Rhinoceros Serum microRNAs: Identification, Characterization, and Evaluation of Potential Iron Overload Biomarkers

Iron overload disorder (IOD) in critically endangered Sumatran (Dicerorhinus sumatrensis) and black (Diceros bicornis) rhinoceros is an over-accumulation of iron in organs which may exacerbate other diseases and indicate metabolic disturbances. IOD in rhinos is not well understood and diagnostics and therapeutics are limited in effectiveness. MicroRNAs (miRNAs) are small non-coding RNAs capable of altering protein synthesis. miRNA expression responds to physiological states and could serve as the basis for development of diagnostics and therapeutics. This study aimed to identify miRNAs differentially expressed among healthy rhinos and those afflicted with IOD or other diseases (“unhealthy”), and assess expression of select miRNAs to evaluate their potential as biomarkers of IOD. miRNAs in serum of black (n = 11 samples; five individuals) and Sumatran (n = 7 samples; four individuals) rhinos, representing individuals categorized as healthy (n = 9), unhealthy (n = 5), and afflicted by IOD (n = 3) were sequenced. In total, 715 miRNAs were identified, of which 160 were novel, 131 were specific to black rhinos, and 108 were specific to Sumatran rhinos. Additionally, 95 miRNAs were specific to healthy individuals, 31 specific to unhealthy, and 63 were specific to IOD individuals. Among healthy, unhealthy, and IOD states, 21 miRNAs were differentially expressed (P ≤ 0.01). Five known miRNAs (let-7g, miR-16b, miR-30e, miR-143, and miR-146a) were selected for further assessment via RT-qPCR in serum from black (n = 61 samples; seven individuals) and Sumatran (n = 38 samples; five individuals) rhinos. let-7g, miR-30e, and miR-143 all showed significant increased expression (P ≤ 0.05) during IOD (between 1 and 2 years prior to death) and late IOD (within 1 year of death) compared to healthy and unhealthy individuals. miR-16b expression increased (P ≤ 0.05) in late IOD, but was not different among IOD, healthy, and unhealthy states (P > 0.05). Expression of miR-146a increased in IOD and late IOD as compared to unhealthy samples (P ≤ 0.05) but was not different from the healthy state (P > 0.05). Selected serum miRNAs of black and Sumatran rhinos, in particular let-7g, miR-30e, and miR-143, could therefore provide a tool for advancing rhino IOD diagnostics that should be further investigated.

Serum MicroRNA Expression Patterns in Subjects After the 5-km Exercise Are Strongly Associated With Cardiovascular Adaptation

Circulating microRNAs (miRNAs) have been reported dysregulated during exercise. However, the changes of specific serum miRNAs during the 5-km run test with intensity of 51–52% maximum oxygen uptake (V̇O2max) and their association with traditional cardiovascular-related indicators remain well-characterized. Levels of miR-1, miR-21, miR-146a, miR-155, miR-181, and miR-210 were detected in 120 young subjects before and after the exercise training by quantitative reverse-transcription PCR (RT-qPCR). Besides, the levels of cardiac troponin I (cTNI), myoglobin (Myo), creatine kinase (CK), creatine kinase-MB (CK-MB), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), ischemia-modified albumin (IMA), interleukin-6 (IL-6), and C-reactive protein (CRP) were measured and the correlation between levels of serum miRNAs and biochemical parameters was also analyzed. Compared with resting state, the serum levels of miR-1, miR-146a, miR-155, miR-181, and miR-210 were significantly increased after exercise training. Serum levels of miR-146a, miR-155, and miR-210 after exercise training were positively correlated with Myo, CK-MB, and LDH, respectively, while miR-1, miR-146a, miR-181, and miR-155 were positively correlated with the levels of IL-6. Additionally, all the five miRNAs were negatively correlated with IMA levels. The multivariate logistic regression analysis showed that high levels of miR-146a, AST, LDH, and IL-6 in serum were risk factors, while low IMA contents were a protective factor for cardiovascular adaptation during exercise. In conclusion, the dynamic changes of miRNAs under the condition of the 5-km continuous running contribute to the adaptive regulation of the cardiovascular function of the body.

Serum miRNA-based signature indicates radiation exposure and dose in humans: a multicenter diagnostic biomarker study

Rationale: Mouse and non-human primate models showed that serum miRNAs may be used to predict the biological impact of radiation doses. We hypothesized that these results can be translated to humans treated with total body irradiation (TBI), and that miRNAs may be used as clinically feasible biodosimeters. Methods: To test this hypothesis, serial serum samples were obtained from 25 patients who underwent allogeneic stem-cell transplantation and profiled for miRNA expression using next-generation sequencing. Circulating exosomes were extracted, their miRNA content sequenced and cross-referenced with the total miRNA fraction. Finally, miRNAs with diagnostic potential were quantified with qPCR and an artificial neural network model was created and validated on an independent group of 12 patients with samples drawn under the same protocol. Results: Differential expression results were largely consistent with previous studies and allowed us to build an 8-miRNA-based model that showed AUC of 0.97 (95%CI 0.89-1.00) and validate it using qPCR in an independent validation set where it showed accuracy >91% for detecting exposure and 87.5% for differentiating between lethal and non-lethal doses. MiRNAs used in the model were miR-150-5p, miR-126-5p, miR-375, miR-215-5p, miR-144-5p, miR-122-5p, miR- 320d and miR-10b-5p. Additionally, miRNAs with detectable expression in this and two prior animal sets almost perfectly separated the irradiated from non-irradiated samples in mice, macaques and humans, validating the miRNAs as radiation-responsive through evolutionarily conserved transcriptional regulation mechanisms. Conclusions: We conclude that serum miRNAs reflect radiation exposure and dose for humans undergoing TBI and may be used as functional biodosimeters for precise identification of people exposed to clinically significant radiation doses. Funding: This work was supported by Foundation for Polish Science grant First TEAM/2016- 2/11 (WF), National Science Centre grant 2019/33/B/NZ5/00536 (WF) and National Science Center grant 2018/29/N/NZ5/02422 (KS). BT gratefully acknowledges financial support provided by the Polish National Agency for Academic Exchange (the Walczak Programme).

Identification of Four Serum miRNAs as Potential Markers to Screen for Thirteen Cancer Types

Introduction: Cancer has consistently remained one of the top causes of death in the United States every year, with many cancer deaths preventable if detected early. Circulating serum miRNAs pose as a promising, minimally invasive supplement, or even alternative, to many current screening procedures. Many studies have shown that different serum miRNAs can discriminate healthy individuals from those with certain types of cancer. Although many of those miRNAs are often reported to be significant in one cancer type, they are also altered in other types of cancers. Currently, very few studies have investigated serum miRNA biomarkers for multiple different cancer types simultaneously for general cancer screening.Method: To identify serum miRNAs that would be useful in simultaneously screening multiple types of cancers, microarray cancer datasets were curated, yielding 13 different types of cancer with a total of 3352 cancer samples and 2809 non-cancer samples. The samples were then divided into discovery and validation sets. A hundred random forest models were built using the discovery set to select candidate miRNAs. The selected miRNAs were then used in the validation set to see how well they can differentiate cancer from normal samples in an independent dataset. Further analysis of the interactions between these miRNAs and their target mRNAs were investigated as well.Result: The random forest models achieved an average of 97% accuracy in the discovery set with 95% bootstrap confidence interval from 0.9544 to 0.9778. The selected miRNAs were hsa-miR-663a, hsa-miR-6802-5p, hsa-miR-6784-5p, hsa-miR-3184-5p, and hsa-miR-8073. Each individual miRNA exhibited high area under the curve (AUC) value using receiver operating characteristic analysis. Moreover, the combination of four out of the five miRNAs achieved the highest AUC value of 0.9815 with perfect sensitivity, indicating that these miRNAs have a high potential for cancer screening. miRNA-mRNA interaction and protein-protein interaction analysis provided insights into how these miRNAs may play a role in cancer in general.

A novel combination of serum microRNAs for the detection of early gastric cancer

Background The aim of this study was to identify serum miRNAs that discriminate early gastric cancer (EGC) samples from non-cancer controls using a large cohort. Methods This retrospective case–control study included 1417 serum samples from patients with EGC (seen at the National Cancer Center Hospital in Tokyo between 2008 and 2012) and 1417 age- and gender-matched non-cancer controls. The samples were randomly assigned to discovery and validation sets and the miRNA expression profiles of whole serum samples were comprehensively evaluated using a highly sensitive DNA chip (3D-Gene®) designed to detect 2565 miRNA sequences. Diagnostic models were constructed using the levels of several miRNAs in the discovery set, and the diagnostic performance of the model was evaluated in the validation set. Results The discovery set consisted of 708 samples from EGC patients and 709 samples from non-cancer controls, and the validation set consisted of 709 samples from EGC patients and 708 samples from non-cancer controls. The diagnostic EGC index was constructed using four miRNAs (miR-4257, miR-6785-5p, miR-187-5p, and miR-5739). In the discovery set, a receiver operating characteristic curve analysis of the EGC index revealed that the area under the curve (AUC) was 0.996 with a sensitivity of 0.983 and a specificity of 0.977. In the validation set, the AUC for the EGC index was 0.998 with a sensitivity of 0.996 and a specificity of 0.953. Conclusions A novel combination of four serum miRNAs could be a useful non-invasive diagnostic biomarker to detect EGC with high accuracy. A multicenter prospective study is ongoing to confirm the present observations.

Serum microRNA Levels in Diabetes Mellitus

The aim of our study is to evaluate the serum circulating levels of some miRNA, such as hsa-let-7b-5p, hsa-let-7a-5p, hsa-miR-320b, hsa-miR-23a-3p, hsa-miR-27a-3p, hsa-miR-15a-5p, and hsa-miR-495-3, in diabetic patients without diabetic retinopathy (DR), diabetic patients with DR, and, healthy subjects in order to find reliable and reproducible biomarkers for DR. A total of 45 subjects underwent serum sampling for miRNAs evaluation and a complete ophthalmologic examination, including microperimetry and widefield swept source optical coherence tomography angiography (OCTA). Total circulating RNA was isolated from patients using the miRNeasy Serum/Plasma Kit. Serum miRNA expression levels were significantly different in the three groups. In detail, circulating hsa-miR-15a-5p levels were significantly reduced in both diabetic patients without DR and diabetic patients with DR (p = 0.027). Serum hsa-miR-495-3p was lower in diabetic patients with DR and diabetic patients without DR (p = 0.049). Hsa-miR-23a-3p serum expression levels were significantly lower in diabetic patients with DR and diabetic patients without DR (p = 0.013). Significant associations of miRNAs with anatomical/perfusion parameters and functional parameters were observed in the diabetic groups. We find evidence of damage in progression biomarkers in DR that are evidently early in patients with diabetes without DR. Serum miRNAs levels are considered to have strong potential as a novel biomarker for the early detection of DR in subjects suffering from diabetes and could represent noninvasive target therapies to block the progression of the disease at the early stages.

Significant Changes in Serum MicroRNAs after High Tibial Osteotomy in Medial Compartmental Knee Osteoarthritis: Potential Prognostic Biomarkers

High tibial osteotomy (HTO) is an effective alternative for medial compartmental knee osteoarthritis (OA). Circulating microRNAs (miRNAs) are known to serve as OA-related biomarkers. The present study investigated the differential expression of serum miRNAs before and after HTO to identify potential miRNAs as prognostic biomarkers. miRNA-polymerase chain reaction (PCR) arrays were used to screen for miRNAs in the serum at preoperative and 6-month postoperative time points from six patients, and the differentially expressed miRNAs identified in the profiling stage were validated using real-time PCR at post-operative months 6 and 18 in 27 other HTO-treated patients. Among 84 miRNAs involved in the inflammatory process, three (miR-19b-3p, miR-29c-3p, and miR-424-5p) showed differential expression patterns in the profiling stage (p = 0.011, 0.015, and 0.021, respectively). Levels of these three and four other miRNAs (miR-140-3p, miR-454-3p, miR-let-7e-5p, and miR-885-5p) known to be related to OA progression were evaluated in the serum of 27 patients. Only four miRNAs (miR-19b-3p, miR-140-3p, miR-454-3p, and miR-let-7e-5p) were significantly upregulated at postoperative month 6 (p = 0.003, 0.005, 0.004, and 0.004, respectively), and only miR-140-3p was significantly upregulated up to 18 months after operation (p = 0.003). Together, this study reveals the significantly upregulated serum miRNAs after HTO as potential prognostic biomarkers; however, further studies are warranted to elucidate their clinical implications.

Circadian Misalignment Leads to Changes in Cortisol Rhythms, Blood Biochemical Variables and Serum miRNA Profiles

Background: Circadian clock plays a critical role in synchronizing the inner molecular, metabolic and physiological processes to the environmental cues which cycle with a period of 24 h. Misalignment in the circadian rhythms leads to decrease in adaptation, performance and increased risk of associated disorders. The non-24-h schedules and shift schedules are commonly used in maritime operations, both of which could result in disturbance in circadian rhythms. The comprehensive effects of these schedules remain to be further elucidated.Methods: In this study, we firstly conducted an experiment in which the volunteers followed a 3-d rotary schedule with consecutive shift in sleep time (rotatory schedule), and analyzed the changes in salivary cortisol rhythms and blood variables. Next we conducted another experiment in which the volunteers followed an 8 h on and 4 h off schedule (non-24-h schedule) to compare the changes in blood/serum variables. Furthermore, the plasma miRNAs from the volunteers following the rotatory schedule were subject for miRNA sequencing. Results: The rotatory schedule led to elevated levels of serum cortisol during the early stage, and the phase became delayed during the early and stages. The results show both of the schedules caused comprehensive changes in blood/serum biochemical variable, and consistently, the increase in phosphate levels. In addition, a subset of serum miRNAs targeting genes involved in circadian rhythms, sleep homeostasis and multiple important physiological processes or pathways were identified in the experiment with rotatory schedule.Conclusions: This study revealed an increased stress during the rotatory schedule. Circadian misalignment caused by either non-24-h or rotatory schedule lead to extensive changes in blood/serum variables. As to the rotatory schedule, altered expression of serum miRNAs may account for the consequences of circadian misalignment. These findings would help understand the deleterious effects of shift schedules and optimize to enhance performance and welfare of the personnel working with similar schedules.

Circulating MicroRNA 29a: An Evolving Biomarker for Diagnosis of Pulmonary Tuberculosis

Introduction: Tuberculosis (TB) is global health problem threatening millions every year. There is urgent need of effective biomarkers for its diagnostics and circulating microRNAs (miRNAs) have recently emerged as novel and non-invasive molecular markers in blood. Aim: This study was done to evaluate serum miRNAs as a potential biomarker for the diagnosis of pulmonary mycobacterium tuberculosis infection. Materials and Methods: In this prospective cross-sectional study, acute pulmonary TB patients were recruited based on positive Mycobacterium tuberculosis Polymerase Chain Reaction (PCR) in sputum samples and Microarray expression profiling was performed on blood of these patients to study differentially expressed miRNAs. The results were validated by Real-Time Polymerase Chain Reaction (RT-PCR) on pulmonary TB cases and healthy controls. Student’s t-tests and analysis variance tests were used for statistical analysis. The p-value <0.05 was considered statistically significant. Results: Results of microarray expression profiling showed 75 differentially expressed miRNAs in cases of pulmonary TB; among these 5 upregulated and 2 downregulated miRNAs were evaluated by RT-PCR. miRNA 29a exhibited good distinguishing efficiency; followed by miRNA 384. ROC plot of expression data for miRNA in cases and control groups showed that AUC of miRNA-29a was 0.8268 (sensitivity=80%, specificity=82%) . Conclusion: This study suggests that altered levels of serum miRNAs have great potential to serve as non-invasive biomarkers for detection of Mycobacterium tuberculosisinfection. miRNA-29a can be used as an effective biomarker for diagnosis of pulmonary TB.