MicroRNAs in obesity-associated disorders.

The emergence of a worldwide obesity epidemic has dramatically increased the prevalence of insulin resistance and metabolic syndrome, predisposing individuals to a greater risk for the development of non-alcoholic fatty liver disease, type II diabetes and atherosclerotic cardiovascular diseases. Current available pharmacological interventions combined with diet and exercise-based managements are still poorly effective for weight management, likely in part due to an incomplete understanding of regulatory mechanisms and pathways contributing to the systemic metabolic abnormalities under disturbed energy homeostasis. MicroRNAs, small non-coding RNAs that regulate posttranscriptional gene expression, have been increasingly described to influence shifts in metabolic pathways under various obesity-related disease settings. Here we review recent discoveries of the mechanistic role that microRNAs play in regulating metabolic functions in liver and adipose tissues involved in obesity associated disorders, and briefly discusses the potential candidates that are being pursued as viable therapeutic targets.

Casein kinase 1δ-dependent Wee1 protein degradation.

Eukaryotic mitotic entry is controlled by Cdk1, which is activated by the Cdc25 phosphatase and inhibited by Wee1 tyrosine kinase, a target of the ubiquitin proteasome pathway. Here we use a reporter of Wee1 degradation, K328M-Wee1-luciferase, to screen a kinase-directed chemical library. Hit profiling identified CK1δ-dependent Wee1 degradation. Small-molecule CK1δ inhibitors specifically disrupted Wee1 destruction and arrested HeLa cell proliferation. Pharmacological inhibition, siRNA knockdown, or conditional deletion of CK1δ also reduced Wee1 turnover. Thus, these studies define a previously unappreciated role for CK1δ in controlling the cell cycle.

Altered Circulating microRNAs in Patients with Diabetic Neuropathy and Corneal Nerve Loss: A Pilot Study.

An alteration in circulating miRNAs may have important diagnostic and therapeutic relevance in diabetic neuropathy. Patients with type 2 diabetes mellitus (T2DM) underwent an assessment of neuropathic symptoms using Douleur Neuropathique 4 (DN4), the vibration perception threshold (VPT) using a Neurothesiometer, sudomotor function using the Sudoscan, corneal nerve morphology using corneal confocal microscopy (CCM) and circulating miRNAs using high-throughput miRNA expression profiling. Patients with T2DM, with (n = 9) and without (n = 7) significant corneal nerve loss were comparable in age, gender, diabetes duration, BMI, HbA1c, eGFR, blood pressure, and lipid profile. The VPT was significantly higher (p < 0.05), and electrochemical skin conductance (p < 0.05), corneal nerve fiber density (p = 0.001), corneal nerve branch density (p = 0.013), and corneal nerve fiber length (p < 0.001) were significantly lower in T2DM patients with corneal nerve loss compared to those without corneal nerve loss. Following a q-PCR-based analysis of total plasma microRNAs, we found that miR-92b-3p (p = 0.008) was significantly downregulated, while miR-22-3p (p = 0.0001) was significantly upregulated in T2DM patients with corneal nerve loss. A network analysis revealed that these miRNAs regulate axonal guidance and neuroinflammation genes. These data support the need for more extensive studies to better understand the role of dysregulated miRNAs' in diabetic neuropathy.

Association of microRNAs With Embryo Development and Fertilization in Women Undergoing Subfertility Treatments: A Pilot Study.

Objective: Small non-coding RNAs, known as microRNAs (miRNAs), have emerging regulatory functions within the ovary that have been related to fertility. This study was undertaken to determine if circulating miRNAs reflect the changes associated with the parameters of embryo development and fertilization. Methods: In this cross-sectional pilot study. Plasma miRNAs were collected from 48 sequentially presenting women in the follicular phase prior to commencing in vitro fertilization (IVF). Circulating miRNAs were measured using locked nucleic acid (LNA)-based quantitative PCR (qPCR), while an updated miRNA data set was used to determine their level of expression. Results: Body mass index and weight were associated with the miRNAs let7b-3p and miR-375, respectively (p < 0.05), with the same relationship being found between endometrium thickness at oocyte retrieval and miR-885-5p and miR-34a-5p (p < 0.05). In contrast, miR-1260a was found to be inversely associated with anti-Mullerian hormone (AMH; p = 0.007), while miR-365a-3p, miR122-5p, and miR-34a-5p correlated with embryo fertilization rates (p < 0.05). However, when omitting cases of male infertility (n = 15), miR122-5p remained significant (p < 0.05), while miR-365a-3p and miR-34a-5p no longer differed; interestingly, however, miR1260a and mir93.3p became significant (p = 0.0087/0.02, respectively). Furthermore, age was negatively associated with miR-335-3p, miR-28-5p, miR-155-5p, miR-501-3p, and miR-497-5p (p < 0.05). Live birth rate was negatively associated with miR-335-3p, miR-100-5p, miR-497-5p, let-7d, and miR-574-3p (p < 0.05), but these were not significant when age was accounted for.However, with the exclusion of male factor infertility, all those miRNAs were no longer significant, though miR.150.5p emerged as significant (p = 0.042). A beta-regression model identified miR-1260a, miR-486-5p, and miR-132-3p (p < 0.03, p = 0.0003, p < 0.00001, respectively) as the most predictive for fertilization rate. Notably, changes in detectable miRNAs were not linked to cleavage rate, top quality embryos (G3D3), and blastocyst or antral follicle count. An ingenuity pathway analysis showed that miRNAs associated with age were also associated with the variables found in reproductive system diseases. Conclusion: Plasma miRNAs prior to the IVF cycle were associated with differing demographic and IVF parameters, including age, and may be predictive biomarkers of fertilization rate.

Increased MicroRNA Levels in Women With Polycystic Ovarian Syndrome but Without Insulin Resistance: A Pilot Prospective Study.

Background: Small noncoding microRNA (miRNA) have regulatory functions in polycystic ovary syndrome (PCOS) that differ to those in women without PCOS. However, little is known about miRNA expression in women with PCOS who are not insulin resistant (IR). Methods: Circulating miRNAs were measured using quantitative polymerase chain reaction (qPCR) in 24 non-obese BMI and age matched women with PCOS and 24 control women. A miRNA data set was used to determine miRNA levels. Results: Women with PCOS showed a higher free androgen index (FAI) and anti-mullerian hormone (AMH) but IR did not differ. Four miRNAs (miR-1260a, miR-18b-5p, miR-424-5p, and miR let-7b-3p) differed between control and PCOS women that passed the false discovery rate (FDR) out of a total of 177 circulating miRNAs that were detected. MiRNA let-7b-3p correlated with AMH in PCOS (p < 0.05). When the groups were combined, miR-1260a correlated with FAI and let-7b-3p correlated with body mass index (BMI) (p < 0.05). There was no correlation to androgen levels. Ingenuity pathway analysis showed that nine of the top 10 miRNAs reported were associated with inflammatory pathways. Conclusion: When IR did not differ between PCOS and control women, only four miRNA differed significantly suggesting that IR may be a driver for many of the miRNA changes reported. Let-7b-3p was related to AMH in PCOS, and to BMI as a group, whilst miR-1260a correlated with FAI. Androgen levels, however, had no effect upon circulating miRNA profiles. The expressed miRNAs were associated with the inflammatory pathway involving TNF and IL6.

Corrigendum: microRNA Expression in Women With and Without Polycystic Ovarian Syndrome Matched for Body Mass Index.

[This corrects the article DOI: 10.3389/fendo.2020.00206.].

microRNA Expression in Women With and Without Polycystic Ovarian Syndrome Matched for Body Mass Index.

Background: Despite several authors who have hypothesized that alterations of small noncoding RNAs (miR) are implicated in the etiopathogenesis of polycystic ovarian syndrome (PCOS), contrasting findings have been reported so far. Discrepancies in body mass index (BMI) levels may account for these differences; therefore, the aim of the present study was to determine whether miR differed in serum samples collected from age- and BMI-matched control and PCOS women. Methods: In a cross-sectional study, miR were measured using quantitative polymerase chain reaction in 29 women with anovulatory PCOS women and 29 control women who were in the follicular phase of their menstrual cycle, from the local biobank. Results: One hundred seventy-six miR were detected, of which 15 miR passed the false discovery rate (FDR; p < 0.05) that differed between PCOS and control women. There was no association of the top 9 miR (p < 0.02) (miR-486-5p, miR-24-3p, miR-19b-3p, miR-22-3p, miR-19a-3p, miR-339-5p, miR-185-5p, miR-101-3p, miR-let-7i-5p) with BMI, androgen levels, insulin resistance, or antimullerian hormone (AMH) in either PCOS or normal women. Ingenuity pathway assessment showed the pathways were interrelated for abnormalities of the reproductive system. Conclusion: When the confounding influence of weight was accounted for, miR levels differed between anovulatory PCOS women and control women in the follicular phase of the menstrual cycle. Interestingly, the differing miR were associated with the pathways of reproductive abnormalities but did not associate with AMH or metabolic parameters.

miRNA-dependent regulation of STIM1 expression in breast cancer.

Store-operated Ca2+ entry (SOCE) has been shown to be important for breast cancer metastasis in xenograft mouse models. The ER Ca2+ sensor STIM1 and Orai plasma membrane Ca2+ channels molecularly mediate SOCE. Here we investigate the role of the microRNA machinery in regulating STIM1 expression. We show that STIM1 expression is regulated post-transcriptionally by the miRNA machinery and identify miR-223 and miR-150 as regulators of STIM1 expression in the luminal non-aggressive MCF7 breast cancer cell line. In contrast, STIM1 expression in the more aggressive basal triple-negative MDA-MB-231 cell line is not significantly modulated by a single miRNA species but is rather upregulated due to inhibition of the miRNA machinery through downregulation of Ago2. Consistently, overexpression of Ago2 results in decreased STIM1 protein levels in MDA-MB-231 cells. Clinically, STIM1 and Ago2 expression levels do not correlate with breast cancer progression, however in the basal subtype high STIM1 expression is associated with poorer survival. Our findings show that STIM1 expression is differentially regulated by the miRNA machinery in different cell types and argue for a role for this regulation in breast cancer.

Expression of microRNA in follicular fluid in women with and without PCOS.

Several studies have shown the expression of small non-coding microRNA (miRNA) changes in PCOS and their expression in follicular fluid has been described, though the number of studies remains small. In this prospective cohort study, miRNA were measured using quantitative polymerase chain reaction (qPCR) in 29 weight and aged matched anovulatory women with PCOS and 30 women without from follicular fluid taken at the time of oocyte retrieval who were undergoing in vitro fertilization (IVF); miRNA levels were determined from a miRNA data set. 176 miRNA were detected, of which 29 differed significantly between normal women and PCOS women. Of these, the top 7 (p < 0.015) were miR-381-3p, miR-199b-5p, miR-93-3p, miR-361-3p, miR-127-3p, miR-382-5p, miR-425-3p. In PCOS, miR-382-5p correlated with age and free androgen index (FAI), miR-199b-5p correlated with anti-mullerian hormone (AMH) and miR-93-3p correlated with C-reactive protein (CRP). In normal controls, miR-127-3p, miR-382-5p and miR-425-3p correlated with the fertilisation rate; miR-127-3p correlated with insulin resistance and miR-381-3p correlated with FAI. Ingenuity pathway assessment revealed that 12 of the significantly altered miRNA related to reproductive pathways, 12 miRNA related to the inflammatory disease pathway and 6 were implicated in benign pelvic disease. MiRNAs differed in the follicular fluid between PCOS and normal control women, correlating with age, FAI, inflammation and AMH in PCOS, and with BMI, fertilization rate (3 miRNA), insulin resistance, FAI and inflammation in control women, according to Ingenuity Pathway Analysis.

Association of childhood traumatization and neuropsychiatric outcomes with altered plasma micro RNA-levels.

Childhood traumatization (CT) is associated with the development of several neuropsychiatric disorders in later life. Experimental data in animals and observational data in humans revealed evidence for biological alterations in response to CT that may contribute to its long-term consequences. This includes epigenetic changes in miRNA levels that contribute to complex alterations of gene expression. We investigated the association between CT and 121 miRNAs in a target sample of N = 150 subjects from the general population and patients from the Department of Psychiatry. We hypothesized that CT exhibits a long-term effect on miRNA plasma levels. We supported our findings using bioinformatics tools and databases. Among the 121 miRNAs 22 were nominally significantly associated with CT and four of them (let-7g-5p, miR-103a-3p, miR-107, and miR-142-3p) also after correction for multiple testing; most of them were previously associated with Alzheimer's disease (AD) or depression. Pathway analyses of target genes identified significant pathways involved in neurodevelopment, inflammation and intracellular transduction signaling. In an independent general population sample (N = 587) three of the four miRNAs were replicated. Extended analyses in the general population sample only (N = 687) showed associations of the four miRNAs with gender, memory, and brain volumes. We gained increasing evidence for a link between CT, depression and AD through miRNA alterations. We hypothesize that depression and AD not only share environmental factors like CT but also biological factors like altered miRNA levels. This miRNA pattern could serve as mediating factor on the biological path from CT to adult neuropsychiatric disorders.

Changes in Blood microRNA Expression and Early Metabolic Responsiveness 21 Days Following Bariatric Surgery.

Background: Early metabolic responses following bariatric surgery appear greater than expected given the initial weight loss and coincide with improvement in diabetes. We hypothesized that small non-coding microRNA changes might contribute to regulating mechanisms for metabolic changes and weight loss in patients with severe obesity and diabetes. Methods: Twenty-nine type 2 patients with severe obesity (mean BMI 46.2 kg/m2) and diabetes underwent Roux-en-Y gastric bypass (RYGB) surgery. Clinical measurements and fasting blood samples were taken preoperatively and at day 21 postoperatively. Normalization of fasting glucose and HbA1c following bariatric surgery (short-term diabetes remission) was defined as withdrawal of anti-diabetic medication and fasting glucose < 100 mg/dL (5.6 mmol/L) or HbA1c < 6.0%. MicroRNA expression was determined by quantitative polymerase chain reaction and tested for significant changes after surgery. Results: BMI decreased by 3.8 kg/m2 21 days postoperatively. Eighteen of 29 RYGB (62%) had short-term diabetes remission. Changes from pre- to post-surgery in 32 of 175 microRNAs were nominally significant (p < 0.05). Following multiple comparison adjustment, changes in seven microRNAs remained significant: miR-7-5p, let-7f-5p, miR-15b-5p, let-7i-5p, miR-320c, miR-205-5p, and miR-335-5p. Four pathways were over-represented by these seven microRNAs, including diabetes and insulin resistance pathways. Conclusion: Seven microRNAs showed significant changes 21 days after bariatric surgery. Functional pathways of the altered microRNAs were associated with diabetes-, pituitary-, and liver-related disease, with expression in natural killer cells, and pivotal intestinal pathology suggesting possible mechanistic roles in early diabetes responses following bariatric surgery.

Differentially expressed circulating microRNAs in the development of acute diabetic Charcot foot.

AIM: Charcot foot (CF) is a rare complication of Type 2 diabetes (T2D). MATERIALS & METHODS: We assessed circulating miRNAs in 17 patients with T2D and acute CF (G1), 17 patients with T2D (G2) and equivalent neuropathy and 17 patients with T2D without neuropathy (G3) using the high-throughput miRNA expression profiling. RESULTS: 51 significantly deregulated miRNAs were identified in G1 versus G2, 37 in G1 versus G3 and 64 in G2 versus G3. Furthermore, we demonstrated that 16 miRNAs differentially expressed between G1 versus G2 could be involved in osteoclastic differentiation. Among them, eight are key factors involved in CF pathophysiology. CONCLUSION: Our data reveal that CF patients exhibit an altered expression profile of circulating miRNAs.

Casein kinase 1δ is an APC/C(Cdh1) substrate that regulates cerebellar granule cell neurogenesis.

Although casein kinase 1δ (CK1δ) is at the center of multiple signaling pathways, its role in the expansion of CNS progenitor cells is unknown. Using mouse cerebellar granule cell progenitors (GCPs) as a model for brain neurogenesis, we demonstrate that the loss of CK1δ or treatment of GCPs with a highly selective small molecule inhibits GCP expansion. In contrast, CK1δ overexpression increases GCP proliferation. Thus, CK1δ appears to regulate GCP neurogenesis. CK1δ is targeted for proteolysis via the anaphase-promoting complex/cyclosome (APC/C(Cdh1)) ubiquitin ligase, and conditional deletion of the APC/C(Cdh1) activator Cdh1 in cerebellar GCPs results in higher levels of CK1δ. APC/C(Cdh1) also downregulates CK1δ during cell-cycle exit. Therefore, we conclude that APC/C(Cdh1) controls CK1δ levels to balance proliferation and cell-cycle exit in the developing CNS. Similar studies in medulloblastoma cells showed that CK1δ holds promise as a therapeutic target.

The APC/C Ubiquitin Ligase: From Cell Biology to Tumorigenesis.

The ubiquitin proteasome system (UPS) is required for normal cell proliferation, vertebrate development, and cancer cell transformation. The UPS consists of multiple proteins that work in concert to target a protein for degradation via the 26S proteasome. Chains of an 8.5-kDa protein called ubiquitin are attached to substrates, thus allowing recognition by the 26S proteasome. Enzymes called ubiquitin ligases or E3s mediate specific attachment to substrates. Although there are over 600 different ubiquitin ligases, the Skp1-Cullin-F-box (SCF) complexes and the anaphase promoting complex/cyclosome (APC/C) are the most studied. SCF involvement in cancer has been known for some time while APC/C's cancer role has recently emerged. In this review we will discuss the importance of APC/C to normal cell proliferation and development, underscoring its possible contribution to transformation. We will also examine the hypothesis that modulating a specific interaction of the APC/C may be therapeutically attractive in specific cancer subtypes. Finally, given that the APC/C pathway is relatively new as a cancer target, therapeutic interventions affecting APC/C activity may be beneficial in cancers that are resistant to classical chemotherapy.

Cyclin A degradation employs preferentially used lysines and a cyclin box function other than Cdk1 binding.

Cyclin A is targeted for mitotic destruction by the anaphase promoting complex/cyclosome (APC/C) and degradation proceeds even when proteolysis of other APC/C substrates are blocked by the spindle assembly checkpoint. Instead of a simple destruction box, a complex N-terminal destruction signal has been implicated in Cyclin A. We show here that Drosophila Cyclin A destruction employs both N- and C-terminal residues, which emphasize that a synergistic action by different parts of the protein facilitates recognition and degradation. The first KEN box, first D-box and an aspartic acid at position 70 are required at the N-terminus and they make additive contributions when the spindle checkpoint is active. From the C-terminal region, the cyclin box contributes. Single point mutations in these four elements abolish mitotic destruction. Additionally, eight lysines in the neighborhood of the N-terminal signals, which could serve as potential ubiquitin acceptor sites, are preferentially used for proteolysis. Mutations in these lysines and the N-terminal signals cause mitotic stability. However, mutating the lysines alone, only delays mitotic progression. Thus, presumably, lysines elsewhere in the protein are used when the preferred ones are absent and this requires the N-terminal signals. Furthermore, our results suggest that some function of the cyclin box other than Cdk1 binding promotes spindle checkpoint-independent recognition of Cyclin A by the APC/C.