The microRNA-29 family: role in metabolism and metabolic disease.

The microRNA-29 family members miR-29a-3p, miR-29b-3p, and miR-29c-3p are ubiquitously expressed and consistently increased in various tissues and cell types in conditions of metabolic disease, obesity, insulin resistance, and type 2 diabetes. In pancreatic ß cells, miR-29a is required for normal exocytosis, but increased levels are associated with impaired ß-cell function. Similarly, in liver, miR-29 species are higher in models of insulin resistance and type 2 diabetes, and either knock-out or depletion using a microRNA inhibitor improves hepatic insulin resistance. In skeletal muscle, miR-29 family upregulation is associated with insulin resistance and altered substrate oxidation, and similarly, in adipocytes, overexpression of miR-29a leads to insulin resistance. Blocking miR-29a using nucleic acid antisense therapeutics show promising results in preclinical animal models of obesity and type 2 diabetes, although the widespread expression pattern of miR-29 family members complicates the exploration of single target tissues. However, in fibrotic diseases, such as in late complications of diabetes and metabolic disease (diabetic kidney disease, nonalcoholic steatohepatitis), miR-29 species expression is suppressed by TGF-ß allowing increased extracellular matrix collagen to form. In the clinical setting, circulating levels of miR-29a and miR-29b are consistently increased in type 2 diabetes and in gestational diabetes and are also possible prognostic markers for deterioration of glucose tolerance. In conclusion, miR-29 family miRNAs play an essential role in various organs relevant to intermediary metabolism and its upregulation contributes to impaired glucose metabolism, whereas it suppresses fibrosis development. Thus, a correct balance of levels of miR-29 family miRNA seems important for cellular and organ homeostasis in metabolism.

Human leukocyte antigen-G and regulatory T cells during specific immunotherapy for pollen allergy.

BACKGROUND: TH2-biased immune responses are important in allergy pathogenesis. Mechanisms of allergen-specific immunotherapy (SIT) might include the induction of regulatory T cells (Tregs) and immunoglobulin (Ig) G4 blocking antibodies, a reduction in the number of effector cells, and skewing of the cytokine profile towards a TH1-polarized immune response. We investigated the effects of SIT on T cells, on immunomodulation of human leukocyte antigen (HLA)-G, which has been associated with allergy, on regulatory cytokine expression, and on serum allergen-specific antibody subclasses (IgE and IgG4). METHODS: Eleven birch and/or grass pollen-allergic patients and 10 healthy nonatopic controls were studied before and during SIT. Tregs, chemokine receptors, soluble HLA-G (sHLA-G), Ig-like transcript (ILT) 2, specific IgE, and IgG4 were studied. Peripheral blood mononuclear cells (PBMCs) were stimulated with pollen extract in vitro and immune factors were evaluated. RESULTS: During SIT, the main changes in the peripheral blood were an increase in CXCR3(+)CD4(+)CD25(+)CD127(low/-) Tregs and a decrease in CCR4(+)CD4(+)CD25(+)CD127(low/-) Tregs, an increase in allergen-specific IgG4, and a decrease in sHLA-G during the first half of the treatment period. In the PBMC in vitro experiments, the following changes were observed upon allergen-stimulation: an increase in CD4(+)CD25(+)CD127(low/-) Tregs and ILT2(+)CD4(+)CD25(+)CD127(low/-) Tregs, an increase in IL-10 and IL-2 levels, and an increase in sHLA-G that was most pronounced at the start of SIT. CONCLUSIONS: The changes in CXCR3(+)CD4(+)CD25(+)CD127(low/-) Treg, IgG4, and sHLA-G levels in the peripheral blood and in ILT2(+) Treg, IL-10, IL-2, and sHLA-G levels upon in vitro allergen stimulation suggest an upregulation in immunomodulatory factors and, to some degree, a shift towards TH1 during SIT.

Circulating miRNAs in Women with Polycystic Ovary Syndrome: A Longitudinal Cohort Study.

BACKGROUND: Women with polycystic ovary syndrome (PCOS) often change their metabolic profile over time to decrease levels of androgens while often gaining a propensity for the development of the metabolic syndrome. Recent discoveries indicate that microRNAs (miRNAs) play a role in the development of PCOS and constitute potential biomarkers for PCOS. We aimed to identify miRNAs associated with the development of an impaired metabolic profile in women with PCOS, in a follow-up study, compared with women without PCOS. METHODS AND MATERIALS: Clinical measurements of PCOS status and metabolic disease were obtained twice 6 years apart in a cohort of 46 women with PCOS and nine controls. All participants were evaluated for degree of metabolic disease (hypertension, dyslipidemia, central obesity, and impaired glucose tolerance). MiRNA levels were measured using Taqman® Array cards of 96 pre-selected miRNAs associated with PCOS and/or metabolic disease. RESULTS: Women with PCOS decreased their levels of androgens during follow-up. Twenty-six of the miRNAs were significantly changed in circulation in women with PCOS during the follow-up, and twenty-four of them had decreased, while levels did not change in the control group. Four miRNAs were significantly different at baseline between healthy controls and women with PCOS; miR-103-3p, miR-139-5p, miR-28-3p, and miR-376a-3p, which were decreased in PCOS. After follow-up, miR-28-3p, miR-139-5p, and miR-376a-3p increased in PCOS women to the levels observed in healthy controls. Of these, miR-139-5p correlated with total testosterone levels (rho = 0.50, padj = 0.013), while miR-376-3p correlated significantly with the waist-hip ratio at follow-up (rho = 0.43, padj = 0.01). Predicted targets of miR-103-3p, miR-139-5p, miR-28-3p, and miR-376a-3p were enriched in pathways associated with Insulin/IGF signaling, interleukin signaling, the GNRH receptor pathways, and other signaling pathways. MiRNAs altered during follow-up in PCOS patients were enriched in pathways related to immune regulation, gonadotropin-releasing hormone signaling, tyrosine kinase signaling, and WNT signaling. CONCLUSIONS: These studies indicate that miRNAs associated with PCOS and androgen metabolism overall decrease during a 6-year follow-up, reflecting the phenotypic change in PCOS individuals towards a less hyperandrogenic profile.

Machine learning workflows identify a microRNA signature of insulin transcription in human tissues.

Dicer knockout mouse models demonstrated a key role for microRNAs in pancreatic ß-cell function. Studies to identify specific microRNA(s) associated with human (pro-)endocrine gene expression are needed. We profiled microRNAs and key pancreatic genes in 353 human tissue samples. Machine learning workflows identified microRNAs associated with (pro-)insulin transcripts in a discovery set of islets (n = 30) and insulin-negative tissues (n = 62). This microRNA signature was validated in remaining 261 tissues that include nine islet samples from individuals with type 2 diabetes. Top eight microRNAs (miR-183-5p, -375-3p, 216b-5p, 183-3p, -7-5p, -217-5p, -7-2-3p, and -429-3p) were confirmed to be associated with and predictive of (pro-)insulin transcript levels. Use of doxycycline-inducible microRNA-overexpressing human pancreatic duct cell lines confirmed the regulatory roles of these microRNAs in (pro-)endocrine gene expression. Knockdown of these microRNAs in human islet cells reduced (pro-)insulin transcript abundance. Our data provide specific microRNAs to further study microRNA-mRNA interactions in regulating insulin transcription.

Hyperandrogenism and Metabolic Syndrome Are Associated With Changes in Serum-Derived microRNAs in Women With Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) remains one of the most common endocrine disorder in premenopausal women with an unfavorable metabolic risk profile. Here, we investigate whether biochemical hyperandrogenism, represented by elevated serum free testosterone, resulted in an aberrant circulating microRNA (miRNAs) expression profile and whether miRNAs can identify those PCOS women with metabolic syndrome (MetS). Accordingly, we measured serum levels of miRNAs as well as biochemical markers related to MetS in a case-control study of 42 PCOS patients and 20 Controls. Patients were diagnosed based on the Rotterdam consensus criteria and stratified based on serum free testosterone levels (≥0.034 nmol/l) into either a normoandrogenic (n = 23) or hyperandrogenic (n = 19) PCOS group. Overall, hyperandrogenic PCOS women were more insulin resistant compared to normoandrogenic PCOS women and had a higher prevalence of MetS. A total of 750 different miRNAs were analyzed using TaqMan Low-Density Arrays. Altered levels of seven miRNAs (miR-485-3p, -1290, -21-3p, -139-3p, -361-5p, -572, and -143-3p) were observed in PCOS patients when compared with healthy Controls. Stratification of PCOS women revealed that 20 miRNAs were differentially expressed between the three groups. Elevated serum free testosterone levels, adjusted for age and BMI, were significantly associated with five miRNAs (miR-1290, -20a-5p, -139-3p, -433-3p, and -361-5p). Using binary logistic regression and receiver operating characteristic curves (ROC), a combination panel of three miRNAs (miR-361-5p, -1225-3p, and -34-3p) could correctly identify all of the MetS cases within the PCOS group. This study is the first to report comprehensive miRNA profiling in different subgroups of PCOS women with respect to MetS and suggests that circulating miRNAs might be useful as diagnostic biomarkers of MetS for a different subset of PCOS.

The long noncoding RNA MALAT1 predicts human pancreatic islet isolation quality.

Human islet isolation is a cost-/resource-intensive program generating islets for cell therapy in Type 1 diabetes. However, only a third of cadaveric pancreas get to clinical transplantation due to low quality/number of islets. There is a need to identify biomarker(s) that predict the quality of islets, prior to initiating their isolation. Here, we sequenced transcriptome from 18 human islet preparations stratified into three groups (Gr.1: Best quality/transplantable islets, Gr.2: Intermediary quality, Gr.3: Inferior quality/non-transplantable islets) based on routine measurements including islet purity/viability. Machine-learning algorithms involving penalized regression analyses identified 10 long-non-coding(lnc)RNAs significantly different across all group-wise comparisons (Gr1VsGr2, Gr2vsGr3, Gr1vsGr3). Two variants of Metastasis-Associated Lung Adenocarcinoma Transcript-1(MALAT1) lncRNA were common across all comparisons. We confirmed RNA-seq findings in a "validation set" of 75 human islet preparations. Finally, in 19 pancreas samples, we demonstrate that assessing the levels of MALAT1 variants alone (ROC curve AUC: 0.83) offers highest specificity in predicting post-isolation islet quality and improves the predictive potential for clinical islet transplantation when combined with Edmonton Donor Points/Body Mass Index(BMI)/North American Islet Donor Score(NAIDS). We present this resource of islet-quality-stratified lncRNA transcriptome data and identify MALAT1 as a biomarker that significantly enhances current selection methods for clinical (GMP)-grade islet isolation.

Non-Coding RNA in Pancreas and ß-Cell Development.

In this review, we provide an overview of the current knowledge on the role of different classes of non-coding RNAs for islet and ß-cell development, maturation and function. MicroRNAs (miRNAs), a prominent class of small RNAs, have been investigated for more than two decades and patterns of the roles of different miRNAs in pancreatic fetal development, islet and ß-cell maturation and function are now emerging. Specific miRNAs are dynamically regulated throughout the period of pancreas development, during islet and ß-cell differentiation as well as in the perinatal period, where a burst of ß-cell replication takes place. The role of long non-coding RNAs (lncRNA) in islet and ß-cells is less investigated than for miRNAs, but knowledge is increasing rapidly. The advent of ultra-deep RNA sequencing has enabled the identification of highly islet- or ß-cell-selective lncRNA transcripts expressed at low levels. Their roles in islet cells are currently only characterized for a few of these lncRNAs, and these are often associated with ß-cell super-enhancers and regulate neighboring gene activity. Moreover, ncRNAs present in imprinted regions are involved in pancreas development and ß-cell function. Altogether, these observations support significant and important actions of ncRNAs in ß-cell development and function.

MicroRNAs related to androgen metabolism and polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a frequent endocrine disorder in women. PCOS is associated with altered features of androgen metabolism, increased insulin resistance and impaired fertility. Furthermore, PCOS, being a syndrome diagnosis, is heterogeneous and characterized by polycystic ovaries, chronic anovulation and evidence of hyperandrogenism, as well as being associated with chronic low-grade inflammation and an increased life time risk of type 2 diabetes. A number of androgen species contribute to the symptoms of increased androgen exposure seen in many, though not all, cases of PCOS: Testosterone, androstenedione, dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS), where the quantitatively highest amount of androgen is found as DHEAS. The sulfation of DHEA to DHEAS depends on a number of enzymes, and altered sulfate metabolism may be associated with and contribute to the pathogenesis of PCOS. MicroRNAs (miRNAs) are small, non-coding RNAs that are able to regulate gene expression at the post-transcriptional level. Altered miRNA levels have been associated with diabetes, insulin resistance, inflammation and various cancers. Studies have shown that circulating miRNAs are present in whole blood, serum, plasma and the follicular fluid of PCOS patients and that these might serve as potential biomarkers and a new approach for the diagnosis of PCOS. In this review, recent work on miRNAs with respect to PCOS will be summarized. Our understanding of miRNAs, particularly in relation to PCOS, is currently at a very early stage, and additional studies will yield important insight into the molecular mechanisms behind this complex and heterogenic syndrome.

MicroRNA Species in Follicular Fluid Associating With Polycystic Ovary Syndrome and Related Intermediary Phenotypes.

CONTEXT: Polycystic ovary syndrome (PCOS) has a largely unknown etiology and presents with a clinical heterogeneous patient group. Small noncoding microRNA (miRNA) might prove promising as biomarker candidates for PCOS patient stratification. Altered miRNA expression profiles have been observed in few studies. OBJECTIVE: The aim was to assess the miRNA expression profile in follicular fluid from PCOS patients and healthy, regularly cycling, matched controls. DESIGN AND SETTING: Experimental case-control study including 49 PCOS women (19 of which were hyperandrogenic and 30 normo-androgenic) and 21 healthy matched women all undergoing in vitro fertilization treatment. INTERVENTIONS AND MAIN OUTCOME: Anthropometric and relevant clinical baseline measurements were obtained. Relative expression of miRNA levels were estimated using miRNA quantitative PCR arrays and validated by quantitative RT-PCR. Correlation between miRNAs and clinical relevant measurements was estimated. RESULTS: PCOS women, both normo-androgenic and hyperandrogenic, had decreased levels of miR-24-3p, -29a, -151-3p, and -574-3p compared with controls. Furthermore, miR-518f-3p was differentially expressed within the PCOS group with high levels observed in the hyperandrogenic group compared with the normo-androgenic PCOS patients. Serum levels of total and free T were positively correlated with miR-518f-3p in PCOS subjects (P = .001). Distinction between PCOS and controls could be made using miR-151-3p alone with an area under the curve of 0.91 or a combination of four selected miRNAs (area under the curve, 0.93). Bioinformatic target analysis points to an involvement of these miRNAs in biological pathways involving regulation of cell proliferation, extracellular matrix, and processes in intermediary metabolism. CONCLUSION: Our study provides evidence that the miRNA expression profile in follicular fluid is altered in PCOS and indicates that specific follicular fluid miRNAs are associated with phenotypical traits of PCOS. An altered miRNA profile holds potentials for new methods of PCOS patient stratification and may contribute to and in part explain the heterogeneous nature found within PCOS women.