Ticagrelor downregulates the expression of proatherogenic and proinflammatory miR125-b compared to clopidogrel: A randomized, controlled trial.

BACKGROUND: Platelet P2Y12 antagonist ticagrelor reduces cardiovascular mortality after acute myocardial infarction (AMI) compared to clopidogrel, but the underlying mechanism is unknown. Because activated platelets release proatherogenic and proinflammatory microRNAs, including miR-125a, miR-125b and miR-223, we hypothesized that the expression of these miRNAs is lower on ticagrelor, compared to clopidogrel. OBJECTIVES: We compared miR-125a, miR-125b and miR-223 expression in plasma of patients after AMI treated with ticagrelor or clopidogrel. METHODS: After percutaneous coronary intervention on acetylsalicylic acid and clopidogrel, 60 patients with first AMI were randomized to switch to ticagrelor or to continue with clopidogrel. Plasma expression of miR-223, miR-125a-5p, miR-125b was measured using quantitative polymerase chain reaction at baseline and after 72 h and 6 months of treatment with ticagrelor or clopidogrel in patients and one in 30 healthy volunteers. Multiple electrode aggregometry using ADP test was used to determine platelet reactivity in response to P2Y12 inhibitors. RESULTS: Expression of miR-125b was higher in patients with AMI 72 h and 6 months, compared to healthy volunteers (p = 0.001), whereas expression of miR-125a-5p and miR-223 were comparable. In patients randomized to ticagrelor, expression of miR-125b decreased at 72 h (p = 0.007) and increased back to baseline at 6 months (p = 0.005). Expression of miR-125a-5p and miR-223 was not affected by the switch from clopidogrel to ticagrelor. CONCLUSIONS: Ticagrelor treatment leads to lower plasma expression of miR-125b after AMI, compared to clopidogrel. Higher expression of miR-125b might explain recurrent thrombotic events and worse clinical outcomes in patients treated with clopidogrel, compared to ticagrelor.

Unveiling the role of osteoblastic micro RNAs in the skeleton: from biological functions to therapeutic potential / Rol de los micro-ARN osteoblásticos en el esqueleto: desde las funciones biológicas al potencial terapéutico

MicroRNAs (miRNAs) are small non-coding RNA molecules that play critical roles in post-transcriptional gene regulation. They function by binding to target messenger RNA (mRNA) molecules, leading to their degradation or inhibiting their translation into proteins. In the context of skeletal diseases, such as osteoporosis, osteoarthritis, and bone metastasis, there is growing evidence osteoblastic miRNAs, are involved in the regulation of bone formation and maintenance.Osteoblasts are bone-forming cells responsible for synthesizing and depositing the extracellular matrix, which ultimately mineralizes to form bone tissue. Osteoblastic miRNAs modulate various aspects of osteoblast function, including proliferation, differentiation, mineralization, and apoptosis. Dysregulation of these miRNAs can disrupt the balance between bone formation and resorption, leading to skeletal diseases.The therapeutic implications of targeting osteoblastic miRNAs in skeletal diseases are significant. Modulating the expression levels of specific miRNAs holds promise for developing novel therapeutic strategies to enhance bone formation, prevent bone loss, and promote bone regeneration. Potential therapeutic approaches include the use of synthetic miRNA mimics to restore miRNA expression in diseases associated with miRNA downregulation or the use of anti-miRNA oligonucleotides to inhibit miRNA function in diseases associated with miRNA upregulation.miRNA-based therapies are still in the early stages of development, and further research is needed to fully understand the complexity of miRNA networks. Additionally, the delivery of miRNAs to specific target tissues and cells remains a challenge that needs to be addressed for effective clinical translation. Nonetheless, targeting osteoblastic miRNAs represents a promising avenue for future therapeutic interventions in skeletal diseases. (AU)

Los micro-ARNs (miARNss) son pequeños ARN no codificantes que desempeñan un papel fundamental en la regulación génica postranscripcional. Ejercen su función al unir-se a moléculas de ARN mensajero (ARNm), promoviendo su degradación e inhibiendo su traducción en proteínas. En el contexto de las enfermedades esqueléticas, como la osteoporosis, la osteoartritis y la metástasis ósea existe evidencia de que los miARNs osteoblásticos están involucrados en la regulación de la formación y del mantenimiento óseo. Los osteoblastos son células formadoras de hueso responsables de sintetizar y depositar la matriz extracelular, que finalmente se mineraliza para formar el hueso. Los miARNs derivados de osteoblastos modulan varios aspectos de la función de estas células, incluida la proliferación, diferenciación, mineralización y la apoptosis. La desregulación de estos miARNs puede alterar el equilibrio entre la formación y la resorción ósea, lo que lleva a enfermedades óseas. Las implicaciones terapéuticas de los miARNs osteoblásticos en enfermedades esqueléticas son significativas. La modulación de los niveles de expresión de miARNs específicos es prometedora para desarrollar nuevas estrate-gias terapéuticas a fin de mejorar la formación, prevenir la pérdida y promover la regeneración ósea. Los enfoques terapéuticos potenciales incluyen el uso de miméticos de miARNs para restaurar la expresión de miARNs o el uso de oligonucleótidos anti-miARNs para inhibir su función. Las terapias basadas en miARNs aún se encuentran en las primeras etapas de desarrollo. La administración de miARNs a las células y los tejidos específicos sigue siendo un desafío para lograr una aplicación clínica eficaz. (AU)

Sequence determinant of small RNA production by DICER.

RNA silencing relies on specific and efficient processing of double-stranded RNA by Dicer, which yields microRNAs (miRNAs) and small interfering RNAs (siRNAs)1,2. However, our current knowledge of the specificity of Dicer is limited to the secondary structures of its substrates: a double-stranded RNA of approximately 22 base pairs with a 2-nucleotide 3' overhang and a terminal loop3-11. Here we found evidence pointing to an additional sequence-dependent determinant beyond these structural properties. To systematically interrogate the features of precursor miRNAs (pre-miRNAs), we carried out massively parallel assays with pre-miRNA variants and human DICER (also known as DICER1). Our analyses revealed a deeply conserved cis-acting element, termed the 'GYM motif' (paired G, paired pyrimidine and mismatched C or A), near the cleavage site. The GYM motif promotes processing at a specific position and can override the previously identified 'ruler'-like counting mechanisms from the 5' and 3' ends of pre-miRNA3-6. Consistently, integrating this motif into short hairpin RNA or Dicer-substrate siRNA potentiates RNA interference. Furthermore, we find that the C-terminal double-stranded RNA-binding domain (dsRBD) of DICER recognizes the GYM motif. Alterations in the dsRBD reduce processing and change cleavage sites in a motif-dependent fashion, affecting the miRNA repertoire in cells. In particular, the cancer-associated R1855L substitution in the dsRBD strongly impairs GYM motif recognition. This study uncovers an ancient principle of substrate recognition by metazoan Dicer and implicates its potential in the design of RNA therapeutics.

Structure of the human DICER-pre-miRNA complex in a dicing state.

Dicer has a key role in small RNA biogenesis, processing double-stranded RNAs (dsRNAs)1,2. Human DICER (hDICER, also known as DICER1) is specialized for cleaving small hairpin structures such as precursor microRNAs (pre-miRNAs) and has limited activity towards long dsRNAs-unlike its homologues in lower eukaryotes and plants, which cleave long dsRNAs. Although the mechanism by which long dsRNAs are cleaved has been well documented, our understanding of pre-miRNA processing is incomplete because structures of hDICER in a catalytic state are lacking. Here we report the cryo-electron microscopy structure of hDICER bound to pre-miRNA in a dicing state and uncover the structural basis of pre-miRNA processing. hDICER undergoes large conformational changes to attain the active state. The helicase domain becomes flexible, which allows the binding of pre-miRNA to the catalytic valley. The double-stranded RNA-binding domain relocates and anchors pre-miRNA in a specific position through both sequence-independent and sequence-specific recognition of the newly identified 'GYM motif'3. The DICER-specific PAZ helix is also reoriented to accommodate the RNA. Furthermore, our structure identifies a configuration of the 5' end of pre-miRNA inserted into a basic pocket. In this pocket, a group of arginine residues recognize the 5' terminal base (disfavouring guanine) and terminal monophosphate; this explains the specificity of hDICER and how it determines the cleavage site. We identify cancer-associated mutations in the 5' pocket residues that impair miRNA biogenesis. Our study reveals how hDICER recognizes pre-miRNAs with stringent specificity and enables a mechanistic understanding of hDICER-related diseases.

Expression Levels and Clinical Significance of Serum miR-497, CEA, CA24-2, and HBsAg in Patients with Colorectal Cancer.

The objective of the current study was to look at the levels of blood micro ribonucleic acid- (miR-) 497, carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 24-2, and hepatitis B surface antigen (HBsAg) in patients with colorectal cancer (CRC), as well as the clinical importance of these markers in CRC patients. The serum levels of miR-497, CEA, CA24-2, and HBsAg were compared between 60 patients with CRC (observation group) and another 60 patients with colorectal polyps (control group). The 4 indicators in patients with lymph node metastasis and liver metastasis were compared. The diagnostic effects of 4 detection methods and the combined detection were analyzed, and the influence of 4 indicators on the 5-year cumulative survival rate of patients was discussed. The results showed that the serum levels of miR-497 and HBsAg were lower, and the levels of CEA and CA24-2 were higher in the observation group (P < 0.05). The combined detection had the best diagnostic effect, and CEA alone had the best prediction effect. The serum level of miR-497 was significantly lower in patients with lymphatic metastasis, with the significantly higher levels of CEA and CA24-2 (P < 0.05). The HBsAg level of patients with liver metastases was greatly lower than that of patients without liver metastases (P < 0.05). The 5-year cumulative survival rate of patients with high levels of CEA and CA24-2 was significantly lower than that of patients with low level of CEA. The 5-year cumulative survival rate was lower in patients with low level of HBsAg, but the difference was small. The 5-year cumulative survival rate of patients with elevated serum miR-497 was observably lower. In conclusion, combined detection could diagnose CRC more accurately. Serum miR-497, CEA, and CA24-2 were important in the diagnosis of lymph node metastasis of CRC. HBsAg did a better job of predicting liver metastases in CRC patients. High level of CEA significantly reduced the cumulative survival rate of CRC patients and could predict the long-term survival rate of patients. Serum levels of miR-497, CEA, CA24-2, and HBsAg played a positive role in the diagnosis and evaluation of CRC and could identify lymph node and liver metastases, having a high clinical guidance value.

Diagnosis value of miR-181, miR-652, and CA72-4 for gastric cancer.

PURPOSE: To find a useful disease marker for early diagnosis of gastric cancer, we tried to explore the expression of serum miR-181, miR-652, and carbohydrate antigen 72-4 (CA72-4). PATIENTS AND METHODS: According to clinical pathologic stages, 112 patients with gastric cancer were divided into early gastric cancer group (n = 60) and advanced gastric cancer group (n = 52), stage I-II (n = 65), and stage III-IV (n = 47). Another 50 cases of gastric benign lesions and 40 healthy controls were also selected. Real-time quantitative PCR together with chemiluminescence were applied to detect expression levels. ROC curve was applied to judge their diagnostic efficiency. Pearson's correlation analysis was put into use to investigate the relevance of three indicators. RESULTS: Compared with benign lesions group and control group, significantly higher expression levels were found in patients of gastric cancer (all p < 0.001). Similarly, compared with early gastric cancer group, significantly higher expression levels were found in advanced gastric cancer group (all p < 0.001). The same result was also found in stage III-IV (all p < 0.001). The best cutoff values were 0.93, 2.38, and 16.94 U/ml, respectively. The area under the curve (0.917, 95%CI: 0.856-0.975) of the three combined diagnosis of early gastric cancer was the largest, and its sensitivity and specificity were 92.5% and 86.8%. And miR-181 and miR-652 were positively correlated with CA72-4 (r = 0.772, p < 0.001, r = 0.853, p < 0.001). CONCLUSION: Serum miR-181, miR-652, and CA72-4 are closely linked to the occurrence and development of gastric cancer. Combination of three indicators has diagnostic value for early gastric cancer.

Clinical value of serum miR-320-3p expression in predicting the prognosis of sepsis-induced acute kidney injury.

BACKGROUND: For investigating the expression of miR-320-3p in children with sepsis-induced acute kidney injury (AKI) and its prognostic value. METHODS: A total of 142 patients were grouped into a survival group (n = 95) and death group (n = 47), which was based on their 28-day survival. Serum degrees of miR-320-3p, neutrophil gelatinase-associated lipid carrier protein (NGAL) and kidney injury molecule-1 (KIM-1) were detected. The Acute Physiology and Chronic Health scoring system Ⅱ (APACHE Ⅱ) marks were recorded. Target gene forecast and functional enrichment discussion of miR-320-3p were performed, and a protein-protein interaction (PPI) network diagram was plotted by applying bioinformatics methods. Multivariate logistic regression, ROC curve and Pearson correlation analysis were applied. RESULTS: The death group showed greatly higher serum levels of miR-320-3p, KIM-1 and APACHE Ⅱ scores than the survival group (p < 0.01). Multivariate logistic regression analysis showed that levels of miR-320-3p, NGAL, KIM-1 and APACHE Ⅱ scores were independent risk elements for death in sepsis children with AKI (p < 0.01). According to ROC curve analysis, the region under the curve (0.963, 95% CI: 0.908-0.996) of miR-320-3p, NGAL, KIM-1 levels and APACHE Ⅱ scores combined to forecast the death of kids suffering from sepsis and AKI were the biggest. According to correlation analysis, the expression degree of serum miR-320-3p in the death group was positively correlated with NGAL, KIM-1 and APACHE Ⅱ scores (all p < 0.01). CONCLUSIONS: The expression level of serum miR-320-3p in children with sepsis-induced AKI was significantly increased, and the combination of NGAL, KIM-1 and APACHE Ⅱ scores has good value for prognosis prediction in children.

DNA methylation and miRNA expression in colon adenomas compared with matched normal colon mucosa and carcinomas.

Dysregulation of DNA methylation patterns and non-coding RNA, including miRNAs, has been implicated in colon cancer, and these changes may occur early in the development of carcinoma. In this study, the role of epigenetics as early changes in colon tumorigenesis was examined through paired sample analysis of patient-matched normal, adenoma and carcinoma samples. Global methylation was assessed by genomic 5-methyl cytosine (5-mC) and long interspersed nuclear element-1 (LINE-1) promoter methylation by pyrosequencing. KRAS mutations were also assessed by pyrosequencing. Expression of miRNA, specifically, two microRNA genes-miR-200a and let-7c-was analysed using RT-qPCR. Differences in global methylation in adenomas were not observed, compared with normal tissue. However, LINE-1 methylation was decreased in adenomas (p = .056) and carcinomas (p = .011) compared with normal tissue. Expressions of miRNA, miR-200a and let-7c were significantly higher in adenomas than normal tissues (p = .008 and p = .045 respectively). Thus the significant changes in LINE-1 methylation and microRNA expression in precancerous lesions support an early role for epigenetic changes in the carcinogenic process. Epigenetic characteristics in adenomas may provide potential diagnostic and prognostic therapeutic targets early in cancer development at the adenoma stage.

Elevation of miR-125b-5p is related to improved prognosis in laryngeal squamous cell carcinoma and inhibits the malignancy and glycometabolic disorder by targeting MAP3K9.

Laryngeal squamous cell carcinoma (LSCC) is the most common malignant tumor in the head and neck cancer, with a poor prognosis. As we know, microRNAs (miRNAs) play a vital role in the initiation and development of various cancers including LSCC. In this study, we explored the role of miR-125b-5p and its downstream regulatory pathway in LSCC. Our data demonstrated that miR-125b-5p expression was significantly downregulated in LSCC tissues and cells. LSCC patients with high expression of miR-125b-5p had higher overall survival (OS) and were closely related to the clinical stage. Overexpression of miR-125b-5p impaired viability and glycolysis, and facilitated apoptosis in LSCC cells. And miR-125b-5p silencing had the opposite effects. Bioinformatics website predicted that MAP3K9 was one of the potential target genes of miR-125b-5p. Cell experiments demonstrated that miR-125b-5p repressed the MAP3K9 levels by directly targeting MAP3K9. Additionally, the negative correlation between miR-125b-5p and MAP3K9 was validated in LSCC tissues. Overexpression of MAP3K9 attenuated the inhibitory effect of miR-125b-5p on viability and glycolysis, and the pro-apoptosis effect of miR-125b-5p in LSCC cells. Furthermore, in vivo experiments demonstrated that tumor growth was hampered in AMC-HN-8 cells transfected with miR-125b-5p mimic. In contrast, the knockdown of miR-125b-5p reduced tumor growth in vivo. Meanwhile, the in vivo immunohistochemistry and TUNEL assays suggested that the miR-125b-5p overexpression restrained cell proliferation and promoted apoptosis via targeting MAP3K9. Overall, these above results suggested that miR-125b-5p suppressed proliferation and glycolysis, and promoted apoptosis by directly targeting MAP3K9 in LSCC cells. Thus, miR-125b-5p acts as a tumor suppressor miRNA and the miR-125b-5p/MAP3K9 axis may be a promising candidate for LSCC treatment.

MiR-138-5p targets RUNX2 to inhibit osteogenic differentiation of aortic valve interstitial cells via Wnt/ß-catenin signaling pathway.

BACKGROUND: Human aortic valve interstitial cells (hAVICs) are a key factor in the pathogenesis of calcific aortic valve disease (CAVD). This research examines the role and mechanism of microRNA miR-138-5p in osteogenic differentiation of hAVICs. METHODS: RT-qPCR analysis was applied for detecting miR-138-5p and RUNX2 expression in valve tissues of CAVD patients and controls. On completion of induction of osteogenic differentiation of hAVICs, and after overexpression or interference of miR-138-5p expression, the condition of osteogenic differentiation and calcification of hAVICs was confirmed using alkaline phosphatase staining and alizarin red staining. Subsequently, western blot was utilized to detect the expression of osteogenesis-related proteins OPN and ALP, and Wnt/ß-catenin signaling pathway-related proteins. Finally, the relationship between miR-138-5p and RUNX2 was validated by dual-luciferase reporter assay and Pearson's correlation test. RESULTS: Down-regulation of miR-138-5p was found in CAVD patients and during osteogenic differentiation of hAVICs. Overexpression of miR-138-5p contribute to the inhibition of osteoblast differentiation and calcium deposition in hAVICs, and of ALP and OPN protein expression. RUNX2 was a target gene of miR-138-5p, and it was negatively correlated with miR-138-5p in CAVD. Additionally, overexpression of RUNX2 could reverse the inhibitory effect of miR-138-5p on osteogenic differentiation of hAVICs. CONCLUSION: miR-138-5p can act as a positive regulator of osteogenic differentiation in CAVD patients to involve in inhibiting valve calcification, which is achieved through RUNX2 and Wnt/ß-catenin signaling pathway.

Prediction and validation of GUCA2B as the hub-gene in colorectal cancer based on co-expression network analysis: In-silico and in-vivo study.

BACKGROUND: Several serious attempts to treat colorectal cancer have been made in recent decades. However, no effective treatment has yet been discovered due to the complexities of its etiology. METHODS: we used Weighted Gene Co-expression Network Analysis (WGCNA) to identify key modules, hub-genes, and mRNA-miRNA regulatory networks associated with CRC. Next, enrichment analysis of modules has been performed using Cluepedia. Next, quantitative real-time PCR (RT-qPCR) was used to validate the expression of selected hub-genes in CRC tissues. RESULTS: Based on the WGCNA results, the brown module had a significant positive correlation (r = 0.98, p-value=9e-07) with CRC. Using the survival and DEGs analyses, 22 genes were identified as hub-genes. Next, three candidate hub-genes were selected for RT-qPCR validation, and 22 pairs of cancerous and non-cancerous tissues were collected from CRC patients referred to the Gastroenterology and Liver Clinic. The RT-qPCR results revealed that the expression of GUCA2B was significantly reduced in CRC tissues, which is consistent with the results of differential expression analysis. Finally, top miRNAs correlated with GUCA2B were identified, and ROC analyses revealed that GUCA2B has a high diagnostic performance for CRC. CONCLUSIONS: The current study discovered key modules and GUCA2B as a hub-gene associated with CRC, providing references to understand the pathogenesis and be considered a novel candidate to CRC target therapy.

Association between extract of Euphorbia szovitsii and expression level of microRNAs in MDA-MB-231 cell line.

BACKGROUND: The miRNAs have been shown to be involved in breast cancer. The aim of the present research was to evaluate the impacts of extract from Euphorbia szovitsii Fisch & C.A. Mey on the expression level of microRNAs in triple-negative breast cancer (MDA-MB-231) cell line. METHODS AND RESULT: The alterations in the expression level of miRNAs in MDA-MB-231 cell line exposed to the extract of E. szovitsii were determined exploiting qRT-PCR technique. The expression of MDA-MB-231 cell microRNAs including miR-15, miR-16, miR-21, miR-29, miR-34a, miR-146b, miR-151, miR-155, miR-181b, miR-221, miR-222, and Let-7 was evaluated at 24 and 48 h after treatment with the E. szovitsii extract. The treatment of MDA-MB-231 cells with E. szovitsii caused a significant elevation in the expression of miR-155, miR-146b (P < 0.05), miR-16, miR-21, miR-151 (P < 0.01), and miR-34a (P < 0.001) after 24 h, and also miR-155, Let-7 (P < 0.05), miR-15, miR-29, miR-151 (P < 0. 01), miR-146b and miR-34a (P<0.001) after 48 h. CONCLUSIONS: The qRT-PCR findings at 24 and 48 h after treatment revealed that the MDA-MB-231 cell line in the presence of E. szovitsii extract showed an alteration in the expression profile of miRNAs implicated in the induction of cell proliferation, apoptosis and migration. These results may be helpful in determining the anticancer activity of E. szovitsii in MDA-MB-231 cell line.

Antitumor Effect of Regorafenib on MicroRNA Expression in Hepatocellular Carcinoma Cell Lines.

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and is one of the leading causes of cancer-related deaths worldwide. Regorafenib, a multi-kinase inhibitor, is used as a second-line treatment for advanced HCC. Here, we aimed to investigate the mechanism of the antitumor effect of regorafenib on HCC and evaluate altered microRNA (miRNA) expression. Cell proliferation was examined in six HCC cell lines (HuH-7, HepG2, HLF, PLC/PRF/5, Hep3B, and Li-7) using the Cell Counting Kit-8 assay. Xenografted mouse models were used to assess the effects of regorafenib in vivo. Cell cycle analysis, western blotting analysis, and miRNA expression analysis were performed to identify the antitumor inhibitory potential of regorafenib on HCC cells. Regorafenib suppressed proliferation in HuH-7 cell and induced G0/G1 cell cycle arrest and cyclin D1 downregulation in regorafenib-sensitive cells. During miRNA analysis, miRNA molecules associated with the antitumor effect of regorafenib were found. Regorafenib suppresses cell proliferation and tumor growth in HCC by decreasing cyclin D1 via alterations in intracellular and exosomal miRNAs in HCC.

Circular RNA 0000654 facilitates the growth of gastric cancer cells through absorbing microRNA-149-5p to up-regulate inhibin-beta A.

Circular (circ) RNAs are differentially expressed in gastric cancer (GC) and participate in the biological growth of tumor cells. Given that, investigations were performed to unravel the function of circ_0000654 in GC. GC tissue and normal tissue specimens were obtained, in which circ_0000654, microRNA (miR)-149-5p, and inhibin-beta A (INHBA) levels were examined. GC cell line (BGC-823) was transfected to alter circ_0000654 and miR-149-5p expression, thereby observing cell malignancy. Stably-transfected BGC-823 cells were injected into nude mice to observe tumor growth in vivo. The interaction circ_0000654, miR-149-5p, and INHBA was validated. circ_0000654 and INHBA were up-regulated but miR-149-5p was down-regulated in GC. circ_0000654 absorbed miR-149-5p to target INHBA. Silencing circ_0000654inhibited the progress of GC cell biology. Oppositely, restoring circ_0000654 enhanced the growth of GC cells. Inhibiting miR-149-5p rescued down-regulated circ_0000654-induced anti-tumor effect on GC. circ_0000654 silence or miR-149-5p overexpression limited the growth of GC tumors in vivo. Obviously, circ_0000654 facilitates the growth of GC cells through absorbing miR-149-5p to up-regulate INHBA.

MiRNA-27a mediates insulin resistance in 3T3-L1 cells through the PPARγ.

The biological actions of insulin have been originated by activation of membrane receptors, which trigger a diversity of signaling pathways in facilitating their biological activities. Insulin homeostasis functions in promoting metabolism balance and promotes cell growth and proliferation. If these mechanisms are reformed, this could lead to insulin resistance as a result of defective insulin signaling triggered by mutations in receptors or effector molecules located downstream or by abnormal posttranslational modifications. The purpose of this is to preliminarily investigate the mechanism of miRNA-27a-mediating insulin resistance in 3T3-L1 cells. Insulin resistance in 3T3-L1 adipocytes as a cell model was induced by tumor necrosis factor-alpha (TNF-α) and the miRNA-27a expression in 3T3-L1 adipocytes had been experiential. The regulation of peroxisome proliferator-activated receptor-gamma (PPARγ) mRNA by miRNA-27a had been studied by reverse transcription receptor polymerase chain reaction (RT-PCR). MiRNA-27a was up-regulated in 3T3-L1 cells, miRNA-27a mimics reserved expression of PPARγ mRNA, and miRNA-27a inhibitors up-regulated the expression of PPARγ mRNA. The insulin resistance in 3T3-L1 cells mediated by miRNA-27a may be achieved by targeting PPARγ.

Epithelial microRNA-30a-3p targets RUNX2/HMGB1 axis to suppress airway eosinophilic inflammation in asthma.

BACKGROUND: Type 2-high asthma is a prominent endotype of asthma which is characterized by airway eosinophilic inflammation. Airway epithelial cells play a critical role in the pathogenesis of asthma. Our previous miRNA profiling data showed that miR-30a-3p was downregulated in bronchial epithelial cells from asthma patients. We hypothesize that epithelial miR-30a-3p plays a role in asthma airway inflammation. METHODS: We measured miR-30a-3p expression in bronchial brushings of asthma patients (n = 51) and healthy controls (n = 16), and analyzed the correlations between miR-30a-3p expression and airway eosinophilia. We examined whether Runt-related transcription factor 2 (RUNX2) was a target of miR-30a-3p and whether RUNX2 bound to the promoter of high mobility group box 1 (HMGB1) by using luciferase reporter assay and chromatin immunoprecipitation (ChIP)-PCR. The role of miR-30a-3p was also investigated in a murine model of allergic airway inflammation. RESULTS: We found that miR-30a-3p expression were significantly decreased in bronchial brushings of asthma patients compared to control subjects. Epithelial miR-30a-3p expression was negatively correlated with parameters reflecting airway eosinophilia including eosinophils in induced sputum and bronchial biopsies, and fraction of exhaled nitric oxide in asthma patients. We verified that RUNX2 is a target of miR-30a-3p. Furthermore, RUNX2 bound to the promoter of HMGB1 and upregulated HMGB1 expression. RUNX2 and HMGB1 expression was both enhanced in airway epithelium and was correlated with each other in asthma patients. Inhibition of miR-30a-3p enhanced RUNX2 and HMGB1 expression, and RUNX2 overexpression upregulated HMGB1 in BEAS-2B cells. Intriguingly, airway overexpression of mmu-miR-30a-3p suppressed Runx2 and Hmgb1 expression, and alleviated airway eosinophilia in a mouse model of allergic airway inflammation. CONCLUSIONS: Epithelial miR-30a-3p could possibly target RUNX2/HMGB1 axis to suppress airway eosinophilia in asthma.

Effect of microRNA-181b on the biological characteristics and clinical drug resistance of small-cell lung cancer by targeting angiotensin converting enzyme 2.

OBJECTIVE: To clarify the effect of miR-181b on the biological function of small-cell lung cancer (SCLC) and explore the effect of clinical resistance on SCLC. METHODS: Blood samples were collected from 30 SCLC patients and 30 non-SCLC patients in our department from 2017 to 2019 to detect the expression level of miR-181b.The expression level of miR-181b was detected in SCLC cells by RT-PCR, and screening of downstream target genes by gene chip, verification with luciferase, and Western blotting. In addition, collect the general data of 30 SCLC patients and 30 non-SCLC patients (control group), the patients were diagnosed by pathology and undergoing EC protocol in the Department of Thoracic Surgery and Oncology of our hospital to detect the expression level of mir-181b in different periods. Furthermore, in the SCLC cell line, EC chemotherapy was administered to detect the sensitivity of drug resistance and nondrug resistance. RESULTS: miR-181b in SCLC patients was lower than in normal people as well as the drug-sensitive cell line. ACE2 was verified as a downstream target of miR-181b by gene chip screening. First-line chemotherapy can promote the recovery of miR-181b, but cannot repair to normal levels. miR-181b can enhance the drug sensitivity of SCLC drug-resistant cells. CONCLUSION: miR-181b directly targets ACE2 to affect the biological characteristics of SCLC. The expression level of miR-181b is highly related to the drug resistance of SCLC, which suggests that miR-181b could be a potential biomarker candidate for treatment efficacy of SCLC.

CircRNA_0001795 sponges miRNA-339-5p to regulate yes-associated protein 1 expression and attenuate osteoporosis progression.

Osteoporosis (OP) is one of the most common bone diseases, especially in women after menopause. Increasing evidence shows that non-coding RNAs are implicated in the pathogenesis of OP. In this study, based on the published circular RNA profiling data between OP patients and healthy controls, we found that circRNA_0001795 (circ_0001795) is downregulated in OP samples, which was further validated in the OP samples collected in this study. We therefore investigated the functional role and molecular mechanism of circ_0001795 in the osteogenic differentiation of human bone marrow stromal cells (hBMSCs) hBMSCs by alkaline phosphatase (ALP) activity assay, ALP and Alizarin Red S (ALS) Staining, luciferase reporter assay. Our data revealed that the overexpression of circ_0001795 could significantly promote the osteogenic differentiation of hBMSCs. MiRNA-339-5p (miR-339-5p) was identified as a target of circ_0001795, and miR-339-5p mimic attenuated the effect of circ_0001795 overexpression. MiR-339-5p downregulated yes-associated protein 1 (YAP1), which mediates the effect of circ_0001795 overexpression. Overall, this study uncovered the role of circ_0001795/miR-339-5p/YAP1 axis in regulating osteogenic differentiation, indicating that targeting Circ_0001795 could serve as a novel therapeutic target for OP.

Micro-fragmented adipose tissue regulated the biological functions of osteoarthritis synoviocytes by upregulating MiR-92a-3p expression.

Apart from the treatment potential of micro-fragmented adipose tissue (MF) in joint diseases, what's less clear is the mechanism of MF on Osteoarthritis (OA). Synoviocytes isolated from synovium tissues of 11 knee joint OA patients were identified and co-cultured with MF collected by Lipogems®. Cytokines and mRNA levels in synoviocytes were detected by enzyme-linked immunosorbent assay (ELISA) and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell viability, apoptosis and apoptosis-related protein expression of Tumor Necrosis Factor-α (TNF-α)-activated synoviocytes were detected by cell counting kit-8, flow cytometry and western blot, respectively. The rescue experiments were conducted to verify the causal relationship of MF and miR-92a-3p. The relationship between miR-92a-3p and KLHL29 was verified by bioinformatics analysis, qRT-PCR, dual-luciferase reporter assay and western blot. OA synoviocytes were composed of synovial fibroblasts and synovial macrophages. After co-cultivation of synoviocytes and TNF-α, the levels of Interleukin (IL)-8 and hyaluronic acid (HA) appeared a few changes, and those of chemotactic cytokine ligand (CCL) 2, CCL3, CCL5 and matrix metalloproteinases (MMP)-9 were downregulated, while the levels of Tissue Inhibitor of Metalloproteinases (TIMP)-1, IL-10 and Prostaglandin E2 (PGE2) were up-regulated. Co-culture of MF and activated synoviocytes reversed the above-mentioned effects regulated by TNF-α and reduced the mRNA levels of inflammatory factors. However, miR-92a-3p inhibitor overturned the reversal. KLHL29 was the target gene of miR-92a-3p and its expression was suppressed in activated synoviocytes co-cultured with MF, which was reversed by down-regulated miR-92a-3p. Collectively, MF regulated the biological functions of OA synoviocytes by upregulating miR-92a-3p expression.

Abnormal expression of long non-coding RNA rhabdomyosarcoma 2-associated transcript (RMST) participates in the pathological mechanism of atherosclerosis by regulating miR-224-3p.

Study shows that long non-coding RNA (lncRNA) plays a regulatory role in cardiovascular diseases, and the mechanism of rhabdomyosarcoma 2-associated transcript (RMST) in atherosclerosis (AS) is still unclear. This study aimed to evaluate the expression of RMST and its possible role in the occurrence of AS. RMST and miR-224-3p level in serum and human umbilical vein endothelial cells (HUVECs) were determined by real-time quantitative PCR (RT-qPCR). In vitro atherosclerotic cell model was achieved by treating HUVECs with ox-LDL. Receiver operating characteristic (ROC) curve assessed the diagnostic value of RMST in AS, and Pearson correlation coefficient estimated the correlation of RMST with carotid intima-media thickness (CIMT) and carotid-femoral pulse wave velocity (cfPWV). Cell counting kit-8 (CCK-8) assay and Enzyme-linked immunosorbent assay (ELISA) were performed to evaluate the effect of RMST on cell viability and inflammatory response. The luciferase analysis was used to validate the relationship between RMST and miR-224-3p. The results showed that in serum and HUVECs, RMST levels were increased, while miR-224-3p level was decreased. ROC curve suggested that RMST had clinical diagnostic value for AS. Besides, CIMT and cfPWV were positively correlated with RMST levels, respectively. In HUVECs, RMST-knockdown notably improved the cell viability and inhibited the production of inflammatory factors. Moreover, miR-224-3p was the target of RMST. In conclusion, RMST has the potential to be a diagnostic marker for AS. RMST-knockdown contributes to the enhancement of cell viability and the inhibition of inflammatory response, which may provide new insights into the conquest of AS.