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To find potential biomarkers based on miRNA and their potential targets in splenic monocytes in burn-injured mice. Male Balb/c mice were subjected to sham or scalding injury of 15% total body surface area. Spenic CD11b+ monocytes were purified with magnetic beads. The monocytes were cultured in the presence of lipopolysaccharide. The proliferation of monocytes was detected by MTT assay, and the cytokines in the supernatant were examined by enzyme linked immunosorbent assay. The purified monocytes were also under total RNA extraction. The differential monocytic miRNAs expression between the sham and burn-injured mice was analysed by miRNA microarray. The activity of monocytes was comparable between the two groups (p > 0.05). However, monocytes from burn-injured mice secreted higher levels of tumour necrosis factor (TNF)-α and transforming growth factor-β, but lower level of monocyte chemoattratctant protein-1. A total of 54 miRNAs were differentially expressed in monocytes from burn relative to sham-injured mice (fold >3). Further quantitative reverse transcription polymerase chain reaction confirmed that the expression of miR-146a was significantly down-regulated, while miR-3091-6p was up-regulated after burn injury. Using the combination of Miranda and TargetScan softwares, we found that mir-146a may regulate 180 potential target genes including TNF receptor related factor 6 (TRAF6), interleukin-1 receptor related kinase 1 (IRAK1) and CD28. Mir-3091-6p may regulate 39 potential targets, including SOCS7 (cytokine signal transduction inhibitor 7) and ARRB2 (arrestin, β 2). The miRNAs expressed by monocytes after burn injury may be involved in the regulation of innate immune response in burn injury., (© 2023 The Authors. International Wound Journal published by Medicalhelplines.com Inc and John Wiley & Sons Ltd.)

Fibrosis is a hallmark of atrial structural remodeling. The main aim of this study was to investigate the role of micro-ribonucleic acids (miRNAs) in the modulation of fibrotic molecular mechanisms in response to hypoxic conditions, which may mediate atrial fibrosis. Under a condition of hypoxia induced by a hypoxia chamber, miRNA arrays were used to identify the specific miRNAs associated with the modulation of fibrotic genes. Luciferase assay, real-time polymerase chain reaction, immunofluorescence and Western blotting were used to investigate the effects of miRNAs on the expressions of the fibrotic markers collagen I and III (COL1A, COL3A) and phosphorylation levels of the stress kinase c-Jun N-terminal kinase (JNK) pathway in a cultured HL-1 atrial cardiomyocytes cell line. COL1A and COL3A were found to be the direct regulatory targets of miR-let-7a, miR-let-7e and miR-133a in hypoxic atrial cardiac cells in vitro. The expressions of COL1A and COL3A were influenced by treatment with miRNA mimic and antagomir while hypoxia-induced collagen expression was inhibited by the delivery of miR-133a, miR-let-7a or miR-let-7e. The JNK pathway was critical in the pathogenesis of atrial fibrosis. The JNK inhibitor SP600125 increased miRNA expressions and repressed the fibrotic markers COL1A and COL3A. In conclusion, MiRNA let-7a, miR-let-7e and miR-133a play important roles in hypoxia-related atrial fibrosis by inhibiting collagen expression and post-transcriptional repression by the JNK pathway. These novel findings may lead to the development of new therapeutic strategies.

Objective: Ketamine is an anesthetic that induces neurotoxicity when administered at high doses. In this work, we explored the protective effects of lipoxin A 4 methyl ester (LXA 4 ME) against ketamine-induced neurotoxicity and the underlying protective mechanism in pheochromocytoma (PC12) cells., Methods: PC12 cells were treated with 50 μM of ketamine and different LXA 4 ME concentrations of LXA4 ME (5-50 nM) for 24 h, and their viability, apoptosis, and oxidative status were assessed., Results: Quantitative real-time polymerase chain reaction experiments showed that ketamine downregulated miR-22 expression and upregulated Bcl-2-associated athanogene 5 (BAG5) in PC12 cells in a concentration-dependent manner. LXA 4 ME induced the opposite effects, thus attenuating ketamine-induced neurotoxicity. Further in vitro assays showed that miR-22 directly targeted BAG5, thus promoting cell viability by suppressing cell apoptosis and oxidative stress. Under expression miR-22 or upregulation of BAG5 antagonized the effects of LXA 4 ME., Conclusion: LXA 4 ME can protect PC12 cells from ketamine-induced neurotoxicity by activating the miR-22/BAG5 signaling pathway. Thus, LXA 4 ME can be used as a protective drug against ketamine-induced neural damage.

Cognitive impairment (CI) is not uncommon in dialysis patients. Various factors have been implicated. This study aims to examine mutual interaction of various clinical factors for CI in patients receiving hemodialysis. A total of 48 hemodialysis patients in outpatient clinic were recruited from 2015 to 2017. Demographics, circulating uremic toxin concentrations, miRNA concentrations, and nerve injury protein concentrations were collected. Clinical dementia rating (CDR) scores were used to stratify the functional scores of the patients. Receiver operating characteristic (ROC) analysis was used to evaluate diagnostic test performance for predicting dichotomous results, and cumulative ROC analysis was used to examine the combined contribution of clinical factors. CDR scale 0 included 15 patients (mean age, 59.1 years); CDR > 0.5 included 33 patients (mean age, 64.0 years). On cumulative ROC analysis, the major predictors of mild CI were hemoglobin, age, sex, homocysteine, neuron-specific enolase (NSE), and miR-486. The cumulative area under the curve (AUC) on combining hemoglobin, age, and miR-486 was the highest (0.897, 95% confidence interval 0.806-0.988). Two dichotomized variables reached 81.82% sensitivity and 86.67% specificity, with the likelihood ratio for positive and negative results being 6.14 and 0.21, respectively. In conclusion, hemoglobin, age, and miR-486 display high-degree combined effects on mild CI in patients receiving hemodialysis.

A recurrence of hepatocellular carcinoma (HCC) after living donor liver transplantation (LDLT) is one of the major concerns reflecting the higher mortality of HCC. This study aimed to explore the impact of circulating exosomes on HCC development and recurrence. One-shot transfusion of hepatoma serum to naïve rats induced liver cancer development with gradual elevation of alpha-fetoprotein (AFP), but exosome-free hepatoma serum failed to induce AFP elevation. The microarray analysis revealed miR-92b as one of the highly expressing microribonucleic acids in hepatoma serum exosomes. Overexpression of miR-92b enhanced the migration ability of liver cancer cell lines with active release of exosomal miR-92b. The hepatoma-derived exosomal miR-92b transferred to natural killer (NK) cells, resulting in the downregulation of CD69 and NK cell-mediated cytotoxicity. Furthermore, higher expression of miR-92b in serum exosomes was confirmed in HCC patients before LDLT, and its value at 1 month after LDLT was maintained at a higher level in the patients with posttransplant HCC recurrence. In summary, we demonstrated the impact of circulating exosomes on liver cancer development, partly through the suppression of CD69 on NK cells by hepatoma-derived exosomal miR-92b. The value of circulating exosomal miR-92b may predict the risk of posttransplant HCC recurrence., (© 2019 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Specificity protein1 (Sp1) is required for TGF-β-induced epithelial-to-mesenchymal transition (EMT) which has been demonstrated to aggravate the progression of cancer including lung cancer. microRNA-29c (miR-29c) is identified to inhibit EMT, but the correlation between miR-29c and Sp1 in human lung cancer remain incompletely clarified. Here, we confirmed decreased expression of miR-29c and enhanced expression of Sp1 in lung cancer tissues (n = 20) and found that Sp1 could be targeted and inhibited by miR-29c. Besides, the expression of miR-29c was down-regulated in high-metastatic lung cancer cell lines and TGF-β1-treated cells. The inhibition of miR-29c or overexpression of Sp1 in 95C and A549 cells dramatically enhanced the cell migration and invasion, and also induced the decrease in the expression of epithelial markers, e.g. thyroid transcription factor 1 (TTF-1) and E-cadherin, together with an increase in mesenchymal markers including vimentin, α-smooth muscle actin (α-SMA), which could be restored by overexpression of miR-29c mimics during the TGF-β-induced EMT. Moreover, dual-luciferase reporter assay was performed and the results indicated that miR-29c/Sp1 could form an auto-regulatory loop with TGF-β1, which impaired TGFB1 transcription. Furthermore, miR-29c overexpression could abrogate the tumor progression and inhibit the Sp1/TGF-β expressions in vivo, indicating that miR-29c could be a tumor suppressor and repress the Sp1/TGF-β axis-induced EMT in lung cancer.

We have previously reported that synthetic dsRNA can activate p21 expression by targeting the p21 promoter, thereby suppressing the proliferation of human bladder cancer cells. As complementarity between dsRNA and its target sequences is necessary for RNA activation, miRNAs may also trigger p21 expression through the same mechanism. Here, the expression levels of three miRNAs (miR-370, miR-1180 and miR-1236) decreased in bladder cancer tissues compared to healthy controls and the levels of these mRNAs positively correlated with p21 mRNA levels. The three miRNAs induced nuclear p21 expression through p21-promoter binding. Overexpression of the three miRNAs inhibited the proliferation of bladder cancer cells mainly by regulating p21. Therefore, these miRNAs could be candidates for anti-cancer drugs., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Medulloblastoma is the most frequent malignant central nervous system tumor in children. MicroRNAs (miRs) are small, non-coding RNAs that target protein-coding and non-coding RNAs, and play roles in a variety of cellular processes through regulation of multiple targets. In the present study, we analyzed miR-22 expression and its effect in cell proliferation and apoptosis in medulloblastomas. Quantitative reverse transcription PCR (RT-PCR) revealed significantly lower expression of miR-22 in 19 out of 27 (70%) medulloblastomas, D341, DAOY, ONS-76 medulloblastoma cell lines, compared with normal cerebellum. Forced expression of miR-22 by lentiviral vector transfection reduced cell proliferation and induced apoptosis, while knockdown of miR-22 increased proliferative activity in DAOY and ONS-76 cells. DAOY cells with miR-22 overexpression in nude mice yielded tumors smaller than those originated from control DAOY cells. Microarray analysis in DAOY cells with forced miR-22 expression showed significant changes in expression profiles, PAPST1 being the most significantly (10 folds) downregulated gene. Quantitative RT-PCR revealed PAPST1 mRNA upregulation in 18 out of 27 (67%) medulloblastomas. In addition, a luciferase reporter assay in ONS-76 and DAOY cells suggested that miR-22 directly targets the PAPST1 gene, and lentivirus-mediated knockdown of PAPST1 suppressed proliferation of DAOY and ONS-76 medulloblastoma cells. These results suggest that frequently downregulated miR-22 expression is associated with cell proliferation in medulloblastomas, and this may be at least in part via PAPST1, which is a novel target of miR-22., (© 2014 International Society of Neuropathology.)

Castration-resistant prostate cancer (CRPC) and its treatment are challenging issues in prostate cancer management. Here, we report that miR-663 is upregulated in CRPC tissues. Overexpression of miR-663 in prostate LNCaP cells promotes cell proliferation and invasion, neuroendocrine differentiation, and reduction in dihydrotestosterone-induced upregulation of prostate-specific antigen expression. Furthermore, results of in situ hybridization show that miR-663 expression is correlated with Gleason score and TNM stage and is an independent prognostic predictor of clinical recurrence. Together, these findings suggest that miR-663 is a potential oncomiR for CRPC and may serve as a tumor biomarker for the early diagnosis of CRPC., (© 2013 Wiley Periodicals, Inc.)

NRP1 as multifunctional non-tyrosine-kinase receptors play critical roles in tumor progression. MicroRNAs (miRNAs) are an important class of pervasive genes that are involved in a variety of biological functions, particularly cancer. It remains unclear whether miRNAs can regulate the expression of NRP1. The goal of this study was to identify miRNAs that could inhibit the growth, invasion and metastasis of gastric cancer by targeting NRP1 expression. We found that miR-338 expression was reduced in gastric cancer cell lines and in gastric cancer tissues. Moreover, we found that miR-338 inhibited gastric cancer cell migration, invasion, proliferation and promoted apoptosis by targeting NRP1 expression. As an upstream regulator of NRP1, miR-338 directly targets NRP1. The forced expression of miR-338 inhibited the phosphorylation of Erk1/2, P38 MAPK and Akt; however, the expression of phosphorylated Erk1/2, P38 MAPK and Akt was restored by the overexpression of NRP1. In AGS cells infected with miR-338 or transfected with SiNRP1, the protein levels of fibronectin, vimentin, N-cadherin and SNAIL were decreased, but the expression of E-cadherin was increased. The expression of mesenchymal markers in miR-338-expressing cells was restored to normal levels by the restoration of NRP1 expression. In vivo, miR-338 also decreased tumor growth and suppressed D-MVA by targeting NRP1. Therefore, we conclude that miR-338 acts as a novel tumor suppressor gene in gastric cancer. miR-338 can decrease migratory, invasive, proliferative and apoptotic behaviors, as well as gastric cancer EMT, by attenuating the expression of NRP1.

Small RNA programmed Argonautes are sophisticated cellular effector platforms known to be involved in a diverse array of functions ranging from mRNA cleavage, translational inhibition, DNA elimination, epigenetic silencing, alternative splicing and even gene activation. First observed in human cells, small RNA-induced gene activation, also known as RNAa, involves the targeted recruitment of Argonaute proteins to specific promoter sequences followed by induction of stable epigenetic changes which promote transcription. The existence of RNAa remains contentious due to its elusive mechanism. A string of recent studies in C. elegans provides unequivocal evidence for RNAa's fundamental role in sculpting the epigenetic landscape and maintaining active transcription of endogenous genes and supports the presence of a functionally sophisticated network of small RNA-Argonaute pathways consisting of opposite yet complementary "yin and yang" regulatory elements. In this review, we summarize key findings from recent studies of endogenous RNAa in C. elegans, with an emphasis on the Argonaute protein CSR-1.

microRNAs (miRNAs), defined as 21-24 nucleotide non-coding RNAs, are important regulators of gene expression. Initially, the functions of miRNAs were recognized as post-transcriptional regulators on mRNAs that result in mRNA degradation and/or translational repression. It is becoming evident that miRNAs are not only restricted to function in the cytoplasm, they can also regulate gene expression in other cellular compartments by a spectrum of targeting mechanisms via coding regions, 5' and 3'untransalated regions (UTRs), promoters, and gene termini. In this point-of-view, we will specifically focus on the nuclear functions of miRNAs and discuss examples of miRNA-directed transcriptional gene regulation identified in recent years.

It is largely recognized that microRNAs (miRNAs) function to silence gene expression by targeting 3'UTR regions. However, miRNAs have also been implicated to positively-regulate gene expression by targeting promoter elements, a phenomenon known as RNA activation (RNAa). In the present study, we show that expression of mouse Cyclin B1 (Ccnb1) is dependent on key factors involved in miRNA biogenesis and function (i.e. Dicer, Drosha, Ago1 and Ago2). In silico analysis identifies highly-complementary sites for 21 miRNAs in the Ccnb1 promoter. Experimental validation identified three miRNAs (miR-744, miR-1186 and miR-466d-3p) that induce Ccnb1 expression in mouse cell lines. Conversely, knockdown of endogenous miR-744 led to decreased Ccnb1 levels. Chromatin immunoprecipitation (ChIP) analysis revealed that Ago1 was selectively associated with the Ccnb1 promoter and miR-744 increased enrichment of RNA polymerase II (RNAP II) and trimethylation of histone 3 at lysine 4 (H3K4me3) at the Ccnb1 transcription start site. Functionally, short-term overexpression of miR-744 and miR-1186 resulted in enhanced cell proliferation, while prolonged expression caused chromosomal instability and in vivo tumor suppression. Such phenotypes were recapitulated by overexpression of Ccnb1. Our findings reveal an endogenous system by which miRNA functions to activate Ccnb1 expression in mouse cells and manipulate in vivo tumor development/growth.

MicroRNAs (miRNAs) are a class of small non-coding RNAs (ncRNAs) that regulate gene expression by repressing translation or triggering the degradation of complementary mRNA sequences. Certain miRNAs have been shown to function as integral components of the p53 and/or retinoblastoma (Rb) regulatory networks. As such, miRNA dysregulation can have a profound effect on cancer development. Previous studies have shown that miR-449a is down-regulated in human prostate cancer tissue and possesses potential tumor suppressor function. In the present study, we identify miR-449a-mediated growth arrest in prostate cancer cells is dependent on the Rb protein. We show that mutant Rb prostate cancer cells (DU- 145) are resistant to cell cycle arrest and cellular senescence induced by miR- 449a, while overexpression of wild-type Rb in DU-145 sublines (DU-1.1 and B5) restores miR-449a function. In silico analysis of 3'UTR regions reveal a putative miR-449a target site in the transcript of Cyclin D1 (CCND1); an oncogene involved in directly regulating Rb activity and cell cycle progression. Luciferase 3'UTR reporter constructs and inhibitory oligonucleotides confirm that Cyclin D1 is a direct downstream target of miR-449a. We also reveal that miR-449a suppresses Rb phosphorylation through the knockdown of Cyclin D1 and previously validated target HDAC1. By targeting genes involved in controlling Rb activity, miR- 449a regulates growth and senescence in an Rb-dependent manner. These data indicate that miR-449a is a miRNA component of the Rb pathway and its tumor suppressor-like effects, in part, depends on Rb status in prostate cancer cells.

We have reported previously that adipose tissue-derived stem cells (ADSC) could be induced by isobutylmethylxanthine (IBMX) to differentiate into neuron-like cells and such differentiation was mediated by insulin-like growth factor I (IGF-I) signaling. In the present study we show that both IBMX and IGF-I upregulated neural markers beta-III-tubulin and paired-like homeobox transcription factor (Pitx3) in ADSC. Because Pitx3 is important for the maturation and function of dopaminergic neurons and has been shown to be regulated by microRNA miR-133b, we also examined the effects of IBMX and IGF-I on miR-133b expression. The results show that both IBMX and IGF-I upregulated miR-133b expression. When miR-133b was overexpressed in ADSC through transfection of a synthetic microRNA mimic, it caused a downregulation of beta-III-tubulin as evidenced by immunofluorescence staining and western blot analysis. Overexpression of miR-133b also downregulated Pitx3 and IGF-1 receptor (IGF-IR), and all such downregulation occurred at the protein but not the RNA level. The apparent posttranscriptional regulation was subsequently found to be exerted through a potential miR-133b target in the 3'-untranslated region of IGF-IR, as evidenced by luciferase assay. Thus, IGF-I signaling and miR-133b co-regulate potential neural differentiation of ADSC through a feedback mechanism, in which IGF-I upregulates miR-133b while miR-133b in turn downregulates IGF-IR.

MicroRNAs (miRNAs) are approximately 22 nucleotides long, noncoding RNAs that control cellular function by either degrading mRNAs or arresting their translation. To understand their functional significance in ischemic pathophysiology, we profiled miRNAs in adult rat brain as a function of reperfusion time after transient middle cerebral artery occlusion. Of the 238 miRNAs evaluated, 8 showed increased and 12 showed decreased expression at least at 4 out of 5 reperfusion time points studied between 3 h and 3 days compared with sham. Of those, 17 showed >5 fold change. Bioinformatics analysis indicated a correlation between miRNAs altered to several mRNAs known to mediate inflammation, transcription, neuroprotection, receptors function, and ionic homeostasis. Antagomir-mediated prevention of mir-145 expression led to an increased protein expression of its downstream target superoxide dismutase-2 in the postischemic brain. In silico analysis showed sequence complementarity of eight miRNAs induced after focal ischemia to 877 promoters indicating the possibility of noncoding RNA-induced activation of gene expression. The mRNA expression of the RNases Drosha and Dicer, cofactor Pasha, and the pre-miRNA transporter exportin-5, which modulate miRNA biogenesis, were not altered after transient middle cerebral artery occlusion. Thus, the present studies indicate a critical role of miRNAs in controlling mRNA transcription and translation in the postischemic brain.

Small RNA molecules are now being recognized as master regulators of gene activity. Although known initially for their gene-silencing functions, recent discoveries have revealed that small RNAs can also activate gene expression. These two opposing activities of gene silencing and activation together constitute an integrated gene regulatory mechanism.

Recent studies have shown that microRNA (miRNA) regulates gene expression by repressing translation or directing sequence-specific degradation of complementary mRNA. Here, we report new evidence in which miRNA may also function to induce gene expression. By scanning gene promoters in silico for sequences complementary to known miRNAs, we identified a putative miR-373 target site in the promoter of E-cadherin. Transfection of miR-373 and its precursor hairpin RNA (pre-miR-373) into PC-3 cells readily induced E-cadherin expression. Knockdown experiments confirmed that induction of E-cadherin by pre-miR-373 required the miRNA maturation protein Dicer. Further analysis revealed that cold-shock domain-containing protein C2 (CSDC2), which possesses a putative miR-373 target site within its promoter, was also readily induced in response to miR-373 and pre-miR-373. Furthermore, enrichment of RNA polymerase II was detected at both E-cadherin and CSDC2 promoters after miR-373 transfection. Mismatch mutations to miR-373 indicated that gene induction was specific to the miR-373 sequence. Transfection of promoter-specific dsRNAs revealed that the concurrent induction of E-cadherin and CSDC2 by miR-373 required the miRNA target sites in both promoters. In conclusion, we have identified a miRNA that targets promoter sequences and induces gene expression. These findings reveal a new mode by which miRNAs may regulate gene expression.