The involvement of circulating miR-146a and miR-27a in patients with atherosclerotic cardiovascular disease after SARS-CoV-2 infection.

BACKGROUND: Atherosclerotic cardiovascular disease (ASCVD) is a group of clinical diseases based on pathology of atherosclerosis that is the leading cause of mortality worldwide. There is a bidirectional interaction between ASCVD and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Alterations in circulating miRNAs levels are involved in the development of ASCVD in patients infected with SARS-CoV-2, however, the correlation between ASCVD co-infection with SARS-CoV-2 and alterations of cardiac-specific miRNAs is not well understood. HYPOTHESIS: The circulating miR-146a and miR-27a are involved in bidirectional interactions between ASCVD and SARS-CoV-2 infections. METHODS: Circulating miR-146a and miR-27a levels were measured in serum and PBMCs deriving from ASCVD patients and controls after SARS-CoV-2 infection by qRT-PCR analysis. The levels of neutralizing antibodies-resistant SARS-CoV-2 in human serum was determined by competitive magnetic particle chemiluminescence method. Interleukin (IL)-6 levels were detected by automatic biochemical analyzer using electrochemiluminescence. RESULTS: Significant downregulation of circulating miR-146a and upregulation of miR-27a in ASCVD patients after infection with SARS-CoV-2 compared with controls were observed, among which the alterations were more evident in ASCVD patients comorbid with hyperlipidemia and diabetes mellitus. Consistently, correlation analysis revealed that serum miR-146a and miR-27a levels were associated with the levels of lipids and glucose, inflammatory response, and immune function in ASCVD patients. Remarkably, SARS-CoV-2 S protein RBD stimulation of PBMCs derived from both ASCVD and controls significantly downregulated miR-146a, upregulated miR-27a expression levels, and promoted IL-6 release in vitro. CONCLUSIONS: The circulating miR-146a and miR-27a are involved in metabolism, inflammation, and immune levels in patients with ASCVD after SARS-CoV-2 infection, laying the foundation for the development of strategies to prevent the risk of SARS-CoV-2 infection in ASCVD patients.

Gene Coexpression and miRNA Regulation: A Path to Early Intervention in Colorectal Cancer.

Early diagnosis and intervention are pivotal in reducing colorectal cancer (CRC) incidence and enhancing patient outcomes. In this study, we focused on three genes, AQP8, GUCA2B, and SPIB, which exhibit high coexpression and play crucial roles in suppressing early-stage CRC. Our objective was to identify key miRNAs that can mitigate CRC tumorigenesis and modulate the coexpression network involving these genes. We conducted a comprehensive analysis using large-scale tissue mRNA data from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus to validate the coexpression of AQP8, GUCA2B, and SPIB, and to assess their diagnostic and prognostic significance in CRC. The mRNA-miRNA interactions were examined using MiRNet and the Encyclopedia of RNA Interactomes. Furthermore, using various molecular techniques, we conducted miRNA inhibitor transfection experiments in HCT116 cells to evaluate their effects on cell growth, migration, and gene/protein expression. Our findings revealed that, compared with normal tissues, AQP8, GUCA2B, and SPIB exhibited high coexpression and were downregulated in CRC, particularly during tumorigenesis. OncoMirs, hsa-miR-182-5p, and hsa-miR-27a-3p, were predicted to regulate these genes. MiRNA inhibition experiments in HCT116 cells demonstrated the inhibitory effects of miR-27a-3p and miR-182-5p on GUCA2B mRNA and protein expression. These miRNAs promoted the proliferation of CRC cells, possibly through their involvement in the GUCA2B-GUCY2C axis, which is known to promote tumor growth. While the expressions of AQP8 and SPIB were barely detectable, their regulatory relationship with hsa-miR-182-5p remained inconclusive. Our study confirms that hsa-miR-27a-3p and hsa-miR-182-5p are oncomiRs in CRC. These miRNAs may contribute to GUCY2C dysregulation by downregulating GUCA2B, which encodes uroguanylin. Consequently, hsa-miR-182-5p and hsa-miR-27a-3p show promise as potential targets for early intervention and treatment in the early stages of CRC.

Berberine attenuates obesity-induced insulin resistance by inhibiting miR-27a secretion.

INTRODUCTION: Berberine (BBR) is an alkaloid found in plants. It has neuroprotective, anti-inflammatory and lipid-lowering activity. However, the efficacy of treatment with BBR and the mechanisms through which it acts need further study. AIMS: This study investigated the therapeutic effects and the mechanism of action of BBR on obesity-induced insulin resistance in peripheral tissues. METHODS: High-fat-fed C57BL/6J mice and low-fat-fed C57BL/6J mice with miR-27a overexpression were given BBR intervention (100 mg/kg, po), and the oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were performed. Palmitic acid-stimulated hypertrophic adipocyte models were treated with BBR (10 µM). Related indicators and protein expression levels were examined. RESULTS: The AUCs of the OGTT and the ITT in the BBR intervention group were reduced significantly (p < 0.01) (p < 0.05), and the serum biochemical parameters, including FBG, TC, TG and LDL-C were significantly reduced after BBR intervention. In the in vitro experiments, the triglyceride level and volume of lipid droplets decreased significantly after BBR intervention (p < 0.01) (p < 0.05). Likewise, BBR ameliorates skeletal muscle and pancreas insulin signalling pathways in vivo and in vitro. DISCUSSION: The results showed that BBR significantly ameliorated insulin resistance, reduced body weight and percent body fat and improved serum biochemical parameters in mice. Likewise, BBR reduced triglyceride level and lipid droplet volume in hypertrophic adipocytes, BBR improved obesity effectively. Meanwhile, BBR ameliorated the histomorphology of the pancreas, and skeletal muscle and pancreas insulin related signalling pathways of islets in in vitro and in vivo experiments. The results further demonstrated that BBR inhibited miR-27a levels in serum from obese mice and supernatant of hypertrophic adipocytes. miR-27a overexpression in low-fat fed mice indicated that miR-27a caused insulin resistance, and BBR intervention significantly improved the miR-27a induced insulin resistance status. CONCLUSION: This study demonstrates the important role of BBR in obesity-induced peripheral insulin resistance and suggest that the mechanism of its effect may be inhibition of miR-27a secretion.

Tumour necrosis factor α regulates the miR-27a-3p-Sfrp1 axis in a mouse model of osteoporosis.

Osteoporosis is a metabolic bone disease that involves gradual loss of bone density and mass, thus resulting in increased fragility and risk of fracture. Inflammatory cytokines, such as tumour necrosis factor α (TNF-α), inhibit osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), and several microRNAs are implicated in osteoporosis development. This study aimed to explore the correlation between TNF-α treatment and miR-27a-3p expression in BMSC osteogenesis and further understand their roles in osteoporosis. An osteoporosis animal model was established using ovariectomized (OVX) mice. Compared with Sham mice, the OVX mice had a significantly elevated level of serum TNF-α and decreased level of bone miR-27a-3p, and in vitro TNF-α treatment inhibited miR-27a-3p expression in BMSCs. In addition, miR-27a-3p promoted osteogenic differentiation of mouse BMSCs in vitro, as evidenced by alkaline phosphatase staining and Alizarin Red-S staining, as well as enhanced expression of the osteogenic markers Runx2 and Osterix. Subsequent bioinformatics analysis combined with experimental validation identified secreted frizzled-related protein 1 (Sfrp1) as a downstream target of miR-27a-3p. Sfrp1 overexpression significantly inhibited the osteogenic differentiation of BMSCs in vitro and additional TNF-α treatment augmented this inhibition. Moreover, Sfrp1 overexpression abrogated the promotive effect of miR-27a-3p on the osteogenic differentiation of BMSCs. Furthermore, the miR-27a-3p-Sfrp1 axis was found to exert its regulatory function in BMSC osteogenic differentiation via regulating Wnt3a-ß-catenin signalling. In summary, this study revealed that TNF-α regulated a novel miR-27a-3p-Sfrp1 axis in osteogenic differentiation of BMSCs. The data provide new insights into the development of novel therapeutic strategies for osteoporosis.

[Corrigendum] miR­27a promotes proliferation, migration, and invasion of colorectal cancer by targeting FAM172A and acts as a diagnostic and prognostic biomarker.

Following the publication of this article, an interested reader drew to the authors' attention that the western blots in Fig. 4B on p. 3560 and Fig. 6B on p. 3562 shared remarkably similar data (including both the GAPDH and the FAM172A blots in Fig. 4B), such that these data were likely to have been derived from the same original source. Upon asking the authors to provide an explanation, the authors realized that these errors inadvertently arose during the process of assembling these figures. Due to a mislabelling of the files, representative blots for FAM172A and GAPDH were chosen incorrectly for Fig. 4B. The authors had retained their original data, however, and were also able to present to the Editorial Office for our perusal the uncropped versions of their western blots, which resolved any other potential issues of anomalies associated with the data. The revised version of Fig. 4, now showing alternative data for Fig. 4B, is shown on the next page (note that, in the repeated experiment, relative to the original version of this figure the miR­27a, miR27a­inhibitor and negative control experiments were run on different lanes of the gel). Also note that the errors made in terms of assembling the data in Fig. 4 did not greatly affect either the results or the conclusions reported in this paper, and all the authors agree to the publication of this corrigendum. The authors regret that these errors went unnoticed prior to the publication of their article, are grateful to the Editor of Oncology Reports for granting them this opportunity to publish a corrigendum, and apologize to the readership for any inconvenience caused. [Oncology Reports 37: 3554­3564, 2017; DOI: 10.3892/or.2017.5592].

BMSC-derived Exosomes Ameliorate Peritoneal Dialysis-associated Peritoneal Fibrosis via the Mir-27a-3p/TP53 Pathway.

OBJECTIVE: Peritoneal fibrosis (PF) is the main cause of declining efficiency and ultrafiltration failure of the peritoneum, which restricts the long-term application of peritoneal dialysis (PD). This study aimed to investigate the therapeutic effects and mechanisms of bone marrow mesenchymal stem cells-derived exosomes (BMSC-Exos) on PF in response to PD. METHODS: Small RNA sequencing analysis of BMSC-Exos was performed by second-generation sequencing. C57BL/6J mice were infused with 4.25% glucose-based peritoneal dialysis fluid (PDF) for 6 consecutive weeks to establish a PF model. A total of 36 mice were randomly divided into 6 groups: control group, 1.5% PDF group, 2.5% PDF group, 4.25% PDF group, BMSC-Exos treatment group, and BMSC-Exos+TP53 treatment group. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed to measure the expression level of miR-27a-3p in BMSC-Exos and peritoneum of mice treated with different concentrations of PDF. HE and Masson staining were performed to evaluate the extent of PF. The therapeutic potential of BMSC-Exos for PF was examined through pathological examination, RT-qPCR, Western blotting, and peritoneal function analyses. Epithelial-mesenchymal transition (EMT) of HMrSV5 was induced with 4.25% PDF. Cells were divided into control group, 4.25% PDF group, BMSC-Exos treatment group, and BMSC-Exos+TP53 treatment group. Cell Counting Kit-8 assay was used to measure cell viability, and transwell migration assay was used to verify the capacity of BMSC-Exos to inhibit EMT in HMrSV5 cells. RESULTS: Small RNA sequencing analysis showed that miR-27a-3p was highly expressed in BMSC-derived exosomes compared to BMSCs. The RT-qPCR results showed that the expression of miR-27a-3p was upregulated in BMSC-Exos, but decreased in PD mice. We found that PF was glucose concentration-dependently enhanced in the peritoneum of the PD mice. Compared with the control mice, the PD mice showed high solute transport and decreased ultrafiltration volume as well as an obvious fibroproliferative response, with markedly increased peritoneal thickness and higher expression of α-SMA, collagen-I, fibronectin, and ECM1. The mice with PD showed decreased miR-27a-3p. Peritoneal structural and functional damage was significantly attenuated after BMSC-Exos treatment, while PF and mesothelial damage were significantly ameliorated. Additionally, markers of fibrosis (α-SMA, collagen-I, fibronectin, ECM1) and profibrotic cytokines (TGF-ß1, PDGF) were downregulated at the mRNA and protein levels after BMSC-Exos treatment. In HMrSV5 cells, BMSC-Exos reversed the decrease in cell viability and the increase in cell migratory capacity caused by high-glucose PDF. Western blotting and RT-qPCR analysis revealed that BMSC-Exos treatment resulted in increased expression of E-cadherin (epithelial marker) and decreased expression of α-SMA, Snail, and vimentin (mesenchymal markers) compared to those of the 4.25% PDF-treated cells. Importantly, a dual-luciferase reporter assay showed that TP53 was a target gene of miR-27a-3p. TP53 overexpression significantly reversed the decreases in PF and EMT progression induced by BMSC-Exos. CONCLUSION: The present results demonstrate that BMSC-Exos showed an obvious protective effect on PD-related PF and suggest that BMSC-derived exosomal miR-27a-3p may exert its inhibitory effect on PF and EMT progression by targeting TP53.

[Expression relationship and significance of NEAT1 and miR-27a-3p in serum and cerebrospinal fluid of patients with Alzheimer disease].

OBJECTIVE: To explore the expression relationship and significance of long chain non-coding RNA nuclear-enriched abundant transcript 1 (LncRNA NEAT1) and miR-27a-3p in serum and cerebrospinal fluid of patients with Alzheimer disease (AD). METHODS: Sixty-six AD patients received by the department of neurology of our hospital from October 2019 to September 2021 were gathered, according to the clinical dementia rating scale score, they were grouped into mild group (≤1 point, n=41) and moderate-to-severe group (>1 point, n=25). Another 66 cases of serum and cerebrospinal fluid samples from outpatient physical examination personnel were regarded as the control group. The general information on all subjects was recorded and cognition was assessed; real-time quantitative PCR was performed to measure the expression levels of miR-27a-3p and NEAT1 in serum and cerebrospinal fluid; enzyme-linked immunosorbent assay was performed to measure the protein levels of ß-amyloid precursor protein cleaving enzyme 1 (BACE1), ß-amyloid (Aß) 40 and Aß42 in cerebrospinal fluid; Spearman' s method was performed to analyze the correlation of serum miR-27a-3p and NEAT1 levels with mini-mental state examination (MMSE) and montreal cognitive assessment (MoCA) scores; Pearson method was performed to analyze the correlation between serum miR-27a-3p and NEAT1 levels and Aß deposition standard uptake value ratio (SUVR) and cerebrospinal fluid miR-27a-3p, NEAT1, BACE1, Aß42 and Aß40 levels. RESULTS: The MMSE score [21 (17, 25), 9(7, 11) vs. 27 (21, 34)], MoCA score [17 (12, 21), 10 (7, 13) vs. 27 (21, 31)], serum miR-27a-3p level (0.55±0.13, 0.46±0.06 vs. 0.97±0.22), cerebrospinal fluid miR-27a-3p (0.48±0.10, 0.35±0.10 vs. 1.03±0.31), Aß42 levels [(303.55±36.77) ng/L, (231.45±34.14) ng/L vs. (499.99±53.63) ng/L] and Aß42/Aß40 ratio (0.030±0.008, 0.022±0.007 vs. 0.048±0.010) of AD patients in mild group and moderate-to-severe group were all lower than those in the control group, and the moderate-to-severe group were lower than the mild group (all P < 0.05); the serum NEAT1 level (2.31±0.64, 3.13±0.76 vs. 1.05±0.20), SUVR (1.50±0.29, 1.76±0.52 vs. 0.74±0.15), and cerebrospinal fluid NEAT1 (3.51±1.24, 4.30±1.65 vs. 1.01±0.23) and BACE1 levels [(55.78±5.98) µg/L, (72.32±16.08) µg/L vs. (21.39±3.73) µg/L] were higher than those in the control group, and the moderate-to-severe group were higher than the mild group (all P < 0.05). Serum NEAT1 level in AD patients was positively correlated with SUVR, cerebrospinal fluid NEAT1 and BACE1 (r=0.350, 0.606, 0.341, P < 0.05), and negatively correlated with MMSE score and MoCA score (r=-0.473, -0.482, all P < 0.05); serum miR-27a-3p level was positively correlated with cerebrospinal fluid miR-27a-3p level, MMSE score and MoCA score (r=0.695, 0.424, 0.412, all P < 0.05), and negatively correlated with SUVR and cerebrospinal fluid BACE1 level (r=-0.521, -0.447, all P < 0.05). CONCLUSION: The expression trends of NEAT1 and miR-27a-3p in the serum and cerebrospinal fluid of AD patients are consistent, the level of NEAT1 is increased, and the level of miR-27a-3p is decreased. The levels of the two are negatively correlated, which is related to the degree of Aß deposition in the brain of AD patients and is involved in the progression of AD.

LncRNA BlncAD1 Modulates Bovine Adipogenesis by Binding to MYH10, PI3K/Akt Signaling Pathway, and miR-27a-5p/CDK6 Axis.

Research on adipogenesis will help to improve the meat quality of livestock. Long noncoding RNAs (lncRNAs) are involved in mammalian adipogenesis as epigenetic modulators. In this study, we analyzed lncRNA expression during bovine adipogenesis and detected 195 differentially expressed lncRNAs, including lncRNA BlncAD1, which was significantly upregulated in mature bovine adipocytes. Gain- and loss-of-function experiments confirmed that BlncAD1 promoted the proliferation, apoptosis, and differentiation of bovine preadipocytes. RNA pull-down revealed that the nonmuscle myosin 10 (MYH10) is a potential binding protein of BlncAD1. Then, we elucidated that loss of BlncAD1 caused increased ubiquitination of MYH10, which confirmed that BlncAD1 regulates adipogenesis by enhancing the stability of the MYH10 protein. Western blotting was used to demonstrate that BlncAD1 activated the PI3K/Akt signaling pathway. Bioinformatic analysis and dual-luciferase reporter assays indicated that BlncAD1 competitively absorbed miR-27a-5p. The overexpression and interference of miR-27a-5p in bovine preadipocytes displayed that miR-27a-5p inhibited proliferation, apoptosis, and differentiation. Further results suggested that miR-27a-5p targeted the CDK6 gene and that BlncAD1 controlled the proliferation of bovine preadipocytes by modulating the miR-27a-5p/CDK6 axis. This study revealed the complex mechanisms of BlncAD1 underlying bovine adipogenesis for the first time, which would provide useful information for genetics and breeding improvement of Chinese beef cattle.

miRNA-27a-3p is involved in the plasticity of differentiated hepatocytes.

BACKGROUND: Epigenetic mechanisms, including DNA methylation, histone modifications, and chromatin remodeling, are highly involved in the regulation of hepatocyte viability, proliferation, and plasticity. We have previously demonstrated that repression of H3K27 methylation in differentiated hepatic HepaRG cells by treatment with GSK-J4, an inhibitor of JMJD3 and UTX H3K27 demethylase activity, changed their phenotype, inducing differentiated hepatocytes to proliferate. In addition to the epigenetic enzymatic role in the regulation of the retro-differentiation process, emerging evidence indicate that microRNAs (miRNAs) are involved in controlling hepatocyte proliferation during liver regeneration. Hence, the aim of this work is to investigate the impact of H3K27 methylation on miRNAs expression profile and its role in the regulation of the differentiation status of human hepatic progenitors HepaRG cells. METHODS: A miRNA-sequencing was carried out in differentiated HepaRG cells treated or not with GSK-J4. Target searching and Gene Ontology analysis were performed to identify the molecular processes modulated by differentially expressed miRNAs. The biological functions of selected miRNAs was further investigated by transfection of miRNAs inhibitors or mimics in differentiated HepaRG cells followed by qPCR analysis, albumin ELISA assay, CD49a FACS analysis and EdU staining. RESULTS: We identified 12 miRNAs modulated by GSK-J4; among these, miR-27a-3p and miR- 423-5p influenced the expression of several proliferation genes in differentiated HepaRG cells. MiR-27a-3p overexpression increased the number of hepatic cells reentering proliferation. Interestingly, both miR-27a-3p and miR-423-5p did not affect the expression levels of genes involved in the differentiation of progenitors HepaRG cells. CONCLUSIONS: Modulation of H3K27me3 methylation in differentiated HepaRG cells, by GSK-J4 treatment, influenced miRNA' s expression profile pushing liver cells towards a proliferating phenotype. We demonstrated the involvement of miR-27a-3p in reinducing proliferation of differentiated hepatocytes suggesting a potential role in liver plasticity.

Identification and Validation of Lipid Metabolism Gene FASN-Associated miRNA in Wilms Tumor.

miRNA has been a research hotspot in recent years and its scope of action is very wide, involving the regulation of cell proliferation, differentiation, apoptosis, and other biological behaviors. This study intends to explore the role of miRNA in the lipid metabolism and development of Wilms tumor (WT) by detecting and analyzing the differences in the expression profiles of miRNAs between the tumor and adjacent normal tissue. Gene detection was performed in tumor tissues and adjacent normal tissues of three cases of WT to screen differentially expressed miRNAs (DEMs). According to our previous research, FASN, which participates in the lipid metabolism pathway, may be a target of WT. The starBase database was used to predict FASN-targeted miRNAs. The above two groups of miRNAs were intersected to obtain FASN-targeted DEMs and then GO Ontology (GO) functional enrichment analysis of FASN-targeted DEMs was performed. Finally, the FASN-targeted DEMs were compared and further verified by qRT‒PCR. Through gene sequencing and differential analysis, 287 DEMs were obtained, including 132 upregulated and 155 downregulated miRNAs. The top ten DEMs were all downregulated. Fourteen miRNAs targeted by the lipid metabolism-related gene FASN were predicted by starBase. After intersection with the DEMs, three miRNAs were finally obtained, namely, miR-107, miR-27a-3p, and miR-335-5p. GO enrichment analysis was mainly concentrated in the Parkin-FBXW7-Cul1 ubiquitin ligase complex and response to prostaglandin E. Further experimental verification showed that miR-27a-3p was significantly correlated with WT (P = 0.0018). Imbalanced expression of miRNAs may be involved in the occurrence and development of WT through lipid metabolism. The expression of miR-27a-3p is related to the malignant degree of WT, and it may become the target of diagnosis, prognosis, and treatment of WT in the later stage.

MIR27A rs895819 TC genotype increases risk of fluoropyrimidine-induced severe toxicity independently of DPYD variations.

Aim: MicroRNA 27a (miR-27a) regulates post-transcriptionally DPD activity. We have analyzed the association of MIR27A rs895819T>C variation, that modulates miR-27a expression, with fluropyrimidine-induced toxicity. Materials & methods: MIR27A rs895819T>C genotyping was conducted by TaqMan® allelic discrimination assay in 313 FP-treated cancer patients. Results: In overdominance (TC vs TT + CC), TC genotype was associated with grade 3-4 toxicity (p = 0.002), any grade toxicity (p = 0.052), and delayed drug administration or therapy discontinuation (p = 0.038). Odds of grade 3-4 toxicity were increased by both DPYD deficiency (OR: 8.923; p = 0.006) and MIR27A rs895819 TC genotype (OR: 3.865; p = 0.002). Conclusion: MIR27A rs895819 TC genotype is an independent risk factor for fluoropyrimidine-associated toxicity in the Greek population. Thus, MIR27A rs895819TC patients can be closely monitored for fluoropyrimidine-induced severe toxicity.

What is this summary about? To date, for cancer patients treated with fluoropyrimidines (capecitabine and 5-fluorouracil), analyzing DPYD gene can be used to guide the optimal dose of treatment in order to reduce the incidence of severe, even life threatening toxicity and thus, increase drug safety. However, the frequency of clinically significant DPYD variants is rare, below 5%, and there is therefore an urgent medical need to identify additional genes that can help in predicting response to fluoropyrimidines. Apart from changes in DPYD gene, noncoding RNAs modulate DPD enzymatic activity. MiR-27a is such a modulator. MIR27A rs895819 polymorphism affects miR-27a expression and is a potential candidate of fluoropyrimidine response. In this study, we have analyzed the association of MIR27A rs895819T>C polymorphism with fluoropyrimidine-induced toxicity in cancer patients. What were the results? MIR27A rs895819 TC genotype is associated with fluoropyrimidine-induced grade 3­4 toxicity, any grade toxicity and delayed drug administration or therapy discontinuation. Carrying MIR27A rs895819 TC genotype leads to over of threefold increased risk for grade 3­4 toxicity and can improve sensitivity of DPYD genotyping alone. What do the results mean? MIR27A rs895819 TC carriers should be closely monitored for fluoropyrimidine-induced severe toxicity. Fluoropyrimidine pharmacogenomics can help in improving drug safety and patient response.

Oxygen glucose deprivation-pretreated astrocyte-derived exosomes attenuates intracerebral hemorrhage (ICH)-induced BBB disruption through miR-27a-3p /ARHGAP25/Wnt/ß-catenin axis.

BACKGROUND: Blood brain barrier (BBB) breakdown is one of the key mechanisms of secondary brain injury following intracerebral hemorrhage (ICH). Astrocytes interact with endothelial and regulate BBB integrity via paracrine signaling factors. More and more studies reveal astrocyte-derived extracellular vesicles (ADEVs) as an important way of intercellular communication. However, the role of ADEV in BBB integrity after ICH remains unclear. METHODS: ADEVs were obtained from astrocytes with or without oxygen and glucose deprivation (OGD) pre-stimulation and the role of ADEVs in ICH was investigated using ICH mice model and ICH cell model. The potential regulatory effect of ADEVs on endothelial barrier integrity was identified by TEER, western blot and immunofluorescence in vitro. In vivo, functional evaluation, Evans-blue leakage and tight junction proteins (TJPs) expression were analyzed. MiRNA sequencing revealed that microRNA-27a-3p (miR-27a-3p) was differentially expressed miRNA in the EVs from OGD-pretreated astrocytes compared with normal control. The regulatory mechanism of miR-27a-3p was assessed using Luciferase assay, RT-PCR, western blot and immunofluorescence. RESULTS: OGD-activated astrocytes reduced hemin-induced endothelial hyper-permeability through secreting EVs. OGD-activated ADEVs alleviated BBB dysfunction after ICH in vivo and in vitro. MicroRNA microarray analysis indicated that miR-27a-3p is a major component that was highly expressed miRNA in OGD pretreated-ADEVs. OGD-ADEVs mitigated BBB injury through transferring miR-27a-3p into bEnd.3 cells and regulating ARHGAP25/Wnt/ß-catenin pathway. CONCLUSION: Taken together, these findings firstly revealed that miR-27a-3p, as one of the main components of OGD-pretreated ADEVs, attenuated BBB destruction and improved neurological deficits following ICH by regulating endothelial ARHGAP25/Wnt/ß-catenin axis. OGD-ADEVs might be a novel strategy for the treatment of ICH. this study implicates that EVs from OGD pre-stimulated astrocytes.

Exercise-Intervened Endothelial Progenitor Cell Exosomes Protect N2a Cells by Improving Mitochondrial Function.

We have recently demonstrated that exosomal communication between endothelial progenitor cells (EPCs) and brain endothelial cells is compromised in hypertensive conditions, which might contribute to the poor outcomes of stroke subjects with hypertension. The present study investigated whether exercise intervention can regulate EPC-exosome (EPC-EX) functions in hypertensive conditions. Bone marrow EPCs from sedentary and exercised hypertensive transgenic mice were used for generating EPC-EXs, denoted as R-EPC-EXs and R-EPC-EXET. The exosomal microRNA profile was analyzed, and EX functions were determined in a co-culture system with N2a cells challenged by angiotensin II (Ang II) plus hypoxia. EX-uptake efficiency, cellular survival ability, reactive oxygen species (ROS) production, mitochondrial membrane potential, and the expressions of cytochrome c and superoxide-generating enzyme (Nox4) were assessed. We found that (1) exercise intervention improves the uptake efficiency of EPC-EXs by N2a cells. (2) exercise intervention restores miR-27a levels in R-EPC-EXs. (3) R-EPC-EXET improved the survival ability and reduced ROS overproduction in N2a cells challenged with Ang II and hypoxia. (4) R-EPC-EXET improved the mitochondrial membrane potential and decreased cytochrome c and Nox4 levels in Ang II plus hypoxia-injured N2a cells. All these effects were significantly reduced by miR-27a inhibitor. Together, these data have demonstrated that exercise-intervened EPC-EXs improved the mitochondrial function of N2a cells in hypertensive conditions, which might be ascribed to their carried miR-27a.

Profiling of Serum miRNAs Constructs a Diagnostic 3-miRNA Panel for Clear-Cell Renal Cell Carcinoma.

BACKGROUND: Renal cell carcinoma (RCC) carries significant morbidity and mortality globally with an increasing incidence per year predominantly represented by clear-cell renal cell carcinoma (ccRCC) which accounts for 70-80% of all RCC cases. MicroRNAs(miRNAs) implicate tumor development and progression in epigenetic mechanisms and available profiling of serum miRNAs potentiate them as diagnostic markers for various cancers. MATERIALS AND METHODS: A total of 108 ccRCC patients and 112 normal controls were enrolled. A 3-stage experiment was conducted to identify differentially expressed serum miRNAs in ccRCC and establish a diagnostic miRNAs panel. Additionally, bioinformatic analysis was employed to predict selected miRNAs' target genes, preform functional annotation and explore the roles in ccRCC. RESULTS: MiR-429, miR-10a-5p, miR-154-5p were found to be up-regulated miRNAs. Inversely, miR-27a-3p and miR-221-3p were found to be down-regulated miRNAs. These 5 miRNAs were selected to construct diagnostic panel by backward stepwise logistic regression analysis and ultimately a 3-miRNA panel (miR-429, miR-10a-5p and miR-27a-3p) was established [area under the curve (AUC) = 0.897, sensitivity = 85.0%, specificity = 83.3%]. CONCLUSION: The panel of 3-miRNA holds promise as a novel, convenient, and noninvasive diagnostic method for early detection of ccRCC.

MicroRNA miR-27a as a possible regulator of anti-inflammatory macrophage phenotype in preeclamptic placenta.

INTRODUCTION: The role of the TGFß signaling pathway, an important cascade responsible for the anti-inflammatory polarization of macrophages, in the development of both early- and late-onset preeclampsia (eoPE and loPE), remains poorly understood. In this study, we examined the components of the TGFß signaling cascade and macrophage markers within placental tissue in normal pregnancy and in PE. METHODS: Patients with eoPE, loPE, and normal pregnancy were enrolled in the study (n = 10 in each group). Following techniques were used for the investigation: immunohistochemistry analysis, western blotting, qRT-PCR, isolation of monocytes by magnetic sorting, transfection, microRNA sequencing, and bioinformatic analysis. RESULTS: We observed a significant decrease in the anti-inflammatory macrophage marker CD206 in the loPE group, alongside with a significant down-regulation of CD206 protein production in both eoPE and loPE groups. The level of CD68-positive cells and relative levels of CD163 and MARCO production were comparable across the groups. However, we identified a significant decrease in the TGFß receptor 2 production and its gene expression in the PE group. Further analysis revealed a link between TGFBR2 and MRC1 (CD206) genes through a single miRNA, hsa-miR-27a-3p. Transfecting CD14-derived macrophages with the hsa-miR-27a-3p mimic significantly changed TGFBR2 production, indicating the potential role of this miRNA in regulating the TGFß signaling pathway. We also revealed the up-regulation of hsa-miR-27a-5p and hsa-miR-27a-3p in the trophoblast BeWo b30 cell line under the severe hypoxia condition and the fact that TGFBR2 3' UTR could serve as a potential target for these miRNAs. DISCUSSION: Our findings uncover a novel potential therapeutic target for managing patients with PE, significantly contributing to a deeper comprehension of the underlying mechanisms involved in the development of this pathology.

Knockdown of miR-27a reduces TGFß-induced EMT and H2O2-induced oxidative stress through regulating mitochondrial autophagy.

OBJECTIVES: The present research aimed at clarifying the role played by miR-27a in the context of intrauterine adhesion (IUA) by focusing on its impact on TGFß1-induced epithelial-mesenchymal transition (EMT), migration, oxidative stress, and mitochondrial autophagy in endometrial stromal cells (ESCs). METHODS: We employed the Cell Counting Kit CCK-8/WST-8 assay to assess ESC proliferation, flow cytometric analysis and an Annexin-V-FITCV-FITC Apoptosis Detection kit to determine cell apoptosis, and wound healing and transwell assays to evaluate cell migration. Besides, intracellular reactive oxygen species (ROS) levels measured by the Reactive Oxygen Species Assay Kit were analyzed by flow cytometry, and protein expression levels were quantified by Western blotting analysis. RESULTS: Knockdown of miR-27a inhibited TGFß1-induced EMT and H2O2-induced oxidative stress in ESCs. H2O2-induced miR-27a suppressed PINK1 expression, leading to inhibition of mitophagy. MiR-27a promoted TGFß1 or H2O2-induced EMT through PINK1. CONCLUSIONS: miR-27a plays a crucial role in endometrial fibrosis. It regulates TGFß1-induced EMT, migration, oxidative stress, and apoptosis in ESCs. Additionally, miR-27a impacts mitophagy through PINK1 suppression upon H2O2 induction. Our findings highlight miR-27a as a potential therapeutic target for IUA treatment, shedding light on its multifaceted involvement in the mechanism of intrauterine adhesion fibrosis.

MicroRNA­27a­3p regulates the proliferation and chemotaxis of pulmonary macrophages in non­small cell lung carcinoma tissues through CXCL2.

The present study aimed to investigate microRNA (miRNA)-27a-3p expression in the pulmonary macrophages and peripheral blood of patients with early non-small cell lung carcinoma (NSCLC) and its regulatory effect on the infiltration of pulmonary macrophages into cancer tissues and invasion of NSCLC cells. Blood specimens were withdrawn from 36 patients with NSCLC and 29 healthy subjects. NSCLC tissues and cancer-adjacent tissues were both obtained from patients with NSCLC; furthermore, certain tissue samples were used to extract macrophages. The levels of miRNA-27a-3p and C-X-C motif ligand chemokine 2 (CXCL2) mRNA were detected by reverse transcription-quantitative PCR and the levels of CXCL2 protein were measured by ELISA and western blot analysis. A dual-luciferase reporter assay was performed to determine the interactions between miRNA and mRNA. An MTT assay was employed to examine the viability of transfected cells and macrophages and a Transwell assay was performed to assess chemotaxis. The differential expression of miRNA-27a-3p in NSCLC tissues, pulmonary macrophages and peripheral blood indicated that miRNA-27a-3p exerted different roles in these specimens. CXCL2 was upregulated in NSCLC tissues at both transcriptional and translational levels. In addition, the untranslated region of CXCL2 was confirmed to be directly targeted by miRNA-27a-3p prior to its transcriptional activation. Furthermore, miRNA-27a-3p regulated CXCL2 expression, thereby affecting the proliferation of human pulmonary macrophages. The present study highlights that miRNA-27a-3p expression in the pulmonary macrophages and peripheral blood of patients with NSCLC is downregulated, while its target gene CXCL2 is upregulated. miRNA-27a-3p may regulate the viability and chemotaxis of macrophages in tumor tissues of patients with NSCLC through CXCL2 and is expected to become a genetic marker of this disease.

Genetic Variation in miR-27a Is Associated with Fluoropyrimidine-Associated Toxicity in Patients with Dihydropyrimidine Dehydrogenase Variants after Genotype-Guided Dose Reduction.

Dihydropyrimidine dehydrogenase (DPYD) is the rate-limiting enzyme involved in the metabolism of fluoropyrimidine-based chemotherapy. However, single-nucleotide variants (SNVs) in DPYD only partially explain fluoropyrimidine-induced toxicity. The expression of DPYD has previously been shown to be regulated by microRNA-27a (miR-27a) and a common miR-27a SNV (rs895819) has been associated with an increased risk of toxicity in patients harboring a DPYD variant who received standard fluoropyrimidine dosing. We investigated if the miR-27a rs895819 SNV was associated with toxicity in DPYD wildtype patients and carriers of DPYD variants who received a reduced dose. The regulation of DPYD using miR-27a was investigated in HepG2 cells utilizing a miR-27a mimic. miR-27a overexpression decreased DPYD mRNA expression compared to control cells (p < 0.0001). In a cohort of patients that received pre-emptive DPYD genotyping, 45 patients had a DPYD variant and 180 were wildtype. Patients heterozygous for rs895819 had an increased risk of toxicity, which was seen in both patients who were wildtype for DPYD variants (OR (95%CI) = 1.99 (1.00-3.99)) and DPYD variant carriers (OR (95%CI) = 8.10 (1.16-86.21)). Therefore, miR-27a rs895819 may be a clinically relevant predictor of fluoropyrimidine-associated toxicities. Furthermore, toxicity was more profound in DPYD variant carriers, even after DPYD genotype-guided dose reduction. This suggests that patients may benefit from miR-27a genotyping to guide fluoropyrimidine dosing.

Inhibition of inflammation and infiltration of M2 macrophages in NSCLC through the ATF3/CSF1 axis: Role of miR-27a-3p.

Non-small cell lung cancer (NSCLC) imposes a significant economic burden on patients and society due to its low overall cure and survival rates. Tumour-associated macrophages (TAM) affect tumour development and may be a novel therapeutic target for cancer. We collected NSCLC and tumour-adjacent tissue samples. Compared with the tumour-adjacent tissues, the Activation Transcription Factor 3 (ATF3) and Colony Stimulating Factor 1 (CSF-1) were increased in NSCLC tissues. Levels of ATF3 and CSF-1 were identified in different cell lines (HBE, A549, SPC-A-1, NCI-H1299 and NCI-H1795). Overexpression of ATF3 in A549 cells increased the expression of CD68, CD206 and CSF-1. Moreover, levels of CD206, CD163, IL-10 and TGF-ß increased when A549 cells were co-cultured with M0 macrophages under the stimulation of CSF-1. Using the starbase online software prediction and dual-luciferase assays, we identified the targeting between miR-27a-3p and ATF3. Levels of ATF3, CSF-1, CD206, CD163, IL-10 and TGF-ß decreased in the miR-27a mimics, and the tumour growth was slowed in the miR-27a mimics compared with the mimics NC group. Overall, the study suggested that miR-27a-3p might inhibit the ATF3/CFS1 axis, regulate the M2 polarization of macrophages and ultimately hinder the progress of NSCLC. This research might provide a new therapeutic strategy for NSCLC.