miR-26a Expression Research Articles

Circulating microRNAs correlate to clinical parameters in individuals with allergic and non-allergic asthma

BackgroundAsthma is a chronic airway disease affecting millions of people. Better methods to define asthma subgroups using clinical parameters and molecular biomarkers are crucial in the development of personalized medicine.ObjectiveThe aim of this study was to determine if circulating microRNAs (miRNAs) may be used to distinguish well–defined asthma groups.MethodsBlood serum from 116 well-defined subjects, including healthy controls and individuals with allergic or non-allergic asthma, from the West Sweden Asthma Study were included. Serum was analyzed for circulating miRNA expression of miR-126, − 145, −146a, − 155, − 223, and -374a and eosinophil cationic protein (ECP). Correlations between clinical characteristics and circulating miRNA expression as well as potential miRNA gene targets were investigated.ResultsA subset of miRNAs were differentially expressed between allergic and non-allergic asthmatic individuals. Alterations in expression of miR-155, −146a, −374a and − 145 were observed in allergic asthmatics in response to inhaled corticosteroid usage. Additionally, miR-223 and miR-374a expression varied in non-allergic asthmatics based on blood eosinophil numbers. Numerous clinical parameters, including lung function measurements, correlated with subsets of miRNAs. Finally, pathway analysis revealed a potential role for inhaled corticosteroid induced miRNAs in leukocyte regulation, IL-6 signaling and glucocorticoid response.ConclusionCirculating miRNA expression was altered in subjects with allergic and non-allergic asthma and correlated to clinical parameters including lung function and potential gene targets involved in immune processes. This combination of clinical and molecular data may be a basis for the further, more precise classification of asthma subgroups. Taken together, these findings would further asthma research and benefit future patients through the discovery of molecular mechanisms as well as identifying asthma subgroups contributing to the development of personalized medicine.

NF-κB pathway took part in the development of apoptosis mediated by miR-15a and oxidative stress via mitochondrial pathway in ammonia-treated chicken splenic lymphocytes

Ammonia, a kind of gas with pungent smell, is harmful to livestock and people, and has bad influence on the atmosphere. However, the mechanism of splenic toxicity caused by ammonia is still poorly understood. The aim of present study was to investigate the effect of ammonia on chicken splenic lymphocytes from the perspective of apoptosis. Chicken splenic lymphocytes were divided into the control group and the two ammonium treatment groups (1 mmol/L and 5 mmol/L ammonia), and were cultured for 24 h. CCK-8, flow cytometry (FC), fluorescence microscope, quantitative real-time PCR (qRT-PCR), and Western blot were used to study the differences between different groups. The results showed that ammonia exposure increased the release of calcium (Ca)2+ and reactive oxygen species (ROS) from mitochondrion. Besides, we found an increase in mRNA levels of glutathione peroxidase (GPx), inflammation-related genes (nuclear factor-κB (NF-κB), cyclooxygenase-2 (COX-2), inducible nitric (iNOS), tumor necrosis factor-α (TNF-α), and transforming growth factor-β (TGF-β)), apoptosis-related genes (B-cell lymphoma-2 (BCL-2), Bcl-2 associated X protein (BAX), Cytochrome c (Cytc), apoptotic protease activating factor 1 (APAF1), Caspase-9, and Caspase-3), and an increase in protein levels of NF-κB, iNOS, BAX, Cytc, Caspase-9, and Caspase-3. At the same time, we found a decrease level of GPx protein expression, and a decrease level of glutathione S-transferase (GST) mRNA expression, and a decrease level of heme oxygenase-1 (HO-1) and BCL-2 mRNA and protein expression in splenic lymphocytes exposed to ammonia. Meanwhile, miR-15a expression increased under ammonia exposure. In summary, these results indicated that ammonia induced oxidative stress, promoted the release of Ca2+, Cytc, and ROS from mitochondria, and then induced mitochondria-mediated inflammatory response, finally triggered apoptosis in chicken splenic lymphocytes.

Circulating miR-320a as a Predictive Biomarker for Left Ventricular Remodelling in STEMI Patients Undergoing Primary Percutaneous Coronary Intervention.

Restoration of epicardial coronary blood flow, achieved by early reperfusion with primary percutaneous coronary intervention (PPCI), is the guideline recommended to treat patients with ST-segment-elevation myocardial infarction (STEMI). However, despite successful blood restoration, increasing numbers of patients develop left ventricular adverse remodelling (LVAR) and heart failure. Therefore, reliable prognostic biomarkers for LVAR in STEMI are urgently needed. Our aim was to investigate the role of circulating microRNAs (miRNAs) and their association with LVAR in STEMI patients following the PPCI procedure. We analysed the expression of circulating miRNAs in blood samples of 56 patients collected at admission and after revascularization (at 3, 6, 12 and 24 h). The associations between miRNAs and left ventricular end diastolic volumes at 6 months were estimated to detect LVAR. miRNAs were also analysed in samples isolated from peripheral blood mononuclear cells (PBMCs) and human myocardium of failing hearts. Kinetic analysis of miRNAs showed a fast time-dependent increase in miR-133a, miR-133b, miR-193b, miR-499, and miR-320a in STEMI patients compared to controls. Moreover, the expression of miR-29a, miR-29b, miR-324, miR-208, miR-423, miR-522, and miR-545 was differentially expressed even before PPCI in STEMI. Furthermore, the increase in circulating miR-320a and the decrease in its expression in PBMCs were significantly associated with LVAR and correlated with the expression of miR-320a in human failing myocardium from ischaemic origin. In conclusion, we determined the time course expression of new circulating miRNAs in patients with STEMI treated with PPCI and we showed that miR-320a was positively associated with LVAR.

Expression study of candidate miRNAs and evaluation of their potential use as biomarkers of diabetic neuropathy.

Aim: To evaluate the expression of candidate miRNAs in relation to diabetic polyneuropathy (DPN) and cardiovascular autonomic neuropathy (CAN). Materials & methods: The expression of six candidate miRNAs has been evaluated in 49 Type 2 diabetes patients with neurological evaluation. Results: A higher expression of miR-128a was seen in patients with DPN compared with those without DPN (p = 0.015). miR-155 and miR-499a seemed to be down-expressed in patients with DPN (p = 0.04 and p = 0.05, respectively). A lower expression of miR-155 (p = 0.05) was observed even in patients with CAN with respect to CAN-negative. A higher expression of miR-155 was associated with the rs767649 polymorphism variant allele compared with the wild-type allele (p = 0.03). Conclusion:miR-128a, miR-155 and miR-499a might be involved in diabetic neuropathies development.

Isoliquiritigenin Derivative Regulates miR-374a/BAX Axis to Suppress Triple-Negative Breast Cancer Tumorigenesis and Development.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer that accounts for the largest proportion of breast cancer-related deaths. Thus, it is imperative to search for novel drug candidates with potent anti-TNBC effects. Recent studies suggest that isoliquiritigenin (ISL) can significantly suppress the growth, migration, and invasion of breast cancer cells. We previously synthesized ISL derivatives and found that 3′,4′,5′,4″-tetramethoxychalcone (TMC) inhibits TNBC cell proliferation to a greater degree than ISL. The present study aimed to investigate the mechanisms underlying the anti-TNBC effects of TMC in vitro and in vivo. We show that TMC significantly inhibits the proliferative, migratory, and invasive abilities of MDA-MB-231 and BT549 cells. TMC induces apoptosis through the upregulation of Bax and downregulation of Bcl-2. PCR arrays demonstrate a significant decrease in miR-374a expression in TNBC cells after 24-h TMC treatment. MiR-374a is overexpressed in TNBC cells and has oncogenic properties. Real-time PCR analysis confirmed that TMC inhibits miR-374a in a dose-dependent manner, and luciferase assays confirmed that BAX is targeted by miR-374a. Further, we show that TMC increases Bax protein and mRNA levels by inhibiting miR-374a. TMC also attenuates TNBC tumor volumes and weights in vivo. These results demonstrate that TMC inhibits TNBC cell proliferation, foci formation, migration, invasion, and tumorigenesis, suggesting its potential to serve as a novel drug for treating TNBC through miR-374a repression.

Differential regulation of microRNA-15a by radiation affects angiogenesis and tumor growth via modulation of acid sphingomyelinase

Activation of acid sphingomyelinase (SMPD1) and the generation of ceramide is a critical regulator of apoptosis in response to cellular stress including radiation. Endothelial SMPD1 has been shown to regulate tumor responses to radiation therapy. We show here that the SMPD1 gene is regulated by a microRNA (miR), miR-15a, in endothelial cells (ECs). Standard low dose radiation (2 Gy) upregulates miR-15a and decreases SMPD1 levels. In contrast, high dose radiation (10 Gy and above) decreases miR-15a and increases SMPD1. Ectopic expression of miR-15a decreases both mRNA and protein levels of SMPD1. Mimicking the effects of high dose radiation with a miR-15a inhibitor decreases cell proliferation and increases active Caspase-3 & 7. Mechanistically, inhibition of miR-15a increases inflammatory cytokines, activates caspase-1 inflammasome and increases Gasdermin D, an effector of pyroptosis. Importantly, both systemic and vascular-targeted delivery of miR-15a inhibitor decreases angiogenesis and tumor growth in a CT26 murine colorectal carcinoma model. Taken together, our findings highlight a novel role for miR mediated regulation of SMPD1 during radiation responses and establish proof-of-concept that this pathway can be targeted with a miR inhibitor.

Gastric Cancer Cell-Derived Exosomal microRNA-23a Promotes Angiogenesis by Targeting PTEN.

Hypoxia-exposed lung cancer-released exosomal microRNA-23a (miR-23a) has been shown to enhance angiogenesis as well as vascular permeability, contributing to the close correlation between exosomal miR-23a and tumorigenesis. The current study aimed to investigate whether gastric cancer (GC) cell-derived exosomal miR-23a could induce angiogenesis and to elucidate the potential mechanisms associated with the process. Differentially expressed miRNAs in GC were initially screened from the Gene Expression Omnibus database. Target genes were selected following miRNA-mRNA prediction and subsequently verified by dual luciferase reporter assay. RT-qPCR was conducted to detect miR-23a and PTEN expression in GC tissues, cells and exosomes. Human umbilical venous endothelial cells (HUVECs) were co-cultured with GC cell-derived exosomes to assess the angiogenesis mediated by exosomes in vitro. Additionally, PTEN was overexpressed in HUVECs to analyze the mechanism by which miR-23a regulates angiogenesis. miR-23a was highly expressed in GC tissues and cells and GC cell-derived exosomes. Angiogenesis was promoted by the co-culture of HUVECs and GC cells-derived exosomes, as evidenced by the increased expression of VEGF but decreased expression of TSP-1. PTEN was targeted by miR-23a and was lowly expressed in GC tissues. In a co-culture system, miR-23a carried by GC cells-derived exosomes promoted angiogenesis via the repression of PTEN. Collectively, GC cell-derived exosomal miR-23a could promote angiogenesis and provide blood supply for growth of GC cells. This study contributes to advancement of miRNA-targeted therapeutics.

RNA G-quadruplex regulates microRNA-26a biogenesis and function

RNA G-quadruplexes (RG4s) appear to be important in post-transcriptional gene regulation, but their pathophysiological functions remain unknown. MicroRNA-26a (miR-26a) is emerging as a therapeutic target for various human diseases, however the mechanisms underlying endogenous miR-26a regulation are poorly understood. Herein, we study the role of RG4 in miR-26a expression and function invitro and invivo. Putative RG4s within liver-enriched miRNAs were predicted by bioinformatic analysis, and the presence of an RG4 structure in the miR-26a-1 precursor (pre-miR-26a-1) was further analyzed by biophysical and biochemical methods. RG4 stabilizers, pre-miR-26a-1 overexpression plasmids, and luciferase reporter assays were used to assess the effect of RG4 on pre-miR-26a-1 maturation. Both miR-26a knock-in and knockout mouse models were employed to investigate the influence of this RG4 on miR-26a expression and function. Moreover, the interaction between RG4 in pre-miR-26a-1 and DEAH-box helicase 36 (DHX36) was determined by biophysical and molecular methods. Finally, miR-26a processing and DHX36 expression were quantified in the livers of obese mice. We identify a guanine-rich sequence in pre-miR-26a-1 that can fold into an RG4 structure. This RG4 impairs pre-miR-26a-1 maturation, resulting in a decrease in miR-26a expression and subsequently an increase in miR-26a cognate targets. In line with known miR-26a functions, this RG4 can regulate hepatic insulin sensitivity and lipid metabolism invitro and invivo. Furthermore, we reveal that DHX36 can bind and unwind this RG4 structure, thereby enhancing miR-26a maturation. Intriguingly, there is a concordant decrease of miR-26a maturation and DHX36 expression in obese mouse livers. Our findings define a dynamic DHX36/RG4/miR-26a regulatory axis during obesity, highlighting an important role of RG4 in physiology and pathology. Specific RNA sequences called G-quadruplexes (orRG4) appear to be important in post-transcriptional gene regulation. Obesity leads to the formation of these RG4 structures in pre-miR-26a-1 molecules, impairing the maturation and function of miR-26a, which has emerged as a therapeutic target in several diseases. This contributes to hepatic insulin resistance and the dysregulation of liver metabolism.

Increased Expression of Micro-RNA-23a Mediates Chemoresistance to Cytarabine in Acute Myeloid Leukemia.

Resistance to chemotherapy is one of the primary obstacles in acute myeloid leukemia (AML) therapy. Micro-RNA-23a (miR-23a) is frequently deregulated in AML and has been linked to chemoresistance in solid cancers. We, therefore, studied its role in chemoresistance to cytarabine (AraC), which forms the backbone of all cytostatic AML treatments. Initially, we assessed AraC sensitivity in three AML cell lines following miR-23a overexpression/knockdown using MTT-cell viability and soft-agar colony-formation assays. Overexpression of miR-23a decreased the sensitivity to AraC, whereas its knockdown had the opposite effect. Analysis of clinical data revealed that high miR-23a expression correlated with relapsed/refractory (R/R) AML disease stages, the leukemic stem cell compartment, as well as with inferior overall survival (OS) and event-free survival (EFS) in AraC-treated patients. Mechanistically, we demonstrate that miR-23a targets and downregulates topoisomerase-2-beta (TOP2B), and that TOP2B knockdown mediates AraC chemoresistance as well. Likewise, low TOP2B expression also correlated with R/R-AML disease stages and inferior EFS/OS. In conclusion, we show that increased expression of miR-23a mediates chemoresistance to AraC in AML and that it correlates with an inferior outcome in AraC-treated AML patients. We further demonstrate that miR-23a causes the downregulation of TOP2B, which is likely to mediate its effects on AraC sensitivity.

Bone marrow mesenchymal stem cells-derived exosomes improve injury of hippocampal neurons in rats with depression by upregulating microRNA-26a expression

ObjectiveBone marrow mesenchymal stem cells (BMSCs)-derived exosomes have been widely applied in disease therapies. However, the role of BMSCs-derived exosomes in depression remains obscure. This study aims to explore the mechanism of BMSCs-derived exosomal microRNA-26a (miR-26a) on hippocampal neuronal injury of depressed rats. MethodsBMSCs and their exosomes were obtained and identified. Rat models of depression were established by corticosterone injection, then injected with BMSCs-derived exosomes. The contents of superoxide dismutase (SOD), imalondialdehyde (MDA), lactate dehydrogenase (LDH), tumor necrosis factor α (TNF-α), and interleukin-1β (IL-1β) in rats’ serum, hippocampal tissues and neurons were determined. The expression of miR-26a in hippocampal tissues and neurons was detected by RT-qPCR. The injury models of rat hippocampal neurons were established to figure out the role of BMSCs-derived exosomes and miR-26a in neuron apoptosis and proliferation. ResultsIn hippocampal tissues of depressed rats, miR-26a was lowly expressed, and BMSCs-derived exosomes upregulated miR-26a expression. BMSCs-derived exosomes restrained apoptosis in hippocampal tissues of depressed rats. BMSCs-derived exosomes and upregulated miR-26a elevated SOD level, lessened MDA, LDH, TNF-α and IL-1β levels, boosted hippocampal neuron proliferation and suppressed apoptosis in depressed rats. ConclusionCollectively, our study reveals that miR-26a is lowly expressed in depressed rats, and BMSCs-derived exosomes improve hippocampal neuron injury of rat with depression by upregulating miR-26a.

Abstract P1-05-12: Norcantharidin suppressed cell migration and invasion by modulating the β-catenin/miR-301a/ZBTB4 signaling axis in triple-negative breast cancer

Abstract Purpose: Breast cancer is the leading malignant tumor in women worldwide. Since not responding to endocrine therapy and anti-human epidermal growth factor receptor 2 (HER2) therapy, triple-negative breast cancer (TNBC) has the highest recurrence rate among all subtypes of breast cancer. MicroRNAs have been proved to be involved in the progression of various tumors. It has been demonstrated that miR-301a promotes cell proliferation, migration and invasion in breast cancer. However, the functions and underlying mechanism of miR-301a in TNBC are undefined. Methods: Bioinformatics analysis was conducted to analyze the expression level of miR-301a. RT-qPCR was used to confirm the level of miR-301a in breast cancer patients. Migration and invasion assay was performed to analyze the function of miR-301a. Western blot was carried out to compare the protein level of genes. A luciferase reporter assay was used to verify the downstream target gene of miR-301a. Immunohistochemistry was conducted to evaluate the protein level of ZBTB4. Immunofluorescence was performed to assess the influence of norcantharidin (NCTD) on β-catenin. Finally, lung metastasis model was established in vivo through tail vein injection of breast cancer cells to evaluate the anti-tumor effect of NCTD. Results: In this study, the expression signatures of miRNAs were analyzed using the high-throughput sequencing data from The Cancer Genome Atlas (TCGA). The results showed that miR-301a was dysregulated in TNBC, and the expression of miR-301a was upregulated in breast cancer, especially in TNBC. In order to analyze the clinical relevance and prognostic correlation of miR-301a in breast cancer, complete data with clinicopathology features and overall survival (OS) of 897 breast cancer patients were collected from the TCGA database. The result indicated a high expression level of miR-301a is an unfavorable prognostic factor for breast cancer patients. Moreover, the migration and invasion of TNBC cells were enhanced by miR-301a mimics and weakened by miR-301a inhibitors. Furthermore, ZBTB4 was identified as a downstream target gene of miR-301a by the Western blot and luciferase reporter assay, and overexpression of ZBTB4 reduced the effect of miR-301a on cell migration and invasion. The level of miR-301a was decreased and that of ZBTB4 was elevated following knockdown of β-catenin by siRNA. The result of immunohistochemistry showed that ZBTB4 was downregulated in breast cancer tissues and the result of RT-qPCR of 77 breast cancer tissue samples indicated that the mRNA level of ZBTB4 was significantly lower in TNBC samples than in non-TNBC samples. Then the plasmid of ZBTB4 was constructed and successfully transfected into TNBC cells. However, overexpression of ZBTB4 had no effect on the level of β-catenin. Importantly, we found that NCTD, the demethylated analog of cantharidin, suppressed the migration and invasion of TNBC cells by downregulating miR-301a and upregulating ZBTB4 through the β-catenin signaling pathway. Then lung metastasis model was established by tail vein injection of cancer cells and NCTD decreased the number of lung metastatic lesions in vivo. Conclusion: Collectively, our findings illustrate that miR-301a plays a role in the carcinogenesis of TNBC by targeting ZBTB4, and NCTD may be used as a potential adjuvant treatment for TNBC. Citation Format: Qiang He, Xinrong Luo, Guosheng Ren, Hongyuan Li. Norcantharidin suppressed cell migration and invasion by modulating the β-catenin/miR-301a/ZBTB4 signaling axis in triple-negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-05-12.

The protective effects of microRNA-26a in steroid-induced osteonecrosis of the femoral head by repressing EZH2

ABSTRACT Recently, the role of microRNAs (miRs) in human diseases has been verified. This study was determined to explore the protective effects of microRNA-26a (miR-26a) in steroid-induced osteonecrosis of the femoral head (SONFH) with the involvement of enhancer of zeste homologue 2 (EZH2). Femoral head (FH) samples from SONFH patients and patients with femoral neck fracture were collected, and rat SONFH models were established by Escherichia coli endotoxin combining with large dose steroid pulse assay. The hemorheology, blood lipid, inflammatory factors, and pathologic changes were measured by a series of experiments. Moreover, the detection of osteoblasts, osteoclasts, miR-26a expression, EZH2 expression, osteoprotegerin (OPG) and osteoprotegerin ligand (OPGL), and the apoptosis of osteocytes were conducted. The target relation between miR-26a and EZH2 was clarified by bioinformatics and dual-luciferase reporter gene assay. MiR-26a was poorly expressed, while EZH2 was highly expressed in SONFH, and the elevation of miR-26a could repress EZH2 expression. Elevated miR-26a and reduced EZH2 were able to decelerate the apoptosis of osteocytes, increase osteoblasts, and decrease osteoclasts, resulting in a repression of SONFH progression. Additionally, EZH2 was a target gene of miR-26a. Furthermore, the elevation of EZH2 could reverse the repression of SONFH progression that is induced by elevated miR-26a. We found that up-regulation of miR-26a and knockdown of EZH2 could suppress the development of SONFH, which would contribute to the therapy of SONFH.

Examination of the Role of miR-23a in the Development of Thermotolerance.

Thermotolerance is an acquired state of increased heat resistance that occurs following exposure to non-lethal proteotoxic stress. A large body of evidences implicates that molecular chaperon members belonging to the heat shock protein family could be acting as potential mediators of the thermotolerant state. Recent evidence has demonstrated heat shock proteins HSP90, HSP70 and HSP27 have inhibited heat-induced cell death by intervening at various steps in stressinduced apoptotic pathways. Previous studies have shown that HSP70 prevented heatinduced apoptosis by preventing the NOXA dependent decrease in MCL-1 levels leading to both BAX activation and cytochrome c release from mitochondria. We have also demonstrated that HSP70 expressing cells have enhanced levels of miR-23a prevent heat-induced increase in NOXA levels and suppress apoptosis. Stably transfected cell lines expressing either a control shRNA or a miR-23a targeting shRNA are quantified using both RT-PCR and semi-quantitative RT-PCR to determine the effect of different hyperthermic exposure treatment on miR-23a and Noxa mRNA expression levels. This study shows that thermotolerant-induced pre-heat shock treatment is capable of increasing miR-23a levels. Furthermore, stable cell clones expressing a miR- 23a targeting shRNA having reduced miR-23a levels are incapable of developing a thermotolerance state, leading to apoptosis. These results demonstrate the novel finding that miR-23a is an important factor in the development of the thermotolerant state.

MicroRNA-135a Regulates VEGFC Expression and Promotes Luteinized Granulosa Cell Apoptosis in Polycystic Ovary Syndrome.

Androgen is known to regulate microRNA-135a (miR-135a) and can be regulated by androgen, suggesting that it may contribute to polycystic ovary syndrome (PCOS) with hyperandrogenism. However, its roles and mechanisms of action in PCOS are unknown. In this study, the role and molecular mechanisms underlying miR-135a in granulosa cells (GCs) in PCOS were evaluated. miR-135a expression was upregulated in patients with PCOS and in GCs isolated from patients compared with that in the respective controls (P < 0.01), as determined by RT-qPCR. The overexpression of miR-135a inhibited GC proliferation and induced GC apoptosis, as observed by CCK-8 assay and apoptosis assay. Furthermore, miR-135a overexpression increased the expression of double-strand break maker, γH2AX, as confirmed by western blotting. Our results further suggest that these effects were mediated via downregulation of vascular endothelial growth factor C (VEGFC), which was identified as a direct target of miR-135a. Moreover, levels of VEGFC and miR-135a expression showed a negative correlation. These findings indicate that miR-135a promotes apoptosis and the DNA damage response in GCs in PCOS, likely via VEGFC signaling. This study provides novel insights into GC dysregulation in PCOS and suggests that miR-135a is a promising therapeutic target for PCOS treatment.

Study of serum microRNA-99a relative expression as a diagnostic and prognostic noninvasive biomarker of breast cancer in Egyptian females

BackgroundBreast cancer (BC) is the most significant cause of malignancy deaths among females in Egypt as well as in other countries. Despite the evident miR-99a deregulation in many malignancies, including breast cancer, its serum level and clinical significance in breast cancer are still vague. This study assessed the role of dysregulation of serum levels of miR-99a in the diagnosis and prognosis of breast cancer in Egyptian females. MethodsBlood samples were obtained from 60 breast cancer females diagnosed as invasive ductal carcinoma and 40 healthy females of matched age. Samples were evaluated for miR-99a relative expression using SYBR green-based real-time RT-PCR technology. Also, the association between serum miR-99a expression and CA 15–3, the known tumor marker of breast cancer, was investigated. ResultsThe serum expression level of MiR-99a was lower in the breast cancer group than that of the control group (P < 0.001). MiR-99a was 76.7% sensitive and 95% specific in the diagnosis of breast cancer and its level correlated with many clinical parameters of breast cancer, including primary tumor extent (P = 0.021), TNM stage (P < 0.001), and histological grade of cancer (P = 0.003). Regression analysis revealed that lower miRNA 99a expression was considered as an independent predictor for the higher BC tumor stage. ConclusionExpression of serum miR-99a was downregulated in breast cancer offering early diagnosis and indicating poor prognosis of cases.

DietaryAdvancedGlycationEnd Products-InducedCognitive Impairment in Aged ICR Mice: Protective Role of Quercetin.

Dietary advanced glycation products (dAGEs) have been reported to induce cognitive impairment while quercetin possesses potential neuroprotective effects. The aim is to explore whether dAGEs would induce similar cognitive impairment from both young and aged ICR mice, and the protective effects of quercetin. A total of 32 aged ICR mice (15-month-old) and 16 young ICR mice (3-month-old) are randomly assigned into the following six groups: Young mice control group, young mice fed with AGEs diet group, old mice control group, old mice fed with AGEs diet group, old mice with quercetin supplemented diet group, old mice fed with AGE diet supplemented with quercetin group. Dietary AGEs induced cognitive impairment only in aged, but not in young, ICR mice, while quercetin intervention is capable of reversing dAGEs-induced cognitive dysfunction. This may be since quercetin 1) increased miR-219, miR-15a, and miR-132 expression, inhibited p-ERK1/2, and tau phosphorylation; and 2) improved gut microbiota richness and diversity, inhibited phylum Tenericutes and Proteobacteria, and elevated butyric acid from cecum. Prolonged application of quercetin may be beneficial in the elderly, especially for those with high consumption of dAGEs.

MiR-15a Functions as a Diagnostic Biomarker for Coronary Artery Disease.

The present study aimed to explore the diagnostic value of miR-15a in coronary artery disease (CAD). After recruiting all the participants, peripheral blood samples were obtained according to the instructions. miR-15a expression was evaluated using real-time quantitative polymerase chain reaction (RT-qPCR) and IL-6, TNF-α, and hs-CRP expressions were detected using ELISA kits. The results elucidated a significantly decreased expression of miR-15a in peripheral blood samples from CAD patients compared to non-CAD controls (p < 0.01). Meanwhile, miR-15a expression was negatively correlated with LDL-C and Gensini score (p = 0.0059, 0.0243, respectively). Moreover, miR-15a expression was negatively correlated with inflammatory cytokines IL-6, TNF-α, and hs-CRP (p = 0.0009, 0.0178, 0.0005, respectively). The receiver operating curve (ROC) curve analysis demonstrated that miR-15a was a promising biomarker for early diagnosis of CAD with an area under the curve (AUC) of 0.9368. The present study elucidated a decreased miR-15a expression in CAD patients and showed negative correlations with LDL-C, Gensini score, and inflammatory cytokines. miR-15a may serve as a promising biomarker for early diagnosis of CAD.

Silencing of long non-coding RNA PCAT6 restrains gastric cancer cell proliferation and epithelial-mesenchymal transition by targeting microRNA-15a.

Gastric cancer (GC) is a high mortality disease. We studied the function and mechanism of long non-coding RNA prostate cancer-associated transcript 6 (lncRNA PCAT6) on cell proliferation and epithelial-mesenchymal transition (EMT) in GC cells. CCK-8, flow cytometry and colony formation assay were respectively used to detect the cell viability, apoptosis and colony formation. PCAT6 and miR-15a expression were changed by cell transfection. Moreover, the level of Cyclin D1, p53, Bax, Cleaved caspase-3 and relate-proteins of EMT and cell pathways were investigated by Western blot. Besides, the level of miR-15a and PCAT6 was tested by RT-qPCR. Besides, the target relation between miR-15a and PCAT6 were tested by luciferase assay. PCAT6 was highly expressed in GC cells and tissues. Silencing of PCAT6 restrained the relate-proteins of cell proliferation and EMT. Furthermore, PCAT6 reversely regulated miR-15a and miR-15a inhibitor reversed the efficacy of sh-PCAT6 in cell proliferation and EMT. PCAT6 restrained the relate-proteins of RB/E2F and Wnt/β-catenin pathways and miR-15a reverse this progress. Finally, PCAT6 was a target of miR-15a. Silencing of lncRNA PCAT6 restrained proliferation and EMT of GC cells by targeting miR-15a via RB/E2F and Wnt/β-catenin pathways.

Plasma Levels of miR-27a, miR-130b, and miR-301a in Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is a gynecological endocrine disorder in women of reproductive age. There is adequate evidence that suggests several microRNAs (miRNAs) are of great importance for PCOS. It seems that dysregulated expression of miR-27a, miR-130b, and miR-301a are associated with PCOS. The aim of this study was to investigate whether plasma levels of these miRNAs are different between patients with PCOS and healthy controls. Fifty-three women with a definite diagnosis of PCOS, and 53 healthy controls were enrolled. MiRNAs expression levels in plasma were evaluated by real-time PCR. The diagnostic values of each miRNA were calculated by the receiver operating characteristic (ROC) curve and areas under the curves (AUC). The main clinical characteristics were not significantly different between the two groups. The circulating plasma expression levels of miR-27a and miR-301a had a significant increase (P = 0.0008 and P <0.0001, respectively) but miR-130b expression level decreased in the patient group (P <0.0001). The AUC for miR-27a, miR-130b, and miR-301a were 0.71, 0.77, and 0.66, respectively. A positive exponential was observed for miR-27a and miR-301a in multiple logistic regression. Changes in the plasma expressions of the studied miRNAs are likely to be associated with PCOS phenotypes. MiR-27a has a potential to serve as a diagnostic biomarker of PCOS.

The Fas/FasL Signaling Pathway: Its Role in the Metastatic Process and as a Target for Treating Osteosarcoma Lung Metastases.

Understanding how the tumor microenvironment participates in inhibiting or supporting tumor growth is critical for the development of novel therapies. Osteosarcoma (OS) metastasizes almost exclusively to the lung, an organ where Fas ligand (FasL) is constitutively expressed. This chapter focuses on our studies dedicated to the interaction of OS cells with the lung microenvironment. We will summarize our studies conducted over the past 20years showing the importance of the Fas/FasL signaling pathway to the establishment and progression of OS metastases in the lung. We demonstrated that the FasL+ lung microenvironment eliminates Fas-positive (Fas+)OS cells that metastasize to the lungs, through apoptosis induced by Fas signaling following interaction of Fas on the tumor cell surface with FasL on the lung epithelial cells. Expression of the Fas receptor on OS cells inversely correlated with the ability of OS cells to form lung metastases. Blocking this pathway interferes with this process, allowing Fas+ cells to grow in the lung. By contrast, upregulation of Fas on Fas- OS cells inhibited their ability to metastasize to the lung. We demonstrated how the FasL+ lung microenvironment can be leveraged for therapeutic intent through the upregulation of Fas expression. To this end, we demonstrated that the histone deacetylase inhibitor entinostat upregulated Fas expression on OS cells, reduced their ability to form lung metastases, and induced regression of established micrometastases. Fas expression in OS cells is regulated epigenetically by the microRNA miR-20a. We showed that expressions of Fas and miR-20a are inversely correlated, and that delivery of anti-miR-20a in vivo to mice with established osteosarcoma lung metastases resulted in upregulation of Fas and tumor regression. Therefore, targeting the Fas signaling pathway may present therapeutic opportunities, which target the lung microenvironment for elimination of OS lung metastases. We have also shown that in addition to being critically involved in the metastatic potential, the Fas signaling pathway may also contribute to the efficacy of chemotherapy. We demonstrated that the chemotherapeutic agent gemcitabine (GCB) increased Fas expression in both human and mouse OS cells in vitro. In vivo, aerosol GCB therapy induced upregulation of Fas expression and the regression of established osteosarcoma lung metastases. The therapeutic efficacy of GCB was contingent upon a FasL+ lung microenvironment as aerosol GCB had no effect in FasL-deficient mice. Manipulation of Fas expression and the Fas pathway should be considered, as this concept may provide additional novel therapeutic approaches for treating patients with OS lung metastases.