miR-124 Levels Research Articles

49-OR: Association of Gene Polymorphisms and Plasma MiRNAs with Diabetic Retinopathy

Circulating microRNAs (miRNAs/miRs) have emerged as potential disease biomarkers; however, their expression in diabetic retinopathy (DR) has been evaluated only recently in humans. Whilst it is recognized that sequence variants in miRNA genes (miRSNPs) can alter the miRNA-mediated regulation of gene expression, there are almost no studies on the association of miRSNPs with DR. In this case-control study, we investigated whether the functional polymorphisms rs531564 G>C (MIR124-1), rs4636297 A>G (MIR126) and 2910164 C>G (MIR146A) are associated with DR in 1,025 Brazilians with type 2 diabetes. We also evaluated whether the plasma levels of miR-124, miR-126 and miR-146a are associated with DR and with miRSNPs in 182 patients. Blood donors were included to determine the prevalence of miRSNPs (n = 104) and miRNA expression (n = 20) in the general population. Genotyping was done by real-time PCR and plasma levels were quantified by RT-qPCR. Genotype and allele frequencies in diabetic subjects did not significantly differ from those of blood donors, and the minor allele frequencies were similar to those reported in other populations. The MIR126 heterozygous genotype was more frequent in patients without DR than in those with DR (52% versus 44%, respectively, p = 0.040), and remained associated with this complication after controlling for clinical covariates. MiR-126 was markedly downregulated in diabetic subjects as compared to blood donors and even more reduced among those with DR (mean log2 fold-change of -0.7 ± 1.7, -1.1 ± 2.0 and -1.7 ± 1.6 for subjects without DR, with nonproliferative DR or with proliferative DR, p = 0.002). Patients carrying the A allele of the rs4636297 polymorphism had almost twice higher levels of miR-126 than patients with GG genotype. MiR-126 and miR-146a were strongly correlated and the levels of miR-146a were also lower in diabetic subjects than in blood donors. Overall, our findings reinforce that gene variants and expression of miRNAs are associated with DR in type 2 diabetes. Disclosure E.R. Polina: None. R.C. Sbruzzi: None. F.M. de Oliveira: None. D. Crispim: None. M.D. Enoia: None. L.H. Canani: Research Support; Self; Novo Nordisk Inc. Speaker's Bureau; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Sanofi. K.G. Santos: None. Funding Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (1994-2551/13-4); Fundo de Incentivo à Pesquisa e Eventos do Hospital de Clínicas de Porto Alegre (14-0645)

MiR-124 inhibits spinal neuronal apoptosis through binding to GCH1.

To explore the effect of micro ribonucleic acid (miR)-124 on the spinal neuronal apoptosis and to explore its related mechanism. The rat model of spinal cord injury (SCI) was established, agomir-124 was injected intrathecally and the effect of agomir-124 on motor function recovery of rats was evaluated using the Basso-Beattie-Bresnahan (BBB) score. The gene expression levels of miR-124 and GTP-cyclohydrolase 1 (GCH1) in spinal cord tissues were detected via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR), and the correlation between them was detected using the Pearson correlation coefficient. Then, the direct interaction between miR-124 and GCH1 mRNA was detected using the TargetScan software and luciferase reporter assay. The changes in apoptosis in each group were examined via flow cytometry and Western blotting. Moreover, the changes in the tetrahydrobiopterin (BH4) content in each group were detected via high-performance liquid chromatography, and the changes in the nitrite level in the supernatant in each group were detected using the Griess reagent. Finally, the changes in the activity of the inducible nitric oxide synthase (iNOS) protein were detected using the iNOS kit. Compared with that in the model group, the BBB score was significantly increased in agomir-124 group at 21, 28, 35 and 42 d. In the agomir-124 group, the relative expression level of miR 124 in spinal cord tissues was significantly increased at 7-28 d and reached the peak at 21 d, while the mRNA level of GCH1 in spinal cord tissues declined and touched the bottom at 21 d. According to the Pearson correlation coefficient, there was a significant negative correlation between the expression of miR-124 and mRNA expression of GCH1 (r =- 0.87, p = 1.5e-6). It was found in the prediction using TargetScan software that GCH1 might be a potential target for miR-124, which was further verified by the luciferase reporter assay. The results of flow cytometry and Western blotting showed that miR-124 significantly reduced the LPS-induced primary spinal neuronal apoptosis, while the miR-124 inhibitor remarkably increased the primary spinal neuronal apoptosis. Moreover, it was also found that the knockout of GCH1 reduced the LPS-induced spinal neuronal apoptosis. In addition, the GCH1 overexpression assay revealed that miR-124 inhibited spinal neuronal apoptosis by suppressing the GCH1 expression. LPS + miR-124 remarkably decreased the BH4 content, nitrite level, and iNOS activity while LPS + miR-124 + GCH1 remarkably increased the BH4 content, nitrite level, and iNOS activity. MiR-124 inhibits neuronal apoptosis in SCI by binding to GCH1. The results in the present study may provide a new mechanism for the therapeutic effect of miR-124, and miR-124 may have a potential therapeutic value in the treatment of SCI in the future.

MiR-124 changes the sensitivity of lung cancer cells to cisplatin through targeting STAT3.

To investigate the role of micro ribonucleic acid (miR)-124 in drug resistance of non-small cell lung cancer (NSCLC), and to explore its underlying mechanism. The expression levels of miR-124 and signal transducer and activator of transcription 3 (STAT3) in maternal A549 cells and cisplatin-resistant A549/DDP cells were detected via quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting. A549 and A549/DDP cells were transfected with miR-124 mimics and miR-124 negative control (NC), respectively. Changes in the expression of STAT3 were detected via qRT-PCR and Western blotting. Meanwhile, the sensitivity of cells transfected with miR-124 mimics to cisplatin was detected via methyl thiazolyl tetrazolium (MTT) assay. The effects of miR-124 on the apoptosis, invasion and metastasis of cells were detected via flow cytometry, wound healing assay and transwell assay, respectively. Moreover, wild-type and mutant-type STAT3 luciferase reporter plasmids were co-transfected with miR-124 mimics or miR-124 NC. Luciferase activity was analyzed using the dual-luciferase reporter gene assay. QRT-PCR and Western blotting revealed that the expression level of miR-124 in A549/DDP cells was significantly lower than that of A549 cells. However, the expression level of STAT3 in A549/DDP cells was significantly higher than that of A549 cells. Overexpression of miR-124 remarkably reduced the expression level of STAT3 in A549/DDP cells, increased the sensitivity of A549/DDP cells to cisplatin, and inhibited the invasion and metastasis capacities of cells. In addition, luciferase reporter gene assay demonstrated that miR-124 could negatively regulate the protein expression of STAT3 by binding to its 3'-untranslated region (UTR). MiR-124 regulates the sensitivity of NSCLC to cisplatin. Moreover, it inhibits the invasion and metastasis capacities through targeting STAT3, which can serve as a therapeutic target for cisplatin-based chemotherapy resistance of NSCLC.

Upregulation of AKT1 and downregulation of AKT3 caused by dysregulation of microRNAs contributes to pathogenesis of hemangioma by promoting proliferation of endothelial cells.

This study aimed to verify the differentially expressed miRNAs (microRNAs) in hemangioma, and explore their roles in the pathogenesis of hemangioma in vivo and ex vivo. Real-time polymerase chain reaction (PCR) and western blot were used to measure reported differentially expressed miRNAs and their potential targets. In-silicon analysis and luciferase assay were conducted to find the target of miR-15a and miR-205. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and flowcytometry were performed to examine the effect of dysregulation of miR-15a and miR-205 on the proliferation and apoptosis of endothelial cells. Among all candidate miRNAs, only miR-205 level was significantly downregulated whereas miR-15a was evidently upregulated in the hemangioma group. Accordingly, AKT3 was validated to be the direct target of miR-15a and miR-205. Using real-time PCR, the level of AKT1 was much higher in hemangioma group, whereas level of AKT3 was much lower in the hemangioma group, and in general expression level of ATK was upregulated in the hemangioma group. Furthermore, the ATK1 level of cells transfected with miR-205 mimics and ATK1 siRNA was substantially downregulated, and anti-miR-205 mimic significantly improved the level of AKT1, and meanwhile the level of ATK3 and PTEN were remarkably suppressed after transfection with miR-15a mimics and ATK3 siRNA, whereas notably overexpressed after introduction of anti-miR-15a. And miR-15a, AKT3 siRNA and anti-miR-205 evidently induced viability, and miR-205, AKT1 siRNA, and anti-miR-15a obviously promoted apoptosis of cells. CONCLUSION: miR-15a and miR-205 had different expression in hemangioma, may be novel therapeutic targets in the treatment of hemangioma by targeting AKT3 and AKT1.

Association between circulating microRNA-126 expression level and tumour necrosis factor alpha in healthy smokers

Introduction: Smoking contributes to the death of a million people worldwide each year. Smokers experience an alteration in tumour necrosis factor-alpha (TNF-α), and the risk of expected lung cancer. The study aimed at investigating the expression levels of mir-126 and mir-124, as well as TNF-α as possible biomarkers of expected smoking-related diseases.Methods: Twenty-five male smokers’ age and sex-matched with 25 non-smokers were recruited for the present study. Plasma expression levels of mir-126 and mir-124 were evaluated using quantitative real-time PCR. Lipid profile, TNF-α, interleukin-6 and C-reactive protein were assessed in plasma of each participant.Results: Plasma miR-126 was statistically down-regulated in smokers relative to non-smokers; however, mir-124 did not show any significant changes between groups. Among the measured parameters, mir-126 and tumour necrosis factor alpha (TNF-α) displayed a good discrimination and sensitivity between smokers and non-smokers (AUC = 0.809 (95% CI: 0.668–0.95; p < 0.001) and 0.742(95% CI: 0.584–0.9; p < 0.01), respectively. Also, the combined evaluation of miR-126 and TNF-α levels showed high discrimination (AUC= 0.889 (95% CI: 0.779–1.00; p < 0.0001), sensitivity = 85%, and specificity = 80% in the diagnosis of smokers with non-smokers.Conclusions: MiR-126 and TNF-α are potential biomarkers of smoking-related diseases and are important in assessing the expected tobacco-related harm.

CADM1, MAL, and miR124 Promoter Methylation as Biomarkers of Transforming Cervical Intrapithelial Lesions.

Background: Squamous intraepithelial lesions/cervical intraepithelial neoplasias (SIL/CIN) are high-risk human papilloma virus (hrHPV)-related lesions which are considered as high grade (HSIL/CIN2-3) or low grade (LSIL/CIN1) lesions according to their risk of progression to cervical cancer (CC). Most HSIL/CIN2-3 are considered as transforming hrHPV infections, so truly CC precursors, although some clear spontaneously. hrHPV testing has a high sensitivity for the detection of HSIL/CIN2-3 but a relatively low specificity for identifying transforming lesions. We aimed to determine whether the combination of CADM1, MAL and miR124 promoter methylation status assessed in histological samples can be used as a biomarker in the identification of transforming HSIL/CIN lesions. Design: 131 cervical biopsies, including 8 cases with no lesion and a negative hrHPV test result (control group), 19 low-grade (L)SIL/CIN1, 30 HSIL/CIN2, 60 HSIL/CIN3, and 14 CC were prospectively collected. hrHPV was detected and genotyped using the polymerase chain reaction (PCR)-based technique SPF10 HPV LIPA. A multiplex quantitative methylation-specific PCR (qMSP) was used to identify the methylation status of the CADM1, MAL, and miR124 promoter genes. Results: Significantly higher methylation levels of CADM1, MAL and miR-124 were found in HSIL/CIN2-3 and CC compared with normal and LSIL lesions. DNA methylation of at least one gene was detected in 12.5% (1/8) of normal samples, 31.5% (6/19) of LSIL/CIN1, 83.3% (25/30) of HSIL/CIN2, 81.6% (49/60) of HSIL/CIN3 and 100% (14/14) of CC (p < 0.001). The sensitivity and specificity for HSIL/CIN2-3 and CC of having at least one methylated gene were 84.6% and 74.0%, respectively. The sensitivity and specificity of the combination of at least one methylated gene and a positive hrHPV test were 80.7% and 85.1% for HSIL/CIN2-3 and CC, respectively. Conclusions: The methylation rate of CADM1, MAL and miR124 increases with the severity of the lesion. Further research is warranted to evaluate the usefulness of these biomarkers for the identification of transforming HSIL/CIN.

Increased levels of miR-124 in human dental pulp stem cells alter the expression of neural markers

Auditory neuropathy is the particular form of deafness in humans which cannot be treated by replacement therapy. Human dental pulp stem cells (hDPSCs) are derived from an ectomesenchymal neural crest cell population. Therefore, they possess a promising capacity for neuronal differentiation and repair. miR-124, a key regulator of neuronal development in the inner ear, is expressed at high levels in auditory and vestibular neurons. Here, we evaluated the possible effect of miR-124 in alteration of neural protein markers expression. Using quantitative reverse transcription-PCR (qRT-PCR) analyses and immunofluorescence staining, we studied the expression patterns of neural progenitor markers (Nestin, NOTCH1, and SOX2) and neural markers (β-tubulin III, GATA-3, and peripherin) upon transfection of hDPSCs with miR-124. The qRT-PCR results showed that Nestin was upregulated 6 h post-transfection. In contrast, Nestin expression exhibited a decreasing trend 24 h and 48 h post-transfection. Higher levels of β-tubulin III, 6 h and 16 h post transfection in RNA level as compared with control cells, were determined in transfected DPSCs. However, β-tubulin-III expression decreased 48 h post-transfection. The immunoflourescence results indicated that transfection of hDPSCs with miR-124, only affected Nestin among the studied neural progenitor and neural marker expression in protein level.

A pilot study on differential expression of microRNAs in the ventromedial prefrontal cortex and serum of sows in activity restricted crates or activity free pens.

ObjectivePhysical activity restriction in sows may lead to behavioral abnormalities and affective disorders. However, the psychophysiological state of these sows is still unclear. As miRNAs can be used as effective markers of psychopathy, the present study aimed to assess the difference in microRNA expression between the long-term activity restricted sows and activity free sows, thus contributing to the understanding of abnormal sow behavior.MethodsFour dry sows (sixth parity, Large×White genetic line) were selected from activity restricted crates (RC) or activity free pens (FP) separately. microRNAs in the ventromedial prefrontal cortex (vMPFC) and serum were examined using real-time polymerase chain reaction, and the correlation between the miRNAs expressed in the vMPFC and serum was evaluated.ResultsmiR-134 (1.11 vs 0.84) and miR-1202 (1.09 vs 0.85) levels were higher in the vMPFC of the RC sows than in the FP sows (p<0.01). Furthermore, miR-132 (1.27 vs 1.08) and miR-335 (1.03 vs 0.84) levels were also higher in the RC sows than in FP sows (p<0.05); however, miR-135a, miR-135b, miR-16, and miR-124 levels were not different (p>0.05). The relative expression of serum miR-1202 was higher in the RC sows than in the FP sows (1.04 vs 0.54) (p<0.05), and there was a strong correlation (R = 0.757, p<0.05) between vMPFC and Serum levels of miR-1202. However, no significant difference was observed in miR-16 levels in the serum of the RC sows and FP sows (p>0.05).ConclusionThis pilot study demonstrates that long-term activity restriction in sows likely results in autism or other complex psychopathies with depression-like behaviors. These observations may provide new insights for future studies on abnormal behavior in sows and contribute to research on human psychopathy.

MiR-218: a molecular switch and potential biomarker of susceptibility to stress.

Low miR-218 expression in the medial prefrontal cortex (mPFC) is a consistent trait of depression. Here we assessed whether miR-218 in the mPFC confers resilience or susceptibility to depression-like behaviors in adult mice, using the chronic social defeat stress (CSDS) model of depression. We also investigated whether stress-induced variations of miR-218 expression in the mPFC can be detected in blood. We find that downregulation of miR-218 in the mPFC increases susceptibility to a single session of social defeat, whereas overexpression of miR-218 selectively in mPFC pyramidal neurons promotes resilience to CSDS and prevents stress-induced morphological alterations to those neurons. After CSDS, susceptible mice have low levels of miR-218 in the blood as compared to control or resilient groups. We show further that up- and downregulation of miR-218 levels specifically in the mPFC correlates with miR-218 expression in blood. Our results suggest that miR-218 in the adult mPFC might function as a molecular switch that determines susceptibility versus resilience to chronic stress, and that stress-induced variations in mPFC levels of miR-218 could be detected in blood. We propose that blood expression of miR-218 might serve as potential readout of vulnerability to stress and as a proxy of mPFC function.

Peripheral blood microRNA levels in females with cocaine use disorder

BackgroundThere is growing emphasis in the field of psychiatry on the need to identify candidate biomarkers to aid in diagnosis and clinical management of addictive disorders. MicroRNAs (miRNAs) are small nucleotide sequences with the ability to regulate gene expression at the transcriptomic level. However, the role of miRNAs as potential biomarkers for addiction is still underexplored. Based on translational and clinical findings, we compared the expression levels of microRNA-124 (miR-124), microRNA-181 (miR-181), and microRNA-212 (miR-212) between a group of females with cocaine use disorder (CUD; n = 30) and a group of healthy female controls (HC; n = 20). MethodsBlood expression levels of miR-124, miR-181, and miR-212 in the HC and CUD group were determined by qPCR, using two miRNAs as endogenous controls (miR-24 and miR-126). Substance use behavior was assessed by self-report using the Addiction Severity Index (ASI-6) and depressive symptoms severity was measured using the Beck Depressive Inventory (BDI-II). Urine screen test was performed to detect cocaine metabolites. ResultsMir-124 and miR-181 were upregulated in the CUD group (p > 0.01). Furthermore, increased cognitive/affective depression symptoms were identified among a CUD subgroup with the higher miR-181 expression levels (p > 0.05). No significant difference in expression levels was found for miR-212. ConclusionsMiR-124 and miR-181 show promise as biomarkers for CUD when assessed in the peripheral blood. Further investigation is needed to elucidate the molecular mechanisms underlying these associations and to validate target genes regulated by these miRNAs.

Bioengineering of single ncRNA molecule for multi‐targeting against NSCLC

Lung cancer is the second most common cancer among both men and women in the United States. The majority of lung cancer cases are classified as non‐small cell lung cancer (NSCLC). Current NSCLC treatments include resection surgery, radiation, and pharmacotherapy which all provide some benefits and have limitations. As a result, lung cancer caused deaths account for about 27% of all cancer deaths in the US. Therefore, new therapeutic agents are highly demanded for NSCLC. MicroRNAs (miRNA or miR) are a class of genome‐derived, small noncoding RNAs (ncRNA) in cells that govern target gene expression through mRNA degradation or translational inhibition. Some miRNAs such as miR‐34a, miR‐124 and let‐7c are commonly downregulated in NSCLC tissues/cells and target various oncogenes such as CDK6, STAT3, and Ras. Restoring the expression or function of such dysregulated, tumor suppressive miRNAs represents a new strategy for cancer therapy. Aiming at simultaneously introducing multiple miRNAs in NSCLC cells for multi‐targeting, we employed our recently developed novel ncRNA bioengineering technology to produce a single ncRNA molecule bearing multiple miRNAs, namely combination‐bioengineered RNA agent (CO‐BERA). Our data showed that a variety of target CO‐BERA molecules were successfully expressed in E. coli. We were able to purify recombinant CO‐BERAs to a high degree of homogeneity using anion exchange FPLC methods. We further demonstrated that treatment of human NSCLC cells with CO‐BERAs elevated cellular miR‐34a, miR‐124 and let‐7c levels, reduced protein levels of target genes including CDK6, STAT3 and Ras, and consequently inhibited NSCLC cell proliferation. CO‐BERAs were successful in inhibiting NSCLC patient derived xenograft (PDX) tumor growth in mice. Therefore, using single ncRNA agent for multi‐targeting may be more effective for the treatment of NSCLC.Support or Funding InformationThis study was supported by grants R01GM133888 and U01CA175315 from NIH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Expression and correlation of miR-124 and miR-126 in breast cancer.

The expression of micro ribonucleic acid (miR)-124 and miR-126 in different stages of breast cancer (BC) and their correlation were investigated to analyze the role of miR-124 and miR-126 in the occurrence and development of BC. BC tissues of 83 BC patients treated in First Teaching Hospital of Tianjin University of Traditional Chinese Medicine from March 2016 to January 2018 were selected as the research group, while the corresponding para-carcinoma normal tissues were selected as the control group. The relative expression levels of miR-124 and miR-126 in both groups were detected via reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and the miR-124 and miR-126 expression asociated with clinicopathological parameters of patients and the correlation between miR-124 and miR-126 was analyzed. The relative expression levels of miR-124 and miR-126 in the research group were significantly lower than those in the control group (P<0.001). miR-124 and miR-126 in the research group were associated with clinicopathological parameters (clinical stage, degree of pathological differentiation and lymph node metastasis) of patients (P<0.001). According to Pearsons correlation analysis, there was a positive correlation between the relative expression levels of miR-124 and miR-126 in cancer tissues (r=0.497, P<0.001). The expression levels of miR-124 and miR-126 were downregulated in BC tissues, which were associated with clinicopathological parameters (clinical stage, degree of pathological differentiation and lymph node metastasis). There was a positive correlation between the expression levels of miR-124 and miR-126 in BC tissues. Thus, miR-124 and miR-126 may be involved in the occurrence, development, invasion and metastasis of BC, and both can be used as targeted biological indexes for treatment of BC.

Plasma Levels of miR-125b-5p and miR-206 in Acute Ischemic Stroke Patients After Recanalization Treatment: A Prospective Observational Study

Introduction: Multiple microRNAs (miRNAs) participate in the response to hypoxic/ischemic and ischemia-reperfusion events. However, the expression of these miRNAs in circulation from patients with acute ischemic stroke (AIS) receiving recanalization treatment has not been examined, and whether they are associated with the severity and outcome of stroke is still unknown. Materials and methods: In this prospective cohort study, plasma levels of miR-125b-5p, miR-15a-3p, miR-15a-5p, and miR-206 were measured at 24 hours after thrombolysis with or without endovascular treatment in 94 patients with AIS, as determined by qRT-PCR. Stroke severity was assessed based on National Institutes of Health Stroke Scale (NIHSS) score and infarct lesion. Intracranial haemorrhage (ICH) was recorded. An unfavorable outcome was defined as a modified Rankin Scale score greater than 2 at day 90 after stroke. Results: miR-125b-5p and miR-206 levels were correlated with NIHSS scores (P = .014 and P = .002) and cerebral infarction volumes (P = .025 and P = .030). miR-125b-5p levels were significantly higher in patients with an unfavorable outcome than in patients with a favorable outcome (P = .002) and showed good diagnostic accuracy in discriminating the presence of an unfavorable outcome (area under the curve .735, 95% confidence interval .623-.829, P < .001). No association was found between different miRNAs and ICH. Conclusions: In AIS patients after thrombolysis with or without endovascular treatment, miR-125b-5p is a novel prognostic biomarker highly associated with an unfavorable outcome. miR-125b-5p and miR-206 levels are associated with stroke severity.

Overexpression of long non-coding RNA NORAD promotes invasion and migration in malignant melanoma via regulating the MIR-205-EGLN2 pathway.

Growing evidence suggests that long non‐coding RNAs NORAD and miR‐205 play a significant role in regulating cancer progression and metastasis. In this study, high expression of NORAD was firstly observed in melanoma tissues and human malignant melanoma cell lines, our aim was to study the interaction of them in the process of invasion and migration of malignant melanoma cells. NORAD, miR‐205, and EGLN2 mRNA level in MM cells was detected by qRT‐PCR. In situ hybridization (ISH) was performed to detect NORAD expression in MM tissues specimens. Effects of NORAD and miR‐205 on Prolyl hydroxylase 2 (EGLN2) expression was explored by western blot in MM cells line. Dual‐luciferase reporter assay was performed to verify the interaction relationship between NORAD and miR‐205, as well as, miR‐205 and EGLN2. Transwell assay was conducted to explore the effects of NORAD and miR‐205 in vitro. Xenografts in nude mice experiment were used to confirm the role of NORAD and miR‐205 in vivo. In vitro, NORAD knockdown significantly inhibited migration and invasion of malignant melanoma cells and elevated the expression of miR‐205, there was an interaction between miR‐205 and NORAD in the RNA‐induced silencing complex. Upregulation of miR‐205 induced significant inhibition of migratory and invasive ability compared with the scrambled control. However, downregulating NORAD largely reversed this effect. Furthermore, the regulatory effects of miR‐205 on EGLN2 levels and the induction of endoplasmic reticulum stress were reversed by NORAD. In vivo, deletion of miR‐205 induced tumor growth in nude mice. NORAD may play critical roles in tumorigenesis and progression of malignant melanoma by regulating of the miR‐205‐EGLN2 pathway, and may serve as a new therapeutic target.

MiR-335 suppresses cell proliferation and migration by upregulating CRKL in bladder cancer.

microRNAs (miRNAs) abnormal expression was proved to regulate the bladder cancer (BC) development. Here, we aimed to investigate the role of miR-335 played in BC. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot were used to examine the miR-335 and CRKL (CT10 regulator of kinase-like protein) expression level in BC. Methyl thiazolyl tetrazolium (MTT) and RT-qPCR were used to examine cell viability of BC cells. Cell transwell assay was used to assess the migratory ability of BC cells. The direct target of miR-335 in BC was verified by luciferase reporter assay. The results showed that the expression of miR-335 and CRKL in normal and adjacent tissues showed no significant differences. Whereas, miR-335 expression in BC was significantly lower and CRKL expression was observably higher than normal. CRKL was verified as a specific target of miR-335 in BC cells and the relationship between CRKL and miR-335 expression was negatively correlated in BC tissues. Furthermore, CRKL siRNA group in BC cells remarkably inhibited cell proliferation and migration. MiR-335 mimic in BC cells remarkably curbed cell proliferation and migration and CRKL could reverse the proliferative and migratory ability of BC cells regulated by miR-335. miR-335 could suppress BC cell proliferation and migration by upregulating of CRKL.

Effects of microRNA-195 on the Prognosis of Glioma Patients and the Proliferation and Apoptosis of Human Glioma Cells.

Glioma is the most common and aggressive intracranial malignant tumor with poor prognosis. Acts as a tumor suppressor, microRNA-195 (miR-195) plays important roles in a variety of cancers. However, the expression of miR-195 and role of miR-195 in glioma are still not well understood. 186 patients with glioma were enrolled and the follow-up period ranges from 1 to 69months. MiR-195 was exogenously transfected into human glioma U87 cell line. The cell proliferation assay (CCK-8), colony formation assay, cell cycle analysis and cell apoptosis analysis were examined to investigate miR-195 effect on U87 cells. MiR-195 levels were reversely correlated with pathological grades (r = -0.487, p = 0.003). For patients with low miR-195 levels, their median survival time was 15months, whereas the median survival time in patients with high miR-195 levels was 56.53months. Multi-factor Cox regression analysis showed that high level of miR-195 (Odds ratio (OR): 0.347, 95% CI: 0.121-0.992) was associated with decreased mortality risk of patients. Moreover, overexpression of miR-195 inhibits proliferation and colony formation, and induces apoptosis of U87 cells. MiR-195 could block the glioma cells in G0/G1 phase, reducing S phase cells and regulating apoptosis related proteins (Caspase-3, Caspase-8, Caspase-9 and Bcl-2). Downregulation of miR-195 was associated with poor prognosis in human glioma. MiR-195 acted as tumor suppressor through inhibiting cell proliferation and promoting cell apoptosis via blockade of cell cycle and regulation of apoptosis related proteins.

Dynamic changes in miR-124 levels in patients with acute cerebral infarction

Objective: To investigate the changes in serum miR-124 levels in patients with acute cerebral infarction (ACI) and elucidate the underlying mechanism by a dynamic monitor.Methods: Fifty-four patients with ACI and 51 healthy controls were included in our study. Baseline characteristics and blood samples were collected for further analysis. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure the serum miR-124 levels. The dual-luciferase reporter assay was used to evaluate the effect of miR-124 on iASPP, a protein that inhibits apoptosis stimulating proteins in the p53 family.Results: Compared with normal controls, the miR-124 levels in the ACI group rapidly decreased at phase 1 (within 24 h after ischemia) (p < 0.001) and then gradually increased at phase 2 (48 ∼ 72 h after ischemia) (p < 0.001) and phase 3 (the 7th day after ischemia) (p < 0.001). The dual-luciferase reporter assay showed that miR-124 down-regulates iASPP expression in 293T cells.Conclusion: The miR-124 levels are down-regulated in ACI patients. The dynamic changes of miR-124 might provide a possible method for the detection of ischemic stroke.HighlightsThe difference in miR-124 expression levels between ACI patients and normal controls.Dynamic changes of miR-124 expression levels in ACI patients.The down-regulation of miR-124 upon iASPP expression.

Brain Regional and Temporal Changes in BDNF mRNA and microRNA-206 Expression in Mice Exposed to Repeated Cycles of Chronic Intermittent Ethanol and Forced Swim Stress

Brain-derived neurotrophic factor (BDNF) expression and signaling activity in brain are influenced by chronic ethanol and stress. We previously demonstrated reduced Bdnf mRNA levels in the medial prefrontal cortex (mPFC) following chronic ethanol treatment and forced swim stress (FSS) enhanced escalated drinking associated with chronic ethanol exposure. The present study examined the effects of chronic ethanol and FSS exposure, alone and in combination, on Bdnf mRNA expression in different brain regions, including mPFC, central amygdala (CeA), and hippocampus (HPC). Additionally, since microRNA-206 has been shown to negatively regulate BDNF expression, the effects of chronic ethanol and FSS on its expression in the target brain regions were examined. Mice received four weekly cycles of chronic intermittent ethanol (CIE) vapor or air exposure and then starting 72-h later, the mice received either a single or 5 daily 10-min FSS sessions (or left undisturbed). Brain tissue samples were collected 4-h following final FSS testing and Bdnf mRNA and miR-206 levels were determined by qPCR assay. Results indicated dynamic brain regional and time-dependent changes in Bdnf mRNA and miR-206 expression. In general, CIE and FSS exposure reduced Bdnf mRNA expression while miR-206 levels were increased in the mPFC, CeA, and HPC. Further, in many instances, these effects were more robust in mice that experienced both CIE and FSS treatments. These results have important implications for the potential link between BDNF signaling in the brain and ethanol consumption related to stress interactions with chronic ethanol experience.

Regulation of miRNAs by Snail during epithelial-to-mesenchymal transition in HT29 colon cancer cells

Epithelial-to-mesenchymal transition (EMT) in cancer cells, represents early stages of metastasis and is a promising target in colorectal cancer (CRC) therapy. There have been many attempts to identify markers and key pathways induced throughout EMT but the process is complex and depends on the cancer type and tumour microenvironment. Here we used the colon cancer cell line HT29, which stably overexpressed Snail, the key transcription factor in early EMT, as a model for colorectal adenocarcinoma cells with a pro-metastatic phenotype. We investigated miRNA expression regulation during that phenotypic switching. We found that overexpression of Snail in HT29 cells triggered significant changes in individual miRNA levels but did not change the global efficiency of miRNA processing. Snail abundance repressed the expression of miR-192 and miR-194 and increased miR-205, let-7i and SNORD13 levels. These identified changes correlated with the reported transcriptomic alterations in Snail-overexpressing HT29 cells. We also investigated how Snail affected the miRNA content of extracellular vesicles (EVs) released from HT29 cells. Our data suggest that the presence of Snail significantly alters the complex mRNA/miRNA interactions in the early steps of metastasis and also has an impact on the content of EVs released from HT29 cells.

Low let-7d and high miR-205 expression levels positively influence HNSCC patient outcome

IntroductionHead and neck squamous carcinoma (HNSCC) is one of the most invasive types of cancer with high mortality. A previous study has indicated that low levels of let-7d and miR-205 in HNSCC patients are correlated with poor survival. Let-7d and miR-205 are tumor suppressors and regulators of epithelial-to-mesenchymal transition (EMT). However, it is unclear if let-7d and miR-205 together influence cancer cells.AimTo determine if let-7d and miR-205 expression levels influence HNSCC patient outcome.MethodsThe TCGA expression data for let-7d, miR-205 and their targets as well as clinical data were downloaded from cBioPortal and starBase v2.0 for 307 patients. The expression levels of let-7d and miR-205 were verified according to clinicopathological parameters. The let-7d and miR-205 high- and low-expression groups as well as disease-free survival (DFS), overall survival (OS) and expression levels of genes related to EMT, cancer stem cells, metastasis, cell cycle, drug response and irradiation response were investigated.ResultsLet-7d and miR-205 were frequently upregulated in HNSCC compared to normal samples, and ROC analysis showed high discrimination ability for let-7d and miR-205 (area 0.7369 and 0.7739, respectively; p < 0.0001). Differences between expression levels of let-7d or miR-205 and grade, angiolymphatic invasion, perineural invasion and alcohol consumption were indicated. No differences were observed in N-stage, tumor localization, gender or patient age. Patients with lower let-7d levels and higher miR-205 levels had significantly better OS (p = 0.0325) than patients with higher let-7d levels and lower miR-205 levels. In the low let-7d level and high miR-205 level group, a lower percentage of more advanced cancers was observed. The analysis of genes related to EMT, cancer stem cells, metastasis, cell cycle, drug response and irradiation response revealed a distinct phenotype of analyzed groups.ConclusionsThe present findings indicated that let-7d down-regulation and miR-205 overexpression create a unique cell phenotype with different behavior compared to cells with upregulated let-7d and down-regulated miR-205. Thus, let-7d and miR-205 are good candidates for new HNSCC biomarkers.