanism underlying the sustained active status of CAFs is largely unrevealed. We found that in CAFs, DNMT3B was not only a target of miR-200b, miR-200c and miR-221, but was able to induce DNA methylation of miR-200s promoters. DNMT3B eventually reached a stably high level by the counteracting effect of decreasing miR-200b/c and increasing miR-221 in normal fibroblasts (NFs) with long-term exogenous TGF-β1 treatment, and DNMT3B further led to a low level of miR-200s which established CAF activation. Meanwhile, miR-200s/miR-221/DNMT3B signaling sustained autocrine TGF-β1 maintaining active CAF status. Destruction of the autocrine TGF-β1/miR-200s/miR-221/DNMT3B signaling led to demethylation of miR-200s promoters and further restored the NF phenotypes. Moreover, we confirmed that TCF12, the target of miR-141, stimulated c-Myc/Cyclin D1 axis in breast cancer cells to promote cancer growth by enhancing CXCL12 of CAFs. The current study reveals that the TGF-β1/miR-200s/miR-221/DNMT3B regulatory loop is responsible for the maintenance of CAFs status and is also necessary for CAF function in promoting malignance of breast cancer, which provides a potential target for CAF-driven therapeutic strategy., (Copyright © 2019. Published by Elsevier B.V.)