Circulating miR-216a as a biomarker of metabolic alterations and obesity in women.
ABSTRACT: Obesity leads to an amplified risk of disease and contributes to the occurrence of type 2 diabetes, fatty liver disease, coronary heart disease, stroke, chronic kidney disease and various types of cancer. MicroRNAs (miRNAs), small non-coding RNA molecules of 20-25 nucleotides, can remain stable in plasma and have been studied as potential (predictive) biomarkers for obesity and related metabolic disorders. The aim of this study was to identify circulating miRNAs as biomarkers for obesity status and metabolic alterations in women. Circulating miR-216a and miR-155-5p were selected by miRNA expression profiling and validated by real time quantitative PCR in a validation cohort of 60 obese women and 60 normal weight-age-matched control women. This was supplemented by correlation analysis of the candidate miRNA and anthropometric variables, blood biochemistry and lipid profile markers. Circulating miR-216a was validated as a biomarker of obesity status with significantly reduced levels in obese women. Interestingly, this was associated with a negative correlation between the plasma miR-216a content and body mass index (BMI), waist circumference, mean arterial pressure (MAP), triglycerides, ratio of total cholesterol/high density lipoprotein (HDL)-cholesterol and high sensitivity-C reactive protein (hs-CRP).Taken together, we provide evidence for an abnormally expressed circulating miRNA, miR-216a, with additive value as a predictive marker for obesity that correlates with metabolic alterations presented by lipid profile and inflammatory markers.
Project description:Aberrant expression of miRNAs in pancreatic islets is closely related to the development of type 1 diabetes (T1D). The aim of this study was to identify key miRNAs dysregulated in pancreatic islets during T1D progression and to develop a theranostic approach to modify their expression using an MRI-based nanodrug consisting of iron oxide nanoparticles conjugated to miRNA-targeting oligonucleotides in a mouse model of T1D. Isolated pancreatic islets were derived from NOD mice of three distinct age groups (3, 8 and 18-week-old). Total RNA collected from cultured islets was purified and global miRNA profiling was performed with 3D-Gene global miRNA microarray mouse chips encompassing all mouse miRNAs available on the Sanger miRBase V16. Of the miRNAs that were found to be differentially expressed across three age groups, we identified one candidate (miR-216a) implicated in beta cell proliferation for subsequent validation by RT-PCR. Alterations in miR-216a expression within pancreatic beta cells were also examined using in situ hybridization on the frozen pancreatic sections. For in vitro studies, miR-216a mimics/inhibitors were conjugated to iron oxide nanoparticles and incubated with beta cell line, ?TC-6. Cell proliferation marker Ki67 was evaluated. Expression of the phosphatase and tensin homolog (PTEN), which is one of the direct targets of miR-216a, was analyzed using western blot. For in vivo study, the miR-216a mimics/inhibitors conjugated to the nanoparticles were injected into 12-week-old female diabetic Balb/c mice via pancreatic duct. The delivery of the nanodrug was monitored by in vivo MRI. Blood glucose of the treated mice was monitored post injection. Ex vivo histological analysis of the pancreatic sections included staining for insulin, PTEN and Ki67. miRNA microarray demonstrated that the expression of miR-216a in the islets from NOD mice significantly changed during T1D progression. In vitro studies showed that treatment with a miR-216a inhibitor nanodrug suppressed proliferation of beta cells and increased the expression of PTEN, a miR-216a target. In contrast, introduction of a mimic nanodrug decreased PTEN expression and increased beta cell proliferation. Animals treated in vivo with a mimic nanodrug had higher insulin-producing functionality compared to controls. These observations were in line with downregulation of PTEN and increase in beta cell proliferation in that group. Our studies demonstrated that miR-216a could serve as a potential therapeutic target for the treatment of diabetes. miR-216a-targeting theranostic nanodrugs served as exploratory tools to define functionality of this miRNA in conjunction with in vivo MR imaging.
Project description:Oncolytic virotherapy shows promise for pancreatic ductal adenocarcinoma (PDAC) treatment, but there is the need to minimize associated-toxicities. In the current work, we engineered artificial target sites recognized by miR-216a and/or miR-148a to provide pancreatic tumor-selectivity to replication-competent adenoviruses (Ad-miRTs) and improve their safety profile. Expression analysis in PDAC patients identified miR-148a and miR-216a downregulated in resectable (FC(miR-148a) = 0.044, P < 0.05; FC(miR-216a) = 0.017, P < 0.05), locally advanced (FC(miR-148a) = 0.038, P < 0.001; FC(miR-216a) = 0.001, P < 0.001) and metastatic tumors (FC(miR-148a) = 0.041, P < 0.01; FC(miR-216a) = 0.002, P < 0.001). In mouse tissues, miR-216a was highly specific of the exocrine pancreas whereas miR-148a was abundant in the exocrine pancreas, Langerhans islets, and the liver. In line with the miRNA content and the miRNA target site design, we show E1A gene expression and viral propagation efficiently controlled in Ad-miRT-infected cells. Consequently, Ad-miRT-infected mice presented reduced pancreatic and liver damage without perturbation of the endogenous miRNAs and their targets. Interestingly, the 8-miR148aT design showed repressing activity by all miR-148/152 family members with significant detargeting effects in the pancreas and liver. Ad-miRTs preserved their oncolytic activity and triggered strong antitumoral responses. This study provides preclinical evidences of miR-148a and miR-216a target site insertions to confer adenoviral selectivity and proposes 8-miR148aT as an optimal detargeting strategy for genetically-engineered therapies against PDAC.
Project description:Inflammation is the host self-protection mechanism to eliminate pathogen invasion. The excessive inflammatory response can result in uncontrolled inflammation, autoimmune diseases, or pathogen dissemination. Recent studies have widely shown that microRNAs (miRNAs) contribute to the regulation of inflammation in mammals by repressing gene expression at the posttranscriptional level. However, the miRNA-mediated mechanism in the inflammatory response in fish remains hazy. In the present study, the regulatory mechanism of the miR-216a-mediated inflammatory response in teleost fish was addressed. We found that the expression of miR-216a could be significantly upregulated in the miiuy croaker after challenge with Vibrio anguillarum and lipopolysaccharide. Bioinformatics predictions demonstrated a potential binding site of miR-216a in the 3' untranslated region of the p65 gene, and the result was further confirmed by luciferase assay. Moreover, both the mRNA and protein levels of p65 in macrophages were downregulated by miR-216a. Deletion mutant analysis of the miR-216a promoter showed that the Ap1 and Sp1 transcription factor binding sites are indispensable for the transcription of miR-216a. Further study revealed that overexpression of miR-216a suppresses inflammatory cytokine expression and negatively regulates NF-?B signaling, which inhibit an excessive inflammatory response. The collective results indicate that miR-216a plays a role as a negative regulator involved in modulating the bacterium-induced inflammatory response.
Project description:The KIAA0101 protein (also referred to as NS5ATP9 or Paf15) is overexpressed in esophageal squamous cell carcinoma (ESCC) and is associated with disease progression and poor patient survival, but how KIAA0101 expression is regulated remains unknown. The relationship between tumor miR?216a?5p expression and prognosis in patients with ESCC was revealed by survival analyses. Quantitative reverse?transcriptase PCR and western blot analysis were used to evaluate miR?216a?5p and KIAA0101 expression in human ESCC tissues and cell lines. The targeting of KIAA0101 by miR?216a?5p was verified by dual?luciferase reporter assays. The EC9706 and TE1 cell lines were transfected with miR?216a?5p mimics and inhibitor, or KIAA0101?specific shRNA and KIAA0101?expressing plasmids, in order to evaluate the effect of manipulating miR?216a?5p and KIAA0101 expression on ESCC cell proliferation, cell cycle progression, migration, and invasion. miR?216a?5p was lowly expressed and inversely correlated with KIAA0101 protein expression in ESCC tissues and cell lines. Lower miR?216a?5p expression was associated with worse prognosis in patients with ESCC. miR?216a?5p negatively regulated KIAA0101 expression by directly targeting the 3'?untranslated region of the KIAA0101 mRNA. Overexpression of miR?216a?5p suppressed the proliferation, migration, and invasion of the ESCC cell lines, whereas inhibition of miR?216a?5p had the opposite effects. Meanwhile, KIAA0101 promoted ESCC migration and invasion, and its overexpression abolished the antitumor effects of miR?216a?5p mimics. As a tumor suppressor, miR?216a?5p targets KIAA0101 to inhibit the proliferation, migration, and invasion of ESCC. Therefore, the miR?216a?5p/KIAA0101 axis may be a potential target for ESCC treatment.
Project description:The use of circulating microRNAs as biomarkers opens up new opportunities for the diagnosis of cardiovascular diseases because of their specific expression profiles. The aim of the present study was to identify circulating microRNAs in human plasma as potential biomarkers of heart failure and related diseases. We used real-time quantitative PCR to screen microRNA in plasma samples from 62 normal controls and 62 heart failure samples. We found that circulating miR-21-5p, miR-30a-3p, miR-30a-5p, miR-155-5p, miR-216a and miR-217 expressed differently between healthy controls and heart failure patients. Plasma levels of miR-21-5p, miR-30a-3p, miR-30a-5p, miR-155-5p, miR-216a and miR-217 were unaffected by hemolysis. Correlation analysis showed any two of these miRNAs possess a strong correlation, indicating a possibility of combined analysis. MiR-21-5p, miR-30a-3p, miR-30a-5p, miR-155-5p, miR-216a and miR-217 could be combined in two or three or more combinations. The results suggest that miR-21-5p, miR-30a-3p, miR-30a-5p, miR-155-5p, miR-216a and miR-217 may be a new diagnostic biomarker for heart failure and related diseases.
Project description:MicroRNAs (miRNAs), belonging to a class of evolutionarily conserved small noncoding RNA of ?22 nucleotides, are widely involved in skeletal muscle growth and development by regulating gene expression at the post-transcriptional level. While the expression feature and underlying function of miR-216a in mammal skeletal muscle development, especially in cattle, remains to be further elucidated. The aim of this study was to investigate the function and mechanism of miR-216a during bovine primary muscle cells proliferation and differentiation. Herein, we found that the expression level of miR-216a both presented a downward trend during the proliferation and differentiation phases, which suggested that it might have a potential role in the development of bovine skeletal muscle. Functionally, during the cells proliferation phase, overexpression of miR-216a inhibited the expression of proliferation-related genes, reduced the cell proliferation status, and resulted in cells G1 phase arrest. In cells differentiation stages, overexpression of miR-216a suppressed myogenic maker genes mRNA, protein, and myotube formation. Mechanistically, we found that SNIP1 and smad7 were the directly targets of miR-216a in regulating bovine primary muscle cells proliferation and differentiation, respectively. Altogether, these findings suggested that miR-216a functions as a suppressive miRNA in development of bovine primary muscle cells via targeting SNIP1 and smad7.
Project description:Vascular endothelial senescence contributes to atherosclerosis and coronary artery disease (CAD), but the mechanisms are yet to be clarified. We identified that microRNA-216a (miR-216a) significantly increased in senescent endothelial cells. The replicative senescence model of human umbilical vein endothelial cells (HUVECs) was established to explore the role of miR-216a in endothelial ageing and dysfunction. Luciferase assay indicated that Smad3 was a direct target of miR-216a. Stable expression of miR-216a induced a premature senescence-like phenotype in HUVECs with an impairment in proliferation and migration and led to an increased adhesion to monocytes by inhibiting Smad3 expression and thereafter modulating the degradation of NF-?B inhibitor alpha (I?B?) and activation of adhesion molecules. Conversely, inhibition of endogenous miR-216a in senescent HUVECs rescued Smad3 and I?B? expression and inhibited monocytes attachment. Plasma miR-216a was significantly higher in old CAD patients (>50 years) and associated with increased 31% risk for CAD (odds ratio 1.31, 95% confidence interval 1.03-1.66; P = .03) compared with the matched healthy controls (>50 years). Taken together, our data suggested that miR-216a promotes endothelial senescence and inflammation as an endogenous inhibitor of Smad3/I?B? pathway, which might serve as a novel target for ageing-related atherosclerotic diseases.
Project description:Cisplatin is the first-line treatment for ovarian cancer. However, the clinical outcome of cisplatin treatment in ovarian cancer is hindered by cancer resistance. Here we aim to explore the role and mechanism of miR-216a in the cisplatin resistance of ovarian cancer. The effects of miR-216a overexpression and inhibition on ovarian cell proliferation, colony formation, and cisplatin resistance were investigated by MTT assay and soft agar colony formation assay. Bioinformatics analyses using TargetScan and rVista, qPCR, and luciferase assay were also used to explore and verify downstream effectors and regulators of miR-216a Proliferation, colony formation, and cisplatin resistance of ovarian cancer cells are promoted by miR-216a overexpression but inhibited by miR-216a inhibition. PTEN is a direct target of miR-216a and PTEN expression antagonizes the tumor-promoting function of miR-216a STAT3 is a regulator of miR-216a, and PTEN is also regulated by STAT3. miR-216a up-regulation is associated with cisplatin resistance in ovarian cancer and this effect is mediated by PTEN. STAT3 is a regulator of miR-216a Strategies that inhibit miR-216a is a potential strategy for overcoming the cisplatin resistance in ovarian cancer.
Project description:Endothelial dysfunction and impaired autophagic activity have a crucial role in aging-related diseases such as cardiovascular dysfunction and atherosclerosis. We have identified miR-216a as a microRNA that is induced during endothelial aging and, according to the computational analysis, among its targets includes two autophagy-related genes, Beclin1 (BECN1) and ATG5. Therefore, we have evaluated the role of miR-216a as a molecular component involved in the loss of autophagic function during endothelial aging. The inverse correlation between miR-216a and autophagic genes was conserved during human umbilical vein endothelial cells (HUVECs) aging and in vivo models of human atherosclerosis and heart failure. Luciferase experiments indicated BECN1, but not ATG5 as a direct target of miR-216a. HUVECs were transfected in order to modulate miR-216a expression and stimulated with 100??g/ml oxidized low-density lipoprotein (ox-LDL) to induce a stress repairing autophagic process. We found that in young HUVECs, miR-216a overexpression repressed BECN1 and ATG5 expression and the ox-LDL induced autophagy, as evaluated by microtubule-associated protein 1 light chain 3 (LC3B) analysis and cytofluorimetric assay. Moreover, miR-216a stimulated ox-LDL accumulation and monocyte adhesion in HUVECs. Conversely, inhibition of miR-216a in old HUVECs rescued the ability to induce a protective autophagy in response to ox-LDL stimulus. In conclusion, mir-216a controls ox-LDL induced autophagy in HUVECs by regulating intracellular levels of BECN1 and may have a relevant role in the pathogenesis of cardiovascular disorders and atherosclerosis.
Project description:Prader-Willi syndrome (PWS) represents the most common genetic-derived obesity disorder caused by the loss of expression of genes located on the paternal chromosome 15q11.2-q13. The PWS phenotype shows peculiar physical, endocrine and metabolic characteristics compared to those observed in non-syndromic essential obesity. Since miRNAs have now a well-established role in many molecular pathways, including regulatory networks related to obesity, this pilot study was aimed to characterize the expression of circulating miRNAs in PWS compared to essential obesity. The circulating miRNome of 10 PWS and 10 obese subjects, adequately matched for age, BMI and sex, was profiled throughout Genechip miRNA 4.0 microarray analysis. We identified 362 out of 2578 mature miRNAs to be expressed in serum of the studied population. The circulating miRNA signature significantly characterising the two populations include 34 differently expressed RNAs. Among them, miR-24-3p, miR-122 and miR-23a-3p highly differ between the two groups with a FC >10 in obese compared to PWS. In the obese subjects, miR-7107-5p, miR-6880-3p, miR-6793-3p and miR-4258 were associated to the presence of steatosis. A different signature of miRNAs significantly distinguished PWS with steatosis from PWS without steatosis, involving miR-619-5p, miR-4507, miR-4656, miR-7847-3p and miR-6782-5p. The miRNA target GO enrichment analysis showed the different pathway involved in these two different forms of obesity. Although the rarity of PWS actually represents a limitation to the availability of large series, the present study provides novel hints on the molecular pathogenesis of syndromic and non-syndromic obesity.