miR-21 Expression Research Articles

Abstract Tu001: MiR-21 mitigated pulmonary hypertension induced right ventricular dysfunction through pulmonary circulating exosomes

Background: Patients with pulmonary arterial hypertension (PAH) often experience adverse outcomes primarily due to right ventricular (RV) dysfunction. However, the intricate relationship between pulmonary circulation and RV remodeling remains largely unexplored. Exosomal microRNAs (miRNAs) function as paracrine signaling mediators in diverse diseases. Aims: To investigate the ability of exosomal miRNAs derived from pulmonary endothelial cells to transmit signals affecting cardiomyocytes. Methods and Results: Utilizing next-generation sequencing, we identified several target miRNAs by analyzing exosome samples obtained through right-heart catheterization. Notably, exosomal miR-21 exhibited the highest expression in the pulmonary circulation (Figure 1) . Under hypoxic conditions, the expression of miR-21 increased in human pulmonary endothelial cells (HPECs) and in exosomes derived from their conditioned medium. Treatment with these exosomes enhanced miR-21 expression and concurrently mitigated apoptosis in hypoxia-exposed cardiomyocytes. Likewise, miR-21 demonstrated a protective role against hypoxia-induced injuries and apoptosis, involving the SPRY-2/p-ERK and PTEN/Akt pathways. In a murine model of sugen/hypoxia-induced PAH, mice lacking miR-21 (miR-21 -/- ) exhibited more severe RV dysfunction, altered hemodynamics, and increased fibrosis compared to wild-type (WT) mice. Employing a parabiosis model, pairs consisting of WT and miR-21 -/- mice were shielded from RV dysfunction, whereas pairs of miR-21 -/- mice displayed exacerbated RV function (Figure 2) . Conclusion: Hypoxia stimulates the production of exosomal miR-21 in both HPECs and their microenvironment, subsequently mitigating apoptosis in cardiomyocytes (Figure 3) . Our findings propose endothelial cell-derived exosomal miR-21 as a promising therapeutic target for PAH.

MiR-21 attenuated inflammation targeting MyD88 in human chondrocytes stimulated with Hyaluronan oligosaccharides

Inflammation is the body's response to injuries, which depends on numerous regulatory factors. Among them, miRNAs have gained much attention for their role in regulating inflammatory gene expression at multiple levels. In particular, miR-21 is up-regulated during the inflammatory response and reported to be involved in the resolution of inflammation by down-regulating pro-inflammatory mediators, including MyD88. Herein, we evaluated the regulatory effects of miR-21 on the TLR-4/MyD88 pathway in an in vitro model of 6-mer HA oligosaccharides-induced inflammation in human chondrocytes.The exposition of chondrocytes to 6-mer HA induced the activation of the TLR4/MyD88 pathway, which culminates in NF-kB activation. Changes in miR-21, TLR-4, MyD88, NLRP3 inflammasome, IL-29, Caspase1, MMP-9, iNOS, and COX-2 mRNA expression of 6-mer HA-stimulated chondrocytes were examined by qRT-PCR. Protein amounts of TLR-4, MyD88, NLRP3 inflammasome, p-ERK1/2, p-AKT, IL-29, caspase1, MMP-9, p-NK-kB p65 subunit, and IKB-a have been evaluated by ELISA kits. NO and PGE2 levels have been assayed by colorimetric and ELISA kits, respectively.HA oligosaccharides induced a significant increase in the expression of the above parameters, including NF-kB activity. The use of a miR-21 mimic attenuated MyD88 expression levels and the downstream effectors. On the contrary, treatment with a miR-21 inhibitor induced opposite effects. Interestingly, the use of a MyD88 siRNA confirmed MyD88 as the target of miR-21 action.Our results suggest that miR-21 expression could increase in an attempt to reduce the inflammatory response, targeting MyD88.

AB062. Investigating miR-21 as a non-invasive prognostic biomarker in glioma patients.

Glioma prognosis remains a challenge due to its high recurrence and resistance to treatment. Diagnosis and follow-up in resource-constrained regions often lead to significant patient attrition. Serum microRNAs (miRNAs) are seen to be aberrantly expressed in malignancies can be found in tumor tissues and peripheral samples. This offers a pathway for non-invasive liquid biopsies. miR-21 is an established biomarker for glioma prognosis, which needs to be validated in our population. We collected 89 intraoperative tumor tissue samples, and 42 pre- and post-operative serum samples from glioma patients, ten control tissues, eight healthy serum samples and analysed for miR-21 expression through quantitative polymerase chain reaction (qPCR) analysis. Correlational analysis with molecular markers isocitrate dehydrogenase (IDH), Ki-67, ATRX, p53, and survival curves through the Kaplan-Meier method were calculated in high and low miR-21 expression groups. The hazard ratio was quantitatively determined using Cox regression analysis, considering both univariate associations and multivariate correlations with clinical parameters. miR-21 expression in tissue was significantly upregulated with increase of glioma grades (P<0.001) and in patients above 50 years (P=0.003) age group. Whereas no gender bias was seen in its expression pattern. Its expression did not show any correlation with tumor volume (r=0.22, P=0.08). A similar expression pattern of miR-21 was observed in serum samples of glioma. IDH-wildtype (P=2.06e-03) and high Ki-67 (P=2.50e-03) patient group showed significant upregulation of miR-21 expression compared to IDH-mutant and low Ki-67 group. Patients with low miR-21 expression had significantly longer overall survival (OS) than patients with high miR-21 expression (P =0.006). Similarly, quantitative hazard analysis indicates that patients in the high expression group have 2.77 times higher risk of mortality [95% confidence interval (CI): 0.19-0.92], in comparison to patients in the low expression group (P=0.008). Our findings validate the utility of miR-21 as a prognostic serum biomarker to help diagnose and assess treatment response in advancing glioma grades, within our population.

Evaluation of the Plasma Expression Levels of miR-21 and miR-145 as Potential Non-Invasive Biomarkers for Early Detection of Colorectal Cancer.

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers in the world. Early detection would be greatly enhanced if accurate and cost-effective diagnostic biomarkers for CRC were accessible. The development of blood tests would evidently lower the screening cost of CRC detection. The aim of the present study was to examine the prospective of plasma miRNAs as non-invasive biomarkers for CRC screening. The expressions of miR-21 and miR-145 in the plasma of colorectal adenocarcinomas and normal healthy controls were quantified by using TaqMan miRNA assays. MiRNA expression levels were also correlated with commonly used clinicopathological features of CRC. Out of 30 CRC patients, 19 were male and 11 were female. The Mean age of patients was 51.3 ±14.6 years. A statistically significant increase in expression of miR-21 was observed in CRC patients' as compared to healthy controls (p<0.001). A significant association between miR-21 expression and age group (p=0.002) was noticed. Also, a statistically significant difference (p=0.015) between miR-21 expression and tumor location in the proximal and distal sites of the colon was observed in CRC patients. Further, a statistically significant downregulation of miR-145 expression was observed in the plasma of CRC patients as compared to healthy controls (p<0.05). This is the first study to report a significant association between miR-21 expression, age group, and tumor location in CRC patients. The present study thus emphasises that the appraisal of miR-21 and miR-145 plasma levels may serve as a promising non-invasive screening tool for the early detection of colorectal cancer.

Expression of Circulating miR-21 and -29 and their Association with Myocardial Fibrosis in Hypertrophic Cardiomyopathy.

Hypertrophic Cardiomyopathy (HCM) is characterized by myocardial hypertrophy, fibrosis, and sarcomeric disarray. To evaluate the expression levels of circulating miR-21 and -29 in patients with HCM and their association with clinical characteristics and myocardial fibrosis. In this case-control study, 27 subjects with HCM, 13 subjects with hypertensive cardiomyopathy, and 10 control subjects were enrolled. Evaluation of patients' functional capacity was made by the six-minute walk test. Echocardiographic measurements of left ventricle systolic and diastolic function were conducted. Cardiac magnetic resonance late gadolinium enhancement (LGE) -through a semiquantitative evaluation- was used in the assessment of myocardial fibrosis extent in HCM patients. The expression of miR-21 and -29 in peripheral blood samples of all patients was measured via the method of quantitative reverse transcription polymerase chain reaction. Circulating levels of miR-21 were higher in both hypertensive and HCM (p<0.001) compared to controls, while expression of miR-29 did not differ between the three studied groups. In patients with HCM and LGE-detected myocardial fibrosis in more than 4 out of 17 myocardial segments, delta CT miR-21 values were lower than in patients with myocardial LGE in 3 or fewer myocardial segments (2.71 ± 1.06 deltaCT vs. 3.50 ± 0.55 deltaCT, p<0.04), indicating the higher expression of circulating miR-21 in patients with more extensive myocardial fibrosis. MiR-21 was overexpressed in patients with HCM and hypertensive cardiomyopathy. Importantly, in patients with HCM, more extensive myocardial fibrosis was associated with higher levels of miR-21.

M1 Macrophage-Derived Exosomal MiR-155 Enhances Autophagy in Sepsis-Triggered Acute Kidney Injury

Sepsis-induced Acute kidney injury (SA-AKI), a common complication in sepsis, significantly impacts patients’ health and quality of life. M1 macrophages have been demonstrated to release inflammatory mediators that exacerbate kidney injury. MiR-155 has been implicated in promoting inflammation and damage during sepsis while reducing miR-155 levels alleviates SA-AKI. However, the relationship between miR-155 and M1 macrophage-derived exosomes in regulating autophagy during SA-AKI remains unclear. In this study, we aim to investigate the relationship between M1 macrophage-derived exosomes and miR-155 in regulating autophagy during SA-AKI. A mouse model of SA-AKI was established by performing cecal ligation and puncture (CLP) surgery. Additionally, the HK-2 cell line was utilized to establish a sepsis cell model by inducing lipopolysaccharide (LPS). We demonstrated that the mice model of SA-AKI exhibited renal injury along with enhanced autophagy, inflammation response, and macrophage polarization after CLP surgery. M1 macrophages attenuated cell viability and enhanced autophagy in LPS-treated HK-2 cells. Additionally, M1 macrophage-derived exosomes were observed to enhance autophagy in LPS-treated HK-2 cells. Furthermore, we confirmed an increased expression of miR-155 in M1 macrophage-derived exosomes. Furthermore, exosome-mediated miR-155 enhanced autophagy in LPS-treated HK-2 cells. In conclusion, this study provides the first evidence that exosomal miR-155 derived from M1 macrophages enhances autophagy in SA-AKI. These findings suggest that targeting exosomal miR-155 could be a promising therapeutic strategy for SA-AKI.

Targeting miR-29 mitigates skeletal senescence and bolsters therapeutic potential of mesenchymal stromal cells

Mesenchymal stromal cell (MSC) senescence is a key factor in skeletal aging, affecting the potential of MSC applications. Identifying targets to prevent MSC and skeletal senescence is crucial. Here, we report increased miR-29 expression in bone tissues of aged mice, osteoporotic patients, and senescent MSCs. Genetic overexpression of miR-29 in Prx1-positive MSCs significantly accelerates skeletal senescence, reducing cortical bone thickness and trabecular bone mass, while increasing femur cross-sectional area, bone marrow adiposity, p53, and senescence-associated secretory phenotype (SASP) levels. Mechanistically, miR-29 promotes senescence by upregulating p53 via targeting Kindlin-2 mRNA. miR-29 knockdown in BMSCs impedes skeletal senescence, enhances bone mass, and accelerates calvarial defect regeneration, also reducing lipopolysaccharide (LPS)-induced organ injuries and mortality. Thus, our findings underscore miR-29 as a promising therapeutic target for senescence-related skeletal diseases and acute inflammation-induced organ damage.

MiR-29a expression negatively correlates with Bcl-2 levels in colorectal cancer and is correlated with better prognosis

MicroRNAs (miRNAs) are a class of small non-coding RNAs that act as important regulators of gene expression, involved in various biological pathways. Aberrant miRNAs expression is associated with the onset and progression of colorectal cancer (CRC). The aim of this study was to investigate the correlation between five miRNAs (miR-29a, miR-101, miR-125b, miR-146a, and miR-155), found to be deregulated in tissue samples of CRC patients, and clinicopathological characteristics and histological markers. Analysis of histological markers was performed by immunohistochemical staining of tumour tissues with Ki-67, p53, CD34, and Bcl-2. Our findings revealed a significant negative correlation between miR-29a expression and Bcl-2 levels. Furthermore, high miR-29a expression was associated with a lower incidence of distant metastasis in CRC patients. We observed negative correlations between miR-101 expression and the number of lymph nodes with metastasis, as well as the size of the largest metastasis; miR-125b expression and lymphovascular invasion; and miR-155 expression and mucus presence. Our survival analysis demonstrated that high miR-29a expression correlated with better progression-free survival of CRC patients, underscoring its potential as a prognostic marker. Our study unveiled intricate relationships between specific miRNA expressions and clinicopathological features in CRC, highlighting the potential utility of miR-29a as a valuable prognostic biomarker.

The Role of the MiR-181 Family in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the fourth-leading cause of cancer-related death worldwide. Due to the high mortality rate in HCC patients, discovering and developing novel systemic treatment options for HCC is a vital unmet medical need. Among the numerous molecular alterations in HCCs, microRNAs (miRNAs) have been increasingly recognised to play critical roles in hepatocarcinogenesis. We and others have recently revealed that members of the microRNA-181 (miR-181) family were up-regulated in some, though not all, human cirrhotic and HCC tissues-this up-regulation induced epithelial-mesenchymal transition (EMT) in hepatocytes and tumour cells, promoting HCC progression. MiR-181s play crucial roles in governing the fate and function of various cells, such as endothelial cells, immune cells, and tumour cells. Previous reviews have extensively covered these aspects in detail. This review aims to give some insights into miR-181s, their targets and roles in modulating signal transduction pathways, factors regulating miR-181 expression and function, and their roles in HCC.

Hypoxia-preconditioned WJ-MSC spheroid-derived exosomes delivering miR-210 for renal cell restoration in hypoxia-reoxygenation injury

BackgroundRecent advancements in mesenchymal stem cell (MSC) technology have paved the way for innovative treatment options for various diseases. These stem cells play a crucial role in tissue regeneration and repair, releasing local anti-inflammatory and healing signals. However, challenges such as homing issues and tumorigenicity have led to exploring MSC-exosomes as a promising alternative. MSC-exosomes have shown therapeutic potential in conditions like renal ischemia-reperfusion injury, but low production yields hinder their clinical use.MethodsTo address this limitation, we examined hypoxic preconditioning of Wharton jelly-derived MSCs (WJ-MSCs) 3D-cultured in spheroids on isolated exosome yields and miR-21 expression. We then evaluated their capacity to load miR-210 into HEK-293 cells and mitigate ROS production, consequently enhancing their survival and migration under hypoxia-reoxygenation conditions.ResultsMiR-210 overexpression was significantly induced by optimized culture and preconditioning conditions, which also improved the production yield of exosomes from grown MSCs. The exosomes enriched with miR-210 demonstrated a protective effect by improving survival, reducing apoptosis and ROS accumulation in damaged renal cells, and ultimately promoting cell migration.ConclusionThe present study underscores the possibility of employing advanced techniques to maximize the therapeutic attributes of exosomes produced from WJ-MSC spheroid for improved recovery outcomes in ischemia-reperfusion injuries.

A Study on the Role of miR-126 in the Repair Process after Spinal Cord Injury

Background: Spinal Cord Injury (SCI) results in motor, sensory, and autonomic dysfunctions and causes social and economic problems. Surgery, medication, and stem cell transplantation are therapeutic strategies for SCI. The use of endogenous neural stem cells seems preferable due to their lower immune responses. miR-126 serves as a promising microRNA for reducing inflammation after SCI. It can promote angiogenesis and proliferation of neural stem cells Objectives: This study aimed to observe changes in miR-126 expression after SCI in an animal mice model. Methods: A total of 42 healthy adult FVB mice were divided equally into 7 groups (6 SCI model versus 1 control). At different periods following SCI establishment in the model groups, Basso Mouse Scale score (BMS), histopathological changes, and expression levels of miR-126 were evaluated in the model groups compared to the control one. Results:: The BMS score increased to a certain extent as the time after spinal cord injury progressed. HE and Nissl staining showed that the acute period (1-7 days) after spinal cord injury was characterized by neuronal loss, whereas the chronic phase (21st day) was characterized by scar and cavity formation. Compared with the control group, the model group exhibited decreased expression of miR-126 during the acute phase (days 1-7 post-SCI). However, its expression increased by 21th day after SCI. Conclusion: Overexpressed miR-126 can contribute to reduced SCI-related damages, which may result in the promotion of the growth and proliferation of neural stem cells as well as the repair of motor function.

Macrophage-derived exosomal miR-155 regulating hepatocyte pyroptosis in MAFLD

BackgroundPrevious studies have shown that pyroptosis in hepatocyte is essential for the development of MAFLD. Growing evidence has shown that exosomal miRNAs-mediated communication between inflammatory cells and hepatocyte is an important link in MAFLD. In the present study, we aim to elucidate whether macrophage-derived exosomal miRNAs contribute to the hepatocyte pyroptosis in the pathophysiological process of MAFLD. MethodsThe effects of hepatocyte pyroptosis were investigated in an HFD-induced MAFLD mouse model and in the liver tissues from patients with MAFLD using immunohistochemistry, real-time PCR, Western blotting, and luciferase reporter assay, among other techniques. MiR-155 inhibitor tail injections and AAV-FoxO3a-GFP were also administered to respectively inhibit or overexpress its expression in an HFD-induced MAFLD mouse model. ResultsHepatocyte pyroptosis was heightened in the liver tissue of patients with MAFLD or HFD-induced MAFLD mouse. Importantly, treatment with a caspase-1 inhibitor or overexpression of FoxO3a reversed this trend. Our study also demonstrated that miR-155 expression and the number of infiltrated macrophages were increased, and knockdown of miR-155 attenuated hepotocyte pyroptosis and liver fibrosis in HFD-induced mouse. In addition, we demonstrated that macrophage-derived exosomal miR-155 was transferred to hepatocytes, leading to hepatocyte pyroptosis in MAFLD mouse. Furthermore, blockade of exosome secretion improved hepotocyte pyroptosis and liver fibrosis in HFD-induced mouse. On the contrary, macrophage-derived exosomal miR-155 worsened hepotocyte pyroptosis. Moreover, we found that miR-155 promoted hepatocyte pyroptosis in MAFLD by down-regulating FoxO3a. ConclusionsTaken together, our results demonstrated that macrophage-derived exosomal miR-155 promotes hepatocyte pyroptosis and liver fibrosis in MAFLD.

Cyclophosphamide ameliorates membranous nephropathy by upregulating miR-223 expression, promoting M2 macrophage polarization and inhibiting inflammation.

Membranous nephropathy (MN), also known as membranous glomerulonephritis, is a leading cause of adult nephrotic syndrome. The main pathological features encompass the deposition of immune complexes within the glomerular basement membrane epithelial cells, thickening of the basement membrane, and fusion of the foot process. This study aims to investigate the role of the immune and inflammatory modulator miR-223 in the immunosuppressive and anti-inflammatory effects of cyclophosphamide (CTX) on membranous nephropathy (MN). miR-223 mimetics or inhibitors was used to regulate miR-223 levels. LPS induced inflammatory cell model and cell polarization. CTX was used to treat Lipopolysaccharides (LPS) induced inflammatory response and polarization. Cationic bovine serum albumin (c-BSA) induced BALB/c mouse MN model, while CTX was used to treat c-BSA induced MN. The miR-223 level in LPS induced inflammatory model cells was lower than that in control cells. The levels of inflammatory factors in LPS+miR-223 mimetics and CTX+miR-223i cells were lower than those in LPS and miR-223i cells. The protein levels of LPS+miR-223 mimic, CTX+miR-223i macrophage M2 phenotype markers Arginase-1 (Arg1), transforming growth factor β1 (TGF-β1), anti-inflammatory factors interleukin-4 (IL4) and interleukin-13 (IL13) were significantly higher than those of LPS and miR-223i. The effect of CTX was confirmed in a BALB/c mouse MN model induced by cationic bovine serum albumin (c-BSA). CTX upregulates the expression of miR-223, promotes polarization of M2 macrophages, alleviates the inflammatory response and renal injury of MN.

Selective Enrichment of Angiomirs in Extracellular Vesicles Released from Ischemic Skeletal Muscles: Potential Role in Angiogenesis and Neovascularization.

MicroRNAs (miRs) regulate physiological and pathological processes, including ischemia-induced angiogenesis and neovascularization. They can be transferred between cells by extracellular vesicles (EVs). However, the specific miRs that are packaged in EVs released from skeletal muscles, and how this process is modulated by ischemia, remain to be determined. We used a mouse model of hindlimb ischemia and next generation sequencing (NGS) to perform a complete profiling of miR expression and determine the effect of ischemia in skeletal muscles, and in EVs of different sizes (microvesicles (MVs) and exosomes) released from these muscles. Ischemia significantly modulated miR expression in whole muscles and EVs, increasing the levels of several miRs that can have pro-angiogenic effects (angiomiRs). We found that specific angiomiRs are selectively enriched in MVs and/or exosomes in response to ischemia. In silico approaches indicate that these miRs modulate pathways that play key roles in angiogenesis and neovascularization, including HIF1/VEGF signaling, regulation of actin cytoskeleton and focal adhesion, NOTCH, PI3K/AKT, RAS/MAPK, JAK/STAT, TGFb/SMAD signaling and the NO/cGMP/PKG pathway. Thus, we show for the first time that angiomiRs are selectively enriched in MVs and exosomes released from ischemic muscles. These angiomiRs could be targeted in order to improve the angiogenic function of EVs for potential novel therapeutic applications in patients with severe ischemic vascular diseases.

Impact of Human Papillomavirus on microRNA-21 Expression in Oral and Oropharyngeal Cancer-A Systematic Review.

Recently, microRNAs (miR) were identified to have potential links with oral squamous cell carcinoma (OSCC) and oropharyngeal squamous cell carcinoma (OPSCC) oncogenesis, specifically miR-21. Since HPV is a major risk factor for the development of these diseases, we aimed to search the literature regarding miR-21 expression in both HPV-positive and HPV-negative OSCC/OPSCC. The search was performed in the PubMed (MEDLINE), Scopus, Web of Science, and Cochrane electronic databases. The research question was as follows: Is there a difference in the tissue expression of miR-21 between patients with HPV-positive and those with HPV-negative OSCC/OPSCC? After conducting a meticulous search strategy, four studies were included, and they had a pooled sample size of 621 subjects with OSCC and/or OPSCC. Three studies did not find any significant difference in miR-21 expression between HPV-positive and HPV-negative OSCC/OPSCC. The findings of this systematic review showed that there are no differences in miR-21 expression between HPV-positive and HPV-negative OSCC/OPSCC. Nevertheless, it is worth noting that there are still insufficient studies regarding this important subject, because understanding how HPV influences miR-21 expression and its downstream effects can provide insights into the molecular mechanisms underlying OSCC/OPSCC development and progression.

Investigating Expression Dynamics of miR-21 and miR-10b in Glioblastoma Cells In Vitro: Insights into Responses to Hypoxia and Secretion Mechanisms.

Glioblastoma poses significant challenges in oncology, with bevacizumab showing promise as an antiangiogenic treatment but with limited efficacy. microRNAs (miRNAs) 10b and 21 have emerged as potential biomarkers for bevacizumab response in glioblastoma patients. This study delves into the expression dynamics of miR-21 and miR-10b in response to hypoxia and explores their circulation mechanisms. In vitro experiments exposed glioma cells (A172, U87MG, U251) and human umbilical vein endothelial cells (HUVEC) to hypoxic conditions (1% oxygen) for 24 h, revealing heightened levels of miR-10b and miR-21 in glioblastoma cells. Manipulating miR-10b expression in U87MG, demonstrating a significant decrease in VEGF alpha (VEGFA) following miR-10b overexpression under hypoxic conditions. Size exclusion chromatography illustrated a notable shift towards miR-21 and miR-10b exosomal packaging during hypoxia. A proposed model suggests that effective bevacizumab treatment reduces VEGFA levels, heightening hypoxia and subsequently upregulating miR-21 and miR-10b expression. These miRNAs, released via exosomes, might impact various cellular processes, with miR-10b notably contributing to VEGFA level reduction. However, post-treatment increases in miR-10b and miR-21 could potentially restore cells to normoxic conditions through the downregulation of VEGF. This study highlights the intricate feedback loop involving miR-10b, miR-21, and VEGFA in glioblastoma treatment, underscoring the necessity for personalized therapeutic strategies. Further research should explore clinical implications for personalized glioma treatments.

Promoter Hypermethylation Downregulates MiR-125b-5p and MiR-199b-5p Targeting of ΔNp63, Resulting in PI3K/AKT/mTOR Pathway Activation and Keratinocyte Differentiation.

Normal keratinocyte differentiation is important for epidermal wound healing. ΔNp63 is a major gene regulating epidermal formation and differentiation. We identified miRNAs targeting ΔNp63 and studied the association between the miRNAs and DNA methylation in keratinocyte differentiation. This study aimed to explore the mechanisms regulating ΔNp63 expression during keratinocyte differentiation. Bioinformatics analysis was performed to screen the miRNAs targeting ΔNp63 and uncover potential pathway mechanisms. The differentiation model of keratinocytes was established by CaCl2 treatment. Furthermore, the effects of the miRNA transgenic technique on Δ Np63 and keratinocyte differentiation were studied. In addition, the RNA FISH experiment was conducted to detect the location of different miRNAs. A double luciferase reporter experiment was carried out to verify the potential bindings between the miRNAs and ΔNp63. A rescue experiment was also performed to assess the effects of different miRNAs targeting ΔNp63 on keratinocyte differentiation. We analyzed the methylation patterns of the promoter regions of miRNAs using keratinocytes treated with 5-Aza-2'-deoxycytidine. Finally, we designed a methylation rescue experiment to verify the effects of miRNA promoter methylation on keratinocyte differentiation. Bioinformatics analysis showed that the miR-125b-5p and miR-199b-5p binding to the ΔNp63 3'UTR region decreased during skin development. Moreover, such binding may downregulate the PI3K/AKT/mTOR pathway. The expression levels of CK10, Inv, TG1, ΔNp63, and PI3K/AKT/mTOR were all significantly increased during keratinocyte differentiation. Both miR- 125b-5p and miR-199b-5p were localized in the cytoplasm. Luciferase assay results showed that both miR-125b-5p and miR-199b-5p can bind to the 3'UTR region of ΔNp63. Overexpression of ΔNp63 can significantly counteract the inhibitory effect of miRNA mimics on keratinocyte differentiation. Moreover, the promoter regions of both miR-125b-5p and miR-199b-5p had methylation sites, and the methylation levels in those promoter regions were significantly increased during keratinocyte differentiation. 5-Aza-2'-Deoxycytidine treatment increased the expression of miR- 125b-5p and miR-199b-5p and inhibited the differentiation of keratinocytes. Finally, miRNA inhibitors reversed the inhibitory effects of 5-Aza-2'-deoxycytidine on keratinocyte differentiation. Promoter hypermethylation in miR-125b-5p and miR-199b-5p seem to promote keratinocyte differentiation via upregulation of ΔNp63 expression and the activation of the PI3K/AKT/mTOR pathway. The findings of this study are helpful for future research on skin development and clinical scar-free healing.

Circulating microRNAs as potential biomarkers of physical activity in geriatric patients with HCV

BackgroundCirculating microRNAs have been implicated in a diverse array of biological and pathological phenomena. Their potential utility as noninvasive biomarkers for screening and diagnosing various diseases has been proposed.ObjectiveThis study aimed to explore the potential role of the miRNAs miR-122 and miR-486 as molecular biomarkers in the pathogenesis of hepatitis C virus (HCV) infection. Thus, miR-122 and miR-486 were detected in the serum of HCV patients and healthy controls. Moreover, the potential correlations of miR-122 and miR-486 with viral complications, such as physical activity, pain, muscle fatigue, and HCV infection, were identified.MethodsA total of 150 subjects aged 30 to 66 years were included in this study. The patients were classified as patients with chronic hepatitis C virus (CHC) (n = 110) or healthy controls (n = 40). Real-time polymerase chain reaction (PCR) analyses were performed to determine miR-122 and miR-486 expression. Physical activity (PA), pain score, HCV genotyping, viral overload, aspartate transaminase (AST), alanine transaminase (ALT), lactic acid dehydrogenase (LDH), creatine kinase (CK), and antioxidant status were also estimated by using prevalidated questionnaires, PCR, and spectrophotometric analyses.ResultsCompared with those in normal controls, significant increases in the serum levels of miR-122 and miR-486 were reported in patients with CHC. In physically active CHC patients, there was a significant correlation between the expression of miRNAs and increased alanine transaminase (ALT), aspartate transaminase (AST), fibrosis scores, and inflammation activity, but no association was reported for hepatitis C virus (HCV) RNA or viral load. Additionally, significant decreases in LDH, CK, GSSG, and pain scores and increases in TAC, GSH, and the GSH/GSSG ratio were reported. Moreover, the expression of miR-122 and miR-486 was positively correlated with changes in body mass index (BMI) and liver fibrosis stage, as well as negatively correlated with sex, PA, TAC, GSH, GSSG, and the GSH/GSSG ratio.ConclusionMiR-122 and miR-486 expression levels were strongly correlated with physical activity, pain perception, and muscle fatigue biomarkers in HCV-infected patients. These miRNA levels were associated with elevated AST, ALT, fibrosis scores, LDH, CK, and antioxidant status, thus suggesting their potential as biomarkers for disease severity and oxidative stress. However, no correlation was observed with viral load or HCV-RNA expression, thus implying that these miRNAs may impact disease progression and symptoms through host factors, rather than directly affecting viral replication. In summary, the results demonstrated that molecular studies of miR-22 and miR-468 and their associations with PA, pain, adiposity, sex differences, and muscle fatigue, as well as routine biomarkers, could be useful as prognostic nanoninvasive biomarkers, thus providing novel therapeutic targets for CHC infection.

Circ-0006332 stimulates cardiomyocyte pyroptosis via the miR-143/TLR2 axis to promote doxorubicin-induced cardiac damage

ABSTRACT Doxorubicin (DOX)-mediated cardiotoxicity can impair the clinical efficacy of chemotherapy, leading to heart failure (HF). Given the importance of circRNAs and miRNAs in HF, this paper intended to delineate the mechanism of the circular RNA 0006332 (circ -0,006,332)/microRNA (miR)-143/Toll-like receptor 2 (TLR2) axis in doxorubicin (DOX)-induced HF. The binding of miR-143 to circ -0,006,332 and TLR2 was assessed with the dual-luciferase assay, and the binding between miR-143 and circ -0,006,332 was determined with FISH, RIP, and RNA pull-down assays. miR-143 and/or circ -0,006,332 were overexpressed in rats and cardiomyocytes, followed by DOX treatment. In cardiomyocytes, miR-143 and TLR2 expression, cell viability, LDH release, ATP contents, and levels of IL-1β, IL-18, TNF-α, and pyroptosis-related molecules were examined. In rats, cardiac function, serum levels of cardiac enzymes, apoptosis, myocardial fibrosis, and levels of IL-1β, IL-18, TNF-α, TLR2, and pyroptosis-related molecules were detected. miR-143 diminished TLR2 expression by binding to TLR2, and circ -0,006,332 bound to miR-143 to downregulate miR-143 expression. miR-143 expression was reduced and TLR2 expression was augmented in DOX-induced cardiomyocytes. miR-143 inhibited DOX-induced cytotoxicity by suppressing pyroptosis in H9C2 cardiomyocytes. In DOX-induced rats, miR-143 reduced cardiac dysfunction, myocardial apoptosis, myocardial fibrosis, TLR2 levels, and pyroptosis. Furthermore, overexpression of circ -0,006,332 blocked these effects of miR-143 on DOX-induced cardiomyocytes and rats. Circ -0,006,332 stimulates cardiomyocyte pyroptosis by downregulating miR-143 and upregulating TLR2, thus promoting DOX-induced cardiac injury.

Single-cell RNA sequencing reveals that NRF2 regulates vascular smooth muscle cell phenotypic switching in abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a life-threatening disease characterized by extensive membrane destruction in the vascular wall that is closely associated with vascular smooth muscle cell (VSMC) phenotypic switching. A thorough understanding of the changes in regulatory factors during VSMC phenotypic switching is essential for managing AAA therapy. In this study, we revealed the impact of NRF2 on the modulation of VSMC phenotype and the development of AAA based on single-cell RNA sequencing analysis. By utilizing a murine model of VSMC-specific knockout of nuclear factor E2-related factor 2 (NRF2), we observed that the absence of NRF2 in VSMCs exacerbated AAA formation in an angiotensin II-induced AAA model. The downregulation of NRF2 promoted VSMC phenotypic switching, leading to an enhanced inflammatory response. Through genome-wide transcriptome analysis and loss- or gain-of-function experiments, we discovered that NRF2 upregulated the expression of VSMC contractile phenotype-specific genes by facilitating microRNA-145 (miR-145) expression. Our data identified NRF2 as a novel regulator involved in maintaining the VSMC contractile phenotype while also influencing AAA formation through an miR-145-dependent regulatory mechanism.