Serum miRNAs Research Articles

Impact of differing methodologies for serum miRNA-371a-3p assessment in stage I testicular germ cell cancer recurrence.

Current evidence shows that serum miR-371a-3p can identify disease recurrence in testicular germ cell tumour (TGCT) patients and correlates with tumour load. Despite convincing evidence showing the advantages of including miR-371a-3p testing to complement and overcome the classical serum tumour markers limitations, the successful introduction of a serum miRNA based test into clinical practice has been impeded by a lack of consensus regarding optimal methodologies and lack of a universal protocol and thresholds. Herein, we investigate two quantitative real-time PCR (qRT-PCR) based pipelines in detecting disease recurrence in stage I TGCT patients under active surveillance, and compare the sensitivity and specificity for each method. Sequential serum samples collected from 33 stage I TGCT patients undergoing active surveillance were analysed for miR-371a-3p via qRT-PCR with and without an amplification step included. Using a pre-amplified protocol, all known recurrences were detected via elevated miR-371a-3p expression, while without pre-amplification, we failed to detect recurrence in 3/10 known recurrence patients. For pre-amplified analysis, sensitivity and specificity was 90% and 94.4% respectively. Without amplification, sensitivity dropped to 60%, but exhibited 100% specificity. We conclude that incorporating pre-amplification increases sensitivity of miR-371a-3p detection, but produces more false positive results. The ideal protocol for quantification of miR-371a-3p still needs to be determined. TGCT patients undergoing active surveillance may benefit from serum miR-371a-3p quantification with earlier detection of recurrences compared to current standard methods. However, larger cross-institutional studies where samples are processed and data is analysed in a standardised manner are required prior to its routine clinical implementation.

OGC P05 Liquid biopsy in oesophageal cancer: a systematic review of blood biomarkers for early diagnosis

Abstract Background Oesophageal cancer is the 6th leading cause of cancer death worldwide, accounting for 1 in 18 cancer-related mortality in 2020. High mortality rates with oesophagogastric cancer is largely related to late diagnosis. In England and Wales, 35% of oesophagogastric cancers are diagnosed at stage 3 and 42% at stage 4. Early detection of oesophageal cancer improves survival. Currently, the only reliable and effective modality for early diagnosis is oesophagogastroduodenoscopy (OGD). However, OGD is an invasive procedure that carries risks, such as bleeding and visceral perforation. Thus, there is no national screening programme for oesophagogastric cancer, although patients with diagnosed Barrett's oesophagus undergo endoscopic surveillance. Cytological sampling devices such as Cytosponge and EsophaCap are less invasive but still require ingestion of a medical device, and are not without risks, including detachment of device, pharyngeal bleed, and failure to swallow. The discovery and validation of blood biomarkers for the screening and diagnosis of oesophageal cancer may provide advantages of non-invasive, rapid, and cost-effective testing. Yet, currently, there is no clinically validated blood screening biomarker. In this systematic review, we aim to identify developing blood biomarkers that have been described in the literature for early diagnosis of oesophageal cancer. Methods A systematic search was conducted on EMBASE (1974-), Medline (1947-), and Web of Science (1980-) from inception to 3rd November 2021. Exploded subject headings and combinations of keywords were used. Search terms used were [oesophageal cancer or oesophageal ca or oesophageal malignancy or oesophageal neoplasm or Barrett's oesophagus or Barrett's metaplasia] AND [Tumour biomarkers or tumour markers or biological marker] AND [diagnosis or early diagnosis or screening or detection or discrimination] AND [adenocarcinoma or squamous cell carcinoma] AND [blood or serum or plasma] AND [esophagus or oesophagus]. Papers describing blood biomarkers that hold potential in screening and diagnosing oesophageal cancer were included. We focussed on adenocarcinoma, and squamous cell carcinoma affecting the oesophagus and the oesophagogastric junction. Tumour markers with a role in more than one type of cancer were included. We excluded rarer histological types. Biomarkers utilised to prognosticate outcomes of therapy were excluded, as were markers of recurrence and “Pan-cancer” biomarkers. Titles and abstracts identified by electronic search were reviewed independently by two reviewers, using Rayyan. Conflict was resolved by a third independent reviewer. Results The electronic search yielded 2619 records. After removal of duplicates 2267 records remained for screening. Following screening of titles and abstracts, 130 studies remained for full-text review. After full-text review, 53 records fulfilled the criteria for detailed analysis and reporting. 86 additional papers were identified through manual searches and review of reference lists. Studies were published from diverse populations including: USA, China, Japan, Germany, Poland, India, Australia, Ireland, South Africa, Italy, Brazil, Turkey and Netherlands. Blood biomarkers investigated in the literature comprised classical tumour markers, such as CEA, SCC-AG, CA19–9, CA-125; inflammatory markers; metabolites; peptides and glycoproteins; enzymes; cytokines; volatile organic compounds; circulating tumour cells; miRNA; circulating DNA; long non-coding RNAs; circular RNAs; peripheral blood cell DNA/RNA; antibodies and tumour-associated antigens; and spectra from Raman spectroscopy. The most widely described markers were antibodies and tumour-associated antigens (61 papers) and miRNAs (25 papers). Several other panels with combinations of different types of markers are described. The highest area under receiver operating curve (AUC) was 0.99 from two papers. One paper described a panel of serum miRNA ratios (RNU6-1/miR-16-5p, miR-25-3p/miR-320a, let-7e-5p/miR-15b-5p, miR-30a-5p/miR-324-5p, miR-17-5p/miR-194-5p). The other described a serum N-glycan with mass:charge ratio of 2244. Conclusions Several blood biomarkers with a potential role in screening or diagnosis of oesophageal cancer have been described in the literature. The results of some tests suggest promising diagnostic accuracy with two papers having AUCs of 0.99. Despite this however, no single test has come into clinical practice as a non-invasive diagnostic test. The gold standard in diagnosing oesophageal squamous cell carcinoma and oesophageal adenocarcinoma remains to be endoscopy and biopsy. Reasons for these markers not being utilised in clinical practice to date include need for further studies to validate results with larger sample size; challenges in reproducibility of studies; cost and lack of availability of techniques used in hospital; and other challenges to translate scientific studies into clinical application. There seem to be promising candidates liquid biopsy markers in oesophageal cancer, but further work is required to validate and translate the use of some promising markers into clinical practice.

Expression level and function analysis of serum miRNAs in workers with occupational exposure to benzene series

Benzene series are ubiquitous in industrial production and daily life, and can have an impact on health even at low concentrations. miRNAs have been proved to be a biomarker of a variety of diseases and carcinogens. The purpose of this study was to explore the distribution characteristics and biological function of miRNAs in subjects exposed to benzene series. In this study, serum miRNAs were measured in 247 occupationally exposed subjects and 256 controls. The relationship between cumulative exposure dose of benzene series and miRNAs was analyzed by Generalized linear model, Spearman's rank correlation, and chi-square test for trend. The function of MiRNAs target gene was analyzed by means of bioinformatics method. The results showed that the expressions of miR-181a-5p, 221-3p, 223-3p, and 342-3p were down-regulated, whilst the expression of miR-638 was up-regulated in the occupational exposure group. miR-181a-5p, 221-3p, 223-3p, 342-3p, and 638 showed dose-response relationship with benzene series, and were closely related to multiple tumor pathways. miR-181a-5p, 221-3p, 223-3p, 342-3p, and 638 may be involved in the carcinogenic process of benzene series, and can be used to evaluate the early biological effects and monitor the exposure level of benzene series. miRNAs are potential biomarkers of benzene series exposure.

Circulating microRNAs as predictors of response to sofosbuvir + daclatasvir + ribavirin in in HCV genotype-4 Egyptian patients

BackgroundMicroRNAs (miRNAs) play an important role in various diseases, including HCV infection, the aim of the current study was to evaluate the potential use of serum miRNAs as biomarkers for diagnosis, prognosis, and prediction of responses to direct acting antivirals (sofosbuvir + daclatasvir + ribavirin) in HCV-4 patients.MethodsThe serum expression profiles of four liver-associated miRNAs (miRNA‐122, 155, 196 and 29) were assessed in 160 HCV-4 patients and 50 healthy controls using real-time PCR prior to therapy.ResultsmiR-122 and miR-155 showed upregulation in HCV-4 patients compared to healthy controls while miR-196 and miR-29 showed downregulation in HCV-4 patients. ROC curve analyses revealed that the four-studied miRNAs could be valuable biomarkers for predicting response to DAAs with AUC 0.973 for miR-122, 0.878 for miR-155, 0.808 for miR-29 and 0.874 for miR-196 respectively. Univariate logistic regression analysis revealed that miR-196 level is positive predictor for SVR, whereas miR-122,155 levels are negative predictors of response. Multivariate logistic regression analysis revealed that miR-196 is the most significant in predicting response to treatment (p value = 0.011).ConclusionTo the best of our knowledge, the current study provided the first clinical evidence of the potential use of circulating miRNAs (miR; 122, 155, 196 and 29) as biomarkers of CHC in HCV-4 patients receiving the new DAA regimen (SOF/DAV + RIB), which is a strong motivator for further studies.

Analysis of clinical characteristics of centrally mediated abdominal pain syndrome, exploration of diagnostic markers and its relationship with the efficacy of duloxetine treatment

Centrally mediated abdominal pain syndrome (CAPS) is characterized by severe abdominal pain. Diagnosis of CAPS is still an exclusionary diagnosis, there remain no effective diagnostic biomarkers so far. Duloxetine is the major pharmacotherapy of CAPS, while some CAPS patients do not respond to duloxetine treatment. However, there is a lack of molecular markers to predict the efficacy of duloxetine. In our pilot study, we have found differential expression profiles of serum miRNAs between CAPS patients and healthy controls. Our study aims to explore the clinical characteristics, specific miRNAs in serum as diagnostic biomarkers of CAPS and predictive biomarkers of the efficacy of duloxetine. In this prospective cohort study, we plan to enroll 430 participants including 215 CAPS patients and 215 healthy controls. The CAPS group takes duloxetine 30 mg per day as an initial dose. Patients will have 24-week medication period and follow up at week 0, 4, 12, 24 and 36. Blood samples will be obtained from patients at every visits and health controls at the initial visit and a series of questionnaires will be completed by the participants. The primary end points are: The differential expression of miRNAs between CAPS groups and healthy control groups at baseline. The changes in abdominal pain scores before and after duloxetine treatment in patients with CAPS and their relationship with the changes in miRNAs. The secondary end point is the changes in scores of depression, anxiety, sleep quality and quality of life before and after duloxetine treatment in patients with CAPS and their relationship with changes in miRNAs. Findings of study will provide the reliable basis for diagnosis and the predictor of duloxetine efficacy of CAPS. Importantly, findings grant patients a chance to benefit from treatment.

Association of circulating miRNAS in patients with Alstrőm and Bardet-Biedl syndromes with clinical course parameters.

Patients with the rare syndromic forms of monogenic diabetes: Alström syndrome (ALMS) and Bardet-Biedl syndrome (BBS) have multiple metabolic abnormalities, including early-onset obesity, insulin resistance, lipid disorders and type 2 diabetes mellitus. The aim of this study was to determine if the expression of circulating miRNAs in patients with ALMS and BBS differs from that in healthy and obese individuals and determine if miRNA levels correlate with metabolic tests, BMI-SDS and patient age. We quantified miRNA expression (Qiagen, Germany) in four groups of patients: with ALMS (n=13), with BBS (n=7), patients with obesity (n=19) and controls (n=23). Clinical parameters including lipids profile, serum creatinine, cystatin C, fasting glucose, insulin and C-peptide levels, HbA1c values and insulin resistance (HOMA-IR) were assessed in patients with ALMS and BBS. We observed multiple up- or downregulated miRNAs in both ALMS and BBS patients compared to obese patients and controls, but only 1 miRNA (miR-301a-3p) differed significantly and in the same direction in ALMS and BBS relative to the other groups. Similarly, 1 miRNA (miR-92b-3p) was dysregulated in the opposite directions in ALMS and BBS patients, but diverged from 2 other groups. We found eight miRNAs (miR-30a-5p, miR-92b-3p, miR-99a-5p, miR-122-5p, miR-192-5p, miR-193a-5p, miR-199a-3p and miR-205-5p) that significantly correlated with at least of the analyzed clinical variables representing an association with the course of the diseases. Our results show for the first time that serum miRNAs can be used as available indicators of disease course in patients with ALMS and BBS syndromes.

Prediction of tissue-of-origin of early stage cancers using serum miRNomes.

Noninvasive detection of early stage cancers with accurate prediction of tumor tissue-of-origin could improve patient prognosis. Because miRNA profiles differ between organs, circulating miRNomics represent a promising method for early detection of cancers, but this has not been shown conclusively. A serum miRNA profile (miRNomes)-based classifier was evaluated for its ability to discriminate cancer types using advanced machine learning. The training set comprised 7931 serum samples from patients with 13 types of solid cancers and 5013 noncancer samples. The validation set consisted of 1990 cancer and 1256 noncancer samples. The contribution of each miRNA to the cancer-type classification was evaluated, and those with a high contribution were identified. Cancer type was predicted with an accuracy of 0.88 (95% confidence interval [CI] = 0.87 to 0.90) in all stages and an accuracy of 0.90 (95% CI = 0.88 to 0.91) in resectable stages (stages 0-II). The F1 score for the discrimination of the 13 cancer types was 0.93. Optimal classification performance was achieved with at least 100 miRNAs that contributed the strongest to accurate prediction of cancer type. Assessment of tissue expression patterns of these miRNAs suggested that miRNAs secreted from the tumor environment could be used to establish cancer type-specific serum miRNomes. This study demonstrates that large-scale serum miRNomics in combination with machine learning could lead to the development of a blood-based cancer classification system. Further investigations of the regulating mechanisms of the miRNAs that contributed strongly to accurate prediction of cancer type could pave the way for the clinical use of circulating miRNA diagnostics.

Identification of potential microRNA diagnostic panels and uncovering regulatory mechanisms in breast cancer pathogenesis

Early diagnosis of breast cancer (BC), as the most common cancer among women, increases the survival rate and effectiveness of treatment. MicroRNAs (miRNAs) control various cell behaviors, and their dysregulation is widely involved in pathophysiological processes such as BC development and progress. In this study, we aimed to identify potential miRNA biomarkers for early diagnosis of BC. We also proposed a consensus-based strategy to analyze the miRNA expression data to gain a deeper insight into the regulatory roles of miRNAs in BC initiation. Two microarray datasets (GSE106817 and GSE113486) were analyzed to explore the differentially expressed miRNAs (DEMs) in serum of BC patients and healthy controls. Utilizing multiple bioinformatics tools, six serum-based miRNA biomarkers (miR-92a-3p, miR-23b-3p, miR-191-5p, miR-141-3p, miR-590-5p and miR-190a-5p) were identified for BC diagnosis. We applied our consensus and integration approach to construct a comprehensive BC-specific miRNA-TF co-regulatory network. Using different combination of these miRNA biomarkers, two novel diagnostic models, consisting of miR-92a-3p, miR-23b-3p, miR-191-5p (model 1) and miR-92a-3p, miR-23b-3p, miR-141-3p, and miR-590-5p (model 2), were obtained from bioinformatics analysis. Validation analysis was carried out for the considered models on two microarray datasets (GSE73002 and GSE41922). The model based on similar network topology features, comprising miR-92a-3p, miR-23b-3p and miR-191-5p was the most promising model in the diagnosis of BC patients from healthy controls with 0.89 sensitivity, 0.96 specificity and area under the curve (AUC) of 0.98. These findings elucidate the regulatory mechanisms underlying BC and represent novel biomarkers for early BC diagnosis.

MiRNAs for testicular germ cell tumours: Contemporary indications for diagnosis, surveillance and follow-up.

Despite excellent outcomes with modern multidisciplinary care, clinicians caring for patients with testicular germ cell tumour (TGCTs) face clinical dilemmas across the spectrum of disease. Wrong treatment choices can lead to undertreatment or overtreatment of these young men. Unfortunately the currently available biomarkers alpha-fetoprotein and human chorionic gonadotropin, lack sufficient sensitivity and specificity to reliably aid in these clinical dilemmas. Thus, a sensitive and specific biomarker is desperately needed. Serum or plasma miRNA, in particular, miR-371a-3p, has shown great promise in discriminating the presence of TGCT and may represent a breakthrough for this disease. In this review, we discuss the potential role of miRNA across clinical states of TGCTs. We review their discovery, methods of assay, limitations and future potential.

Bone-Metabolism-Related Serum microRNAs to Diagnose Osteoporosis in Middle-Aged and Elderly Women.

Objective: Postmenopausal osteoporosis (PMOP), a chronic systemic metabolic disease prevalent in middle-aged and elderly women, heavily relies on bone mineral density (BMD) measurement as the diagnostic indicator. In this study, we investigated serum microRNAs (miRNAs) as a possible screening tool for PMOP. Methods: This investigation recruited 83 eligible participants from 795 community-dwelling postmenopausal women between June 2020 and August 2021. The miRNA expression profiles in the serum of PMOP patients were evaluated via miRNA microarray (six PMOP patients and four postmenopausal women without osteoporosis (n-PMOP) as controls). Subsequently, results were verified in independent sample sets (47 PMOP patients and 26 n-PMOP controls) using quantitative real-time PCR. In addition, the target genes and main functions of the differentially expressed miRNAs were explored by bioinformatics analysis. Results: Four highly expressed miRNAs in the serum of patients (hsa-miR-144-5p, hsa-miR-506-3p, hsa-miR-8068, and hsa-miR-6851-3p) showed acceptable disease-independent discrimination performance (area under the curve range: 0.747-0.902) in the training set and verification set, outperforming traditional bone turnover markers. Among four key miRNAs, hsa-miR-144-5p is the only one that can simultaneously predict changes in BMD in lumbar spine 1-4, total hip, and femoral neck (β = -0.265, p = 0.022; β = -0.301, p = 0.005; and β = -0.324, p = 0.003, respectively). Bioinformatics analysis suggested that the differentially expressed miRNAs were targeted mainly to YY1, VIM, and YWHAE genes, which are extensively involved in bone metabolism processes. Conclusions: Bone-metabolism-related serum miRNAs, such as hsa-miR-144-5p, hsa-miR-506-3p, hsa-miR-8068, and hsa-miR-6851-3p, can be used as novel biomarkers for PMOP diagnosis independent of radiological findings and traditional bone turnover markers. Further study of these miRNAs and their target genes may provide new insights into the epigenetic regulatory mechanisms of the onset and progression of the disease.

Thyroid Cancer Persistence in Patients with Unreliable Thyroglobulin Measurement: Circulating microRNA as Candidate Alternative Biomarkers.

We aimed to evaluate the role of circulating miRNAs as a biomarker of the persistence of papillary thyroid cancer (PTC) in patients with an "uninformative" thyroglobulin (Tg) measurement. We prospectively enrolled 49 consecutive PTC patients with Tg-positive antibodies (TgAb) who had undergone a (near)-total thyroidectomy and 131I therapy (RIT). The serum thyroid stimulating hormone (TSH), Tg, and TgAb levels were measured before and at 6 and 12 months after RIT, respectively. The serum miRNA (221, 222, 375, 155, and 146b) levels were measured simultaneously. The response to the initial therapy was assessed according to the 2015 ATA criteria. A decrease in 50% or more of serum miRNA over time was observed in 41/49 PTC patients, who showed an excellent response (ER), but six and two patients were classified to have an indeterminate/incomplete biochemical or incomplete structural response to initial therapy. Serum miRNA kinetics emerge as a promising biomarker for the early detection of a persistent disease in PTC patients with uninformative Tg results.

CDX-301 prevents radiation-induced dysregulation of miRNA expression and biogenesis

Risks of radiation exposure necessitate the development ofradioprophylactic drugs. We have reported the efficacy of CDX-301, a recombinantly developed human protein form of Fms-related tyrosine kinase 3 ligand (Flt3L), as a radioprophylactic and radiomitigatory agent. Here, we performed global microRNA profiling to further understand the mechanism of action of CDX-301. We find that CDX-301 administration 24h prior to total body irradiation prevents radiation-induced dysregulation of microRNA biogenesis and expression in murine serum and spleen samples in a time- and tissue-dependent manner. Further analysis shows that activation of the HOTAIR regulatory pathway has a prominent function in radiation-induced injury responses, which is inhibited by pre-treatment with CDX-301. Moreover, CDX-301 attenuates radiation-induced dysregulation of several cellular functions such as inflammatory and immune responses. In corroboration, we also find that pre-treatment with CDX-301 restores the expression of bone marrow aplasia markers and inflammatory cytokines and growth factors, as well as the expression of genes associated with MAP kinase and TGF-β pathways that are altered by radiation. Our findings provide new insights into CDX-301-mediated molecular and cellular mechanisms and point to a possible novel radioprotective drug for the prevention of irradiation-induced injury and hematopoietic acute radiation syndrome.

Serum microRNA Signature Predicting Poor Therapeutic Response to Bortezomib-Based Therapy and Clinical Outcome in Newly Diagnosed Multiple Myeloma: A Result of miRNA Profiling By Deep Sequencing

Introduction Bortezomib-based regimens are the preferred induction therapy for multiple myeloma (MM) in resource limited regions. Response to bortezomib is inconsistent across patients, and impact of disease heterogeneity on treatment failure remains an unpredictable challenge. Circulating/cell-free miRNA (cfmiRNA) have been shown to contribute to disease prognostication and response monitoring in relapse/refractory setting, serving as an auxiliary biomarker. However, data on real world utility of cfmiRNA in prediction of therapeutic response in newly diagnosed MM (NDMM), treated without transplant is scarce. Aim To identify MM-specific cfmiRNA signature and investigate its utility in prediction of initial therapeutic response (ITR) to Bortezomib-based regimens in NDMM. Further, we aimed to study the kinetics of cfmiRNA during induction therapy as a tool for disease monitoring and predicting clinical outcome. Methods We prospectively enrolled 141 NDMM patients diagnosed and treated at Tata Memorial Centre, India. Small RNA sequencing for miRNA profiling was performed in 65 NDMM and 10 age-matched control and validated by quantitative PCR (qPCR). cfmiRNA levels were evaluated by qPCR at diagnosis, at the end of 3 cycles (follow up1-FU1) and 6 cycles (follow up2-FU2) of chemotherapy. Changes in cfmiRNA expression at FU1 and FU2 was represented as fold change (FC) relative to baseline. Samples were grouped as high or low expressors using median FC of individual miRNAs as cut-off. Patients received bortezomib-based treatment. ITR was monitored at the end of FU2. Results Median age of patients was 55 years (range:27-81 years; M:F-2:1). Median follow-up was 35 months (1-71 months). ITR post FU2 included 7.09% CR, 43.97% VGPR, 33.33% PR, 2.84% SD, 2.84% PD and 9.93% deaths during initial therapy. BM clonal plasma cells (CPC) were detected in 134/141 patients (median, range:97.8%, 1.5-100%). Small RNA sequencing identified 36 miRNAs differentially expressed (DEMs) in MM serum (FC≥2; p-adj<0.05) at diagnosis, the greater majority being upregulated (31/37). DEMs with Log2FC≥2 was confirmed by qPCR (n=21). There was significant correlation between miRNA levels measured by RNA-seq and qPCR (R2=0.78; p<0.001), with a near-constant bias of 0.6361 for miRNA with Log2FC≥4 (n=13; Upper, lower limits of agreement:2.463, -1.191). Results were validated in additional 76 NDMM and 10 control samples. Of the 13 DEMs, high expression (Log2FC>6.01; p=0.0047) of miR-4665-5p at diagnosis was significantly associated with poor ITR at FU2 (OR:2.92; p=0.01), however, no association with OS or PFS was observed. We studied changes in cfmiRNA levels at FU1 and FU2 for therapeutic monitoring. Univariate analysis identified 5 miRNA - miR-92b-5p, miR-4775, miR-103a-2-5p, miR-4665-5p and miR-365a-3p significantly associated with poor ITR at FU1 and FU2. Multivariate logistic regression yielded a model (Hosmer-Lemeshow p=0.9) with high expression of miR-92b-5p (Log2FC>2.04; p=0.006; OR:5.9; p<0.0001) and miR-103a-2-5p (median Log2FC=1.65; p=0.0329; OR:2.54; p=0.0106) at FU2 significantly associated with poor ITR (p=0.0049). A combination of these miRNAs could differentiate good and poor responders with area under ROC curve of 0.87, 83.64% sensitivity and 75% specificity. Among these, miR-92b-5p demonstrated an association with poor PFS (27 vs. not reached; HR:2.4; p=0.0005) and OS (52 months vs. not reached; HR:2.88; p=0.0056). Univariate analysis also showed high risk cytogenetics, high LDH and CPC>90% of total plasma cells to be associated with poor PFS. Expression of miR-92b-5p at FU2 was independently associated with poor PFS (HR:1.4; p=0.0005) on Cox proportional hazards regression. Conclusion: We studied the kinetics of serum miRNA and demonstrated that changes in levels of miR-92b-5p and miR-103a-2-5p are useful in prediction of poor response to Bortezomib-based regimen. Increase in levels of miR-92b-5p at follow up time points post induction was found to be an indicator of poor ITR and shorter PFS. The role of miR-92b-5p in the biology of MM is yet to be evaluated. Herein, we highlight the utility of cfmiRNA assays as non-invasive biomarker in prediction of initial therapeutic response to Bortezomib-based protocol. Determining clinical relevance of these findings could expand applications of cfmiRNA as a biomarker. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Diagnostic implication of a circulating serum-based three-microRNA signature in hepatocellular carcinoma.

Owing to the diagnostic dilemma, the prognosis of hepatocellular carcinoma (HCC) remains impoverished, contributing to the globally high mortality rate. Currently, HCC diagnosis depends on the combination of imaging modalities and the measurement of serum alpha-fetoprotein (AFP) levels. Nevertheless, these conventional modalities exhibit poor performance in detecting HCC at early stages. Thus, there is a pressing need to identify novel circulating biomarkers to promote diagnostic accuracy and surveillance. Circulating miRNAs are emerging as promising diagnostic tools in screening various cancers, including HCC. However, because of heterogenous and, at times, contradictory reports, the universality of miRNAs in clinical settings remains elusive. Consequently, we proposed to explore the diagnostic potential of ten miRNAs selected on a candidate-based approach in HCC diagnosis. The expression of ten candidate miRNAs (Let-7a, miR-15a, miR-26a, miR-124, miR-126, miR-155, miR-219, miR-221, miR-222, and miR-340) was investigated in serum and tissue of 66 subjects, including 33 HCC patients and 33 healthy controls (HC), by rt-PCR. Receiver operating characteristic curve (ROC) analysis was used to determine the diagnostic accuracy of the prospective serum miRNA panel. To anticipate the potential biological roles of a three-miRNA signature, the target genes were evaluated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway. The serum and tissue expression of miRNAs (Let-7a, miR-26a, miR-124, miR-155, miR-221, miR-222, and miR-340) were differentially expressed in HCC patients (p < 0.05). The ROC analysis revealed promising diagnostic performance of Let-7a (AUC = 0.801), miR-221 (AUC = 0.786), and miR-2 (AUC = 0.758) in discriminating HCC from HC. Furthermore, in a logistic regression equation, we identified a three-miRNA panel (Let-7a, miR-221, and miR-222; AUC = 0.932) with improved diagnostic efficiency in differentiating HCC from HC. Remarkably, the combination of AFP and a three-miRNA panel offered a higher accuracy of HCC diagnosis (AUC = 0.961) than AFP alone. The functional enrichment analysis demonstrated that target genes may contribute to pathways associated with HCC and cell-cycle regulation, indicating possible crosstalk of miRNAs with HCC development. To conclude, the combined classifier of a three-miRNA panel and AFP could be indispensable circulating biomarkers for HCC diagnosis. Furthermore, targeting predicted genes may provide new therapeutic clues for the treatment of aggressive HCC.

PDA-PEI-Copolymerized Nanodots with Tailorable Fluorescence Emission and Quenching Properties for the Sensitive Ratiometric Fluorescence Sensing of miRNA in Serum.

Dopamine and polyethyleneimine (PEI) copolymerized nanodots (PDA-PEI nanodots) with both fluorescence emission and quenching features were synthesized by a simple one-step reaction at room temperature. By adjusting the dopamine and PEI ratio as well as the chain length of PEI, the fluorescence emission and quenching properties of PDA-PEI nanodots can be controlled well. Under optimal conditions, the nanodots showed strong green fluorescence emission with an absolute quantum yield of 1-2% and a quenching efficiency of more than 99% to several fluorophores with emission wavelengths ranging from blue to red light regions. The nanodots with a large number of functional groups also showed strong affinity to nucleic acid strands, excellent solubility in aqueous solution, long-term stability, and uniform size distribution. Integrating these attractive features with the specific enzymatic digestion reaction of the DSN enzyme, a highly sensitive ratiometric fluorescence nanoprobe for miRNA analysis was developed. Aminomethylcoumarin acetate (AMCA), which possesses the same excitation wavelength but a well-resolved blue fluorescence emission with PDA-PEI nanodots, was selected as the signal-reporting unit for capture probe labeling, while the inherent green fluorescence of PDA-PEI nanodots served as the reference. According to the ratiometric fluorescence signal, the ratiometric fluorescence nanoprobes showed high sensitivity and good accuracy for the miRNA assay. Because of the high and universal quenching efficiency, stable fluorescence emission, easily assembled interface, and uniform morphology, the nanodots may have great application prospects to serve as a universal nanoplatform for the fabrication of ratiometric fluorescence nanoprobes.

MRI Features and Significance of Serum miRNAs and Inflammatory Cytokines in Patients with Temporal Lobe Epilepsy

This study was aimed to investigate the changes of brain MRI features and serum biological parameters in patients with TLE. 30 patients with unilateral TLE confirmed by surgical pathology were selected as study subjects, and 30 subjects without a history of epilepsy who underwent health examinations during the same period were selected as controls. The brain MRI features of the patients were explored and the T2 relaxation time (HCT2) indexes of the bilateral hippocampus were extracted. The differences in levels of peripheral blood T lymphocyte subsets, inflammatory cytokines, and miRNAs were measured. The results showed that the hippocampal volume of TLE patients was significantly reduced, and the HCT2 value of the hippocampus was greater than that of the control group ( P &lt; 0.05 ). CD3+ (77.9 ± 4.4)%, CD4+ (45.6 ± 2.2)%, CD8+ (22.1 ± 1.9)%, and CD3+/CD8+ (2.24 ± 0.22) in peripheral blood T lymphocyte subsets of epileptic patients, compared with control group, CD3+, CD4+, and CD4+/CD8+ levels were significantly increased and CD8+ concentration was significantly decreased in epileptic patients ( P &lt; 0.05 ); inflammatory cytokines TNF-α was (2.63 ± 0.26) pg/mL, IL-1β was (4.61 ± 0.57) pg/mL, IL-2 was (1.59 ± 0.21) pg/mL, IL-6 was (2.28 ± 0.19) pg/mL, and ICAM-1 was (1.89 ± 0.30) pg/mL in peripheral blood of epileptic patients, which was significantly increased compared with control group, while IL-10 was significantly decreased in epileptic patients ( P &lt; 0.05 ); miR-146a was (2.14 ± 0.28) and miR-210 was (1.89 ± 0.31), miR-221 (2.44 ± 0.35), miR-34a (0.59 ± 0.14), miR-135b (10.17 ± 0.16), miR-33 (0.26 ± 0.09) in peripheral blood miRNA levels of epileptic patients, and miR-146a, miR-210, miR-221, and miR-34a levels of epileptic patients were significantly increased compared with control group, while MiR-135b and miR-33 levels of epileptic patients were significantly reduced ( P &lt; 0.05 ). In summary, patients with TLE have hippocampal lesions, which may be related to peripheral blood T lymphocyte subsets imbalance, chronic inflammatory response, and abnormal expression of miRNAs.

Ultrasensitive detection of serum miRNA biomarkers related to papillary thyroid cancer using ligation-initiated phosphorothioated primer-based loop-mediated isothermal amplification

Accurately distinguishing of papillary thyroid cancer (PTC) from benign nodules, at the same time, avoiding overdiagnosis is of great significance. In consideration of the role of miRNA in pathogenesis and progression of tumors, miRNA might also be useful as diagnostic marker for cancer. In this study, a ligation-initiated phosphorothioated primer-based loop-mediated isothermal amplification (PS-LAMP) strategy was established to quantify miRNAs in serum sample, thereby achieving noninvasive diagnosis of PTC. Two linker probes designed from dumbbell-shaped amplicons of LAMP are ligated to obtain an active amplicon only when they are perfectly hybridized with target miRNA, exhibiting high specificity. The introduction of PS modification into inner primers (BIP/FIP) facilitated extra efficient hairpin formation and extension for LAMP, which significantly improved sensitivity. Then, the proposed method was successfully applied in the early diagnosis of PTC, in which circulating serum level of miR-146b-5p and miR-199b-5p could draw a clear distinction between patients and controls. This ligation-initiated PS-LAMP assay opens new avenues for highly sensitive quantification of miRNA biomarkers and thus exhibits a considerable potential utility in clinical diagnosis and prognosis.

Combining serum miR-144-3p and miR-652-3p as potential biomarkers for the early diagnosis and stratification of acute cellular rejection in heart transplantation patients

Abstract Background There is a dire need for specific, noninvasive biomarkers that can accurately and early detect cardiac acute cellular rejection (ACR). Previously, we described miR-144-3p as an excellent candidate to detect Grade ≥2R ACR. Purpose Now, we investigated the combination of miR-144-3p with other differentially expressed serum miRNA that we previously described to improve diagnostic accuracy, mainly in mild rejection to avoid reaching severe stages. Methods We selected miR-652-3p, miR-6747-3p and miR-221-3p from a preliminary RNA-seq study to be validated by RT-qPCR on 212 consecutive serum samples from transplantation recipients undergoing routine endomyocardial biopsies to subsequently combine them with miR-144-3p results and investigate their diagnostic capability. Results We confirmed the miR-652-3p overexpression (p&amp;lt;0.0001) and its capability to discriminate between patients with and without ACR of any grade (p&amp;lt;0.0001), but not miR-6747-3p or miR-221-3p. The combined serum levels of miR-144-3p and miR-652-3p were significantly higher in patients with rejection regardless of posttransplantation time (p&amp;lt;0.0001). This combination resulted in a diagnostic efficacy for 1R (AUC=0.794) and ≥2R (AUC=0.892) (p&amp;lt;0.0001) that was superior to each biomarker alone. Furthermore, it was a strong independent predictor of ACR for 1R (odds ratio of 10.950, p&amp;lt;0.0001) and ≥2R (odds ratio of 14.289, p&amp;lt;0.01). Conclusions We demonstrated that an appropriate combination of blood-based biomarkers can exhibit greater efficiency for cardiac rejection diagnosis. The combined detection of abnormal expression of miR-144-3p and miR-652-3p in the serum of ACR patients can improve the diagnostic sensitivity of rejection at the early stage and contribute to increasing the diagnostic accuracy, mainly in the lower rejection grades. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Institute of Health Carlos III European Regional Development Fund (ERDF)

Evaluation of circulating extracellular vesicles and miRNA in neutered and obese female dogs

Adipose tissue is a metabolic and endocrine organ, and its adipocytes can synthesize and secrete extracellular vesicles (EVs), thus allowing intercellular communication. EVs are nanoparticles that transport lipids, proteins, metabolites, and nucleic acids (mRNA and microRNAs). MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression. miR-132, miR-26b, and miR-155 are associated with obesity, lipid metabolism and adipogenesis. The aim of this study was to evaluate the enriched EVs fraction containing miRNAs (miR-132, miR-26b, and miR-155) in serum from obese female dogs. Thirty-two neutered females in good general condition were recruited, including 21 obese and 11 healthy controls. The initial evaluation of the females included a general physical examination and laboratory tests. Small EVs (sEVs) were isolated from whole blood by serial centrifugation and ultracentrifugation, and nanoparticle analysis was used to determine the size and concentration of serum sEVs. miRNAs were extracted from sEVs enriched fraction and analyzed by real-time polymerase chain reaction. Obese female dogs with hypertriglyceridemia showed an increase in the sEVs concentration and in the expression of miR-132 and miR-26b in sEVs enriched fraction. No changes were observed in the group of obese female dogs with normal serum biochemical profile and in relation to miR-155 expression. These results suggest that obese female dogs with hypertriglyceridemia may present alterations in sEVs and in the expression of miRNAs related to lipid metabolism and adipogenesis.

Identification of the Minimum Combination of Serum microRNAs to Predict the Recurrence of Colorectal Cancer Cases

BackgroundSerum microRNAs (miRNAs) have been recognized as potential stable biomarkers for various types of cancer. Considering the clinical applications, there are certain critical requirements, such as minimizing the number of miRNAs, reproducibility in a longitudinal clinical course, and superiority to conventional tumor markers, such as carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9. This study aimed to identify serum miRNAs that indicate the recurrence of colorectal cancer (CRC), surpassing inter-tumor heterogeneity.MethodsWe conducted an analysis of 434 serum samples from 91 patients with CRC and 71 healthy subjects. miRNAs were obtained from Toray Co., Ltd, and miRNA profiles were analyzed using a three-step approach. miRNAs that were highly expressed in patients with CRC than in the healthy controls in the screening phase, and those that were highly expressed in the preoperative samples than in the 1-month postoperative samples in the discovery phase, were extracted. In the validation phase, the extracted miRNAs were evaluated in 323 perioperative samples, in chronological order.ResultsA total of 12 miRNAs (miR-25-3p, miR-451a, miR-1246, miR-1268b, miR-2392, miR-4480, miR-4648, miR-4732-5p, miR-4736, miR-6131, miR-6776-5p, and miR-6851-5p) were significantly concordant with the clinical findings of tumor recurrence, however their ability to function as biomarkers was comparable with CEA. In contrast, the combination of miR-1246, miR-1268b, and miR-4648 demonstrated a higher area under the curve (AUC) than CEA. These three miRNAs were upregulated in primary CRC tissues.ConclusionWe identified ideal combinatorial miRNAs to predict CRC recurrence.