miRNA Quantification Research Articles

A Framework Nucleic Acid-Mediated Multimodal Tandem Multivariate Signal Amplification Strategy for Simultaneous Absolute Quantification of Three Different MicroRNAs in a Single Cell.

The simultaneous quantification of multiple microRNAs (miRNA) in a single cell can help scientists understand the relationship between different miRNA groups and different types of cancers from an miRNA omics perspective at the single-cell level. However, there currently remains a challenge in developing techniques for the simultaneous absolute quantification of multiple miRNAs in single cells. Herein, we propose a framework nucleic acid (FNA)-mediated multimodal tandem multivariate signal amplification strategy for simultaneous absolute quantification of three different miRNAs in a single cell. In this study, DNA hexahedron FNAs (DHFs) and DNA tetrahedron FNAs (DTFs) were first prepared, multiple DNA hairpins and substrates were then connected to the hexahedron frame nucleic acid as the target recognition units, and three substrates with labeled FAM fluorophores on the tetrahedral frame nucleic acid served as signal output units. After the two types of FNAs entered the cell, they reacted with three different miRNAs (miRNA-155, miRNA-373, and miRNA-21) and multimodal tandem multivariate signal amplification was initiated simultaneously, reducing the detection limit of the three miRNAs to 8 × 10-15, 2 × 10-15, and 1 × 10-15 M, respectively. The detection sensitivity of the three miRNAs was simultaneously increased by six orders of magnitude, reaching the quantitative requirement of trace miRNAs in single cells. Combined with single-cell injection, membrane melting, and intracellular component separation technology on a microchip electrophoresis platform, we achieved the simultaneous absolute quantification of three different miRNAs in a single cell, thereby providing an important novel method that can be used to conduct single-cell research.

Data Normalization of Urine miRNA Profiling from Head and Neck Cancer Patients Treated with Cisplatin.

The microRNA (miRNA) expression profile by qRT-PCR depends directly on the most appropriate normalization strategy adopted; however, currently there is no universally adequate reference gene. Therefore, this study aimed to determine, considering RNA-Seq results, the most adequate endogenous normalizer for use in the relative quantification of urine miRNAs from head and neck cancer patients, treated with cisplatin chemoradiotherapy. The massive sequencing was performed to identify the miRNAs differentially expressed between the group with cisplatin nephrotoxicity (n = 6) and the one without (n = 6). The candidate endogen normalizer was chosen according to four criteria: (1) the miRNA must be expressed in most samples; (2) the miRNA must have a fold change value between 0.99 and 1.01; (3) the miRNA must have a p-value ≥ 0.98; and (4) the miRNA must not be commented on by the final GeneGlobe (Qiagen, Hilden, Germany) analysis. Four miRNAs met all the criteria (hsa-miR-363-5p, hsa-miR-875-5p, hsa-miR-4302, and hsa-miR-6749-5p) and were selected for validation by qRT-PCR in a cohort of 49 patients (including the 12 sequencing participants). Only hsa-miR-875-5p was shown to be an adequate normalizer for the experimental condition under investigation, as it exhibited invariant expression between the two groups.

Double hairpin-assembled probe-mediating catalytic hairpin assembly and primer exchange reaction for sensitive and label-free miRNA analysis in gastric carcinoma

The aberrant expression of microRNA (miRNA) is closely associated with various pathological processes, such as the development of gastric cancer. High-efficiency quantification of miRNAs is significant for the diagnosis, prognosis, and treatment of cancers. However, the sensitive and reliable detection of miRNA remains a huge challenge. We depict here a novel fluorescent approach for sensitive and label-free miRNA detection by exploiting a designed detection scaffold to integrate the catalytic hairpin assembly and primer exchange reaction (PER). In this method, the detection scaffold that is constructed based on the hybridization between two hairpin structure probes (H1 probe and H2 probe), is capable of specifically recognizing target miRNA and activating signal amplification, and the PER process transcribes numerous G-rich sequences to induce ThT-based label-free signal generation. Based on the efficient signal amplification strategy and label-free signal generation mode, the method exhibits a wide detection range of 7 orders of magnitude and a high repeatability (coefficient of variation, 2.76%), implying that the proposed approach will be a robust tool in quantification of miRNA and early diagnosis of disease.Graphical abstract

Screening of reference genes for microRNA analysis in the study of solider caste differentiation of Formosan subterranean termite Coptotermes formosanus Shiraki

The soldier caste differentiation is a complex process that is governed by the transcriptional regulation and post-transcriptional regulation. microRNAs (miRNAs) are noncoding RNAs that control a wide range of activities. However, their roles in solider caste differentiation are barely studied. RT-qPCR is a powerful tool to study the function of genes. A reference gene is required for normalization for the the relative quantification method. However, no reference gene is available for miRNA quantification in the study of solider caste differentiation of Coptotermes formosanus Shiraki. In this research, in order to screen the suitable reference genes for the study of the roles of miRNAs in solider caste differentiation, the expression levels of 8 candidate miRNA genes were quantified in the head and thorax + abdomen during soldier differentiation. The qPCR data were analyzed using geNorm, NormFinder, BestKeeper, ΔCt method and RefFinder. The normalization effect of the reference genes was evaluated using the let-7-3p. Our study showed that novel-m0649-3p was the most stable reference gene, while U6 was the least stable reference gene. Our study has selected the most stable reference gene, and has paved the way for functional analysis of miRNAs in solider caste differentiation.

Multiplexed and accurate quantification strategy for miRNA based on specific terminal-mediated PCR with equivalent amplification.

MicroRNAs (miRNAs) are recognized as potential biomarkers for the early diagnosis and prognosis of different diseases. Multiplexed and accurate miRNA quantification methods with equivalent detection efficiency are particularly crucial due to their complex biological functions and lack of a unified internal reference gene. Here, a unique multiplexed miRNA detection method, named Specific Terminal-Mediated miRNA PCR (STEM-Mi-PCR), was developed. It mainly includes a linear reverse transcription step using tailored-designed target specific capture primers, followed by an exponential amplification process using two universal primers to execute the multiplex assay. For proof of concept, four miRNAs were used as models to develop a multiplexed detection assay within one tube simultaneously and then evaluate the performance of the established STEM-Mi-PCR. The sensitivity of the 4-plexed assay was approximately 100 aM with an equivalent amplification efficiency (95.67±8.58%), and had no cross-reactivity each other with high specificity. Quantification of different miRNAs in twenty patients' tissues shown variation from approximately pM to fM concentration level, demonstrating the possibility of practical application of the established method. Moreover, this method was extraordinarily capable of single nucleotide mutation discrimination in different let-7 family members with no more than 0.7% nonspecific detection signal. Hence, the STEM-Mi-PCR we proposed here paves an easy and promising way for miRNA profiling in future clinical applications.

Identification of Endometrial Cancer-Specific microRNA Biomarkers in Endometrial Fluid.

Abnormal uterine bleeding is a common benign gynecological complaint and is also the most common symptom of endometrial cancer (EC). Although many microRNAs have been reported in endometrial carcinoma, most of them were identified from tumor tissues obtained at surgery or from cell lines cultured in laboratories. The objective of this study was to develop a method to detect EC-specific microRNA biomarkers from liquid biopsy samples to improve the early diagnosis of EC in women. Endometrial fluid samples were collected during patient-scheduled in-office visits or in the operating room prior to surgery using the same technique performed for saline infusion sonohysterography (SIS). The total RNA was extracted from the endometrial fluid specimens, followed by quantification, reverse transcription, and real-time PCR arrays. The study was conducted in two phases: exploratory phase I and validation phase II. In total, endometrial fluid samples from 82 patients were collected and processed, with 60 matched non-cancer versus endometrial carcinoma patients used in phase I and 22 in phase II. The 14 microRNA biomarkers, out of 84 miRNA candidates, with the greatest variation in expression from phase I, were selected to enter phase II validation and statistical analysis. Among them, three microRNAs had a consistent and substantial fold-change in upregulation (miR-429, miR-183-5p, and miR-146a-5p). Furthermore, four miRNAs (miR-378c, miR-4705, miR-1321, and miR-362-3p) were uniquely detected. This research elucidated the feasibility of the collection, quantification, and detection of miRNA from endometrial fluid with a minimally invasive procedure performed during a patient in-office visit. The screening of a larger set of clinical samples was necessary to validate these early detection biomarkers for endometrial cancer.

Encoded Fusion-Mediated MicroRNA Signature Profiling of Tumor-Derived Extracellular Vesicles for Pancreatic Cancer Diagnosis.

MicroRNAs (miRNAs) in tumor-derived extracellular vesicles (tEVs) are important cancer biomarkers for cancer screening and early diagnosis. Multiplex detection of miRNAs in tEVs facilitates accurate diagnosis but remains a challenge. Herein, we propose an encoded fusion strategy to profile the miRNA signature in tEVs for pancreatic cancer diagnosis. A panel of encoded-targeted-fusion beads was fabricated for the selective recognition and fusion of tEVs, with the turn-on fluorescence signals of molecule beacons for miRNA quantification and barcode signals for miRNA identification using readily accessible flow cytometers. Using this strategy, six types of pancreatic-cancer-associated miRNAs can be profiled in tEVs from 2 μL plasma samples (n = 36) in an isolation-free and lysis-free manner with only 2 h of processing, offering a high accuracy (98%) to discriminate pancreatic cancer, pancreatitis, and healthy donors. This encoded fusion strategy exhibits great potential for multiplex profiling of miRNA in tEVs, offering new avenues for cancer diagnosis and screening.

Quantification of miRNAs (miR4743-5p, miR149-5p, and miR331-5p) in the placenta of south Indian pregnant women with Early and Late onset of Preeclampsia

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Sensitive analysis of miRNAs via primer exchange reaction integrated with hairpin catalytic reaction

MicroRNAs (miRNAs) can serve as potential biological targets for early screening, targeted therapy, and prognosis in ovarian cancer (OC). However, sensitive and reliable quantification and identification of miRNA remain a huge challenge. Herein, we proposed a simple and reliable approach for the ultra-sensitive detection of miRNA by integrating endonuclease-III (Exo-III) assisted signal recycle, primer exchange reaction (PER), and hairpin catalytic reaction (HCR). In this method, target miRNA specifically binds with toehold sequence to form a blunt 3’ terminus in the detection probe (dumbbell probe) that can be recognized by Exo-III, and to initiate subsequent signal amplifications. Based on this, the approach is successfully utilized in detecting OC related miRNAs with high sensitivity (limit of detection for miRNA-211 was 13 aM) and stability. By simply changing the toehold sequence in detection probe, the established approach can be easily extended to other miRNA detection. We believe that the platform is robust in detecting OS related biomarkers and is promising in renovating cancer diagnostic tools.

Advances in Electrochemical Biosensor Technologies for the Detection of Nucleic Acid Breast Cancer Biomarkers.

Breast cancer is the second leading cause of cancer deaths in women worldwide; therefore, there is an increased need for the discovery, development, optimization, and quantification of diagnostic biomarkers that can improve the disease diagnosis, prognosis, and therapeutic outcome. Circulating cell-free nucleic acids biomarkers such as microRNAs (miRNAs) and breast cancer susceptibility gene 1 (BRCA1) allow the characterization of the genetic features and screening breast cancer patients. Electrochemical biosensors offer excellent platforms for the detection of breast cancer biomarkers due to their high sensitivity and selectivity, low cost, use of small analyte volumes, and easy miniaturization. In this context, this article provides an exhaustive review concerning the electrochemical methods of characterization and quantification of different miRNAs and BRCA1 breast cancer biomarkers using electrochemical DNA biosensors based on the detection of hybridization events between a DNA or peptide nucleic acid probe and the target nucleic acid sequence. The fabrication approaches, the biosensors architectures, the signal amplification strategies, the detection techniques, and the key performance parameters, such as the linearity range and the limit of detection, were discussed.

MicroRNA qPCR normalization in Nile tilapia (Oreochromis niloticus): Effects of acute cold stress on potential reference targets

The Nile tilapia (Oreochromis niloticus) is one of the most important cultured fish worldwide, but tilapia culture is largely affected by low temperatures. Recent studies suggest that microRNAs (miRNAs) regulate cold tolerance traits in fish. In general, qPCR-based methods are the simplest and most accurate forms of miRNA quantification. However, qPCR data heavily depends on appropriate normalization. Therefore, the aim of the present study is to determine whether the expression of previously tested, stably expressed miRNAs are affected by acute cold stress in Nile tilapia. For this purpose, one small nuclear RNA (U6) and six candidate reference miRNAs (miR-23a, miR-25-3, Let-7a, miR-103, miR-99-5, and miR-455) were evaluated in four tissues (blood, brain, liver, and gills) under two experimental conditions (acute cold stress and control) in O. niloticus. The stability of the expression of each candidate reference miRNA was analyzed by four independent methods (the delta Ct method, geNorm, NormFinder, and BestKeeper). Further, consensual comprehensive ranking of stability was built with RefFinder. Overall, miR-103 was the most stable reference miRNA in this study, and miR-103 and Let-7a were the best combination of reference targets. Equally important, Let-7a, miR-23a, and miR-25-3 remained consistently stable across different tissues and experimental groups. Considering all variables, U6, miR-99-5, and miR-455 were the least stable candidates under acute cold stress. Most important, suitable reference miRNAs were validated in O. niloticus, facilitating further accurate miRNA quantification in this species.

Targeting YAP1 ameliorates progesterone resistance in endometriosis

Does YAP1 inhibition alleviate progesterone resistance in endometriosis? YAP1 inhibition reduces progesterone resistance in vitro and in vivo. Progesterone resistance not only causes treatment failure for endometriosis but also inhibits eutopic endometrial cell proliferation, dysregulates decidualization, and reduces the success rates of pregnancy. Hippo/yes-associated protein 1 (YAP1) signaling pathway plays an important role in the pathogenesis of endometriosis. Paraffin-embedded tissues containing paired endometriotic and endometrial specimens (n = 42) and serum samples isolated from normal controls (n = 15) or endometriotic patients with (n = 25) or without (n = 21) prior dienogest treatment were analyzed. A mouse model of endometriosis was also used to evaluate the effects of YAP1 inhibition on progesterone resistance. Primary endometriotic and endometrial stromal cells treated with YAP1 inhibitor or miR-21 mimic/inhibitor were used for the in vitro studies including decidualization induction, chromatin immunoprecipitation (ChIP), and RNA immunoprecipitation. Tissue specimens and serum from human and mouse were used for immunohistochemistry staining, exosome isolation, and microRNA (miRNA) quantification, respectively. Herein, we report, by using ChIP-PCR and RNA-IP, that YAP1 inhibits progesterone receptor (PGR) expression through upregulation of miR-21-5p. Upregulation of miR-21-5p not only reduces PGR expression but also inhibits endometrial stromal cell decidualization. Indeed, levels of YAP1 and miR-21-5p are inversely correlated with the level of PGR in human endometrial samples. In contrast, knockdown of YAP1 or treatment with verteporfin (VP), a YAP1 inhibitor, reduces miR-21-5p expression, thus leading to an increase in PGR expression in ectopic endometriotic stromal cells. In the mouse model of endometriosis, treatment with VP increases PGR expression and enhances decidualization. More importantly, VP synergistically increases the treatment effect of progestin in causing the regression of endometriotic lesions and improves the decidualization capability of the endometrium. Interestingly, treatment with dienogest, a synthetic progestin, reduces YAP1 and miR-21-5p expression in human cells and in the mouse model of endometriosis. Patients who received dienogest treatment for 6 months show a significant decrease in serum extracellular vesicle-associated miR-21-5p level. A public dataset (GSE51981) containing a large cohort of endometriotic tissues is available from the Gene Expression Omnibus (GEO). A large cohort of clinical samples is needed to verify the current diagnostic value of miR-21-5p in future studies. The reciprocal regulation of YAP1 and PGR suggests that combined YAP1 inhibitor and progestin may be a better therapeutic approach for treating endometriosis. This study was supported by the Ministry of Science and Technology, Taiwan (MOST-111-2636-B-006-012, MOST-111-2314-B-006-075-MY3, and MOST-106-2320-B-006-072-MY3). The authors have no conflict of interest to disclose.

Preanalytic Methodological Considerations and Sample Quality Control of Circulating miRNAs

As miRNAs emerge as potential circulating biomarkers for the diagnosis or prognosis of a wide variety of diseases, the quantification of miRNA necessitates careful preanalytic considerations and sample quality control becomes crucial. This study comprehensively analyzed the profiles of 356 miRNAs by quantitative RT-PCR in various blood sample types, with various processing protocols. The comprehensive analysis investigated the correlations of individual miRNAs with certain confounding factors. On the basis of these profiles, a panel of 7 miRNAs was established for the quality control of samples corresponding to hemolysis and platelet contamination. The panel was used to investigate the confounding impacts based on the size of the blood collection tube, the centrifugation protocol, post-freeze-thaw spinning, and whole blood storage. A standard dual-spin workflow for the processing of blood had been established for optimal sample quality. The real-time stability of 356 miRNAs was also investigated with demonstration of the temperature and time-induced miRNA degradation profile. Stability-related miRNAs were identified from real-time stability study and further incorporated into the quality control panel. This quality control panel enables the assessment of sample quality for more robust and reliable detection of circulating miRNAs.

Assembly of DNA triangular pyramid frustum for ultrasensitive quantification of exosomal miRNA

Early screening of biomarkers benefits therapy and prognosis of cancers. MiRNAs encapsulated in tumor-derived exosomes are emerging biomarkers for early diagnosis of cancers. Nevertheless, traditional methods suffer certain drawbacks, which hamper their wide applications. In this contribution, we have developed a convenient electrochemical approach for quantification of exosomal miRNA based on the assembly of DNA triangular pyramid frustum (TPF) and strand displacement amplification. Four single-stranded DNA helps the formation of primary DNA triangle with three thiols for gold electrode immobilization at the bottom and three amino groups on overhangs for the capture of silver nanoparticles. On the other hand, target miRNA induced strand displacement reaction produces abundant specific DNA strands, which help the DNA structural transition from triangle to TPF. Amino groups are thus hidden and the declined silver stripping current can be used for the evaluation of target miRNA concentration. This biosensor exhibits excellent analytical performances and successfully achieves analysis of exosomal miRNAs from cells and clinical serum samples.

Profiles of lipid, protein and microRNA expression in exosomes derived from intestinal epithelial cells after ischemia-reperfusion injury in a cellular hypoxia model.

Intestinal ischemia-reperfusion injury leads to proinflammatory responses via gut-derived mediators, and accumulating evidence suggests that exosomes secreted by intestinal epithelial cells are involved in the development of systemic inflammation. Studies have reported changes in protein, lipid, and microRNA (miRNA) expression; however, considering the different experimental conditions, information on the relationships among these biomolecules remains insufficient. The aim of this study was to elucidate the multiple changes that simultaneously occur in exosomes after ischemic stimulation. Here, differentiated human intestinal Caco-2 cells were exposed to 95% air (normoxia group) or 5% O2 (hypoxia group) for 6 h. Cells in each group were subsequently incubated for 24 h in an atmosphere of 5% CO2 plus 95% air. The conditioned medium of each group was collected for isolating intestinal epithelial cell-derived exosomes. Together with proteome analyses, lipid analyses, and miRNA quantification, biological functional assays were performed using monocytic NF-κB reporter cells. Lipid metabolism-related protein expression was upregulated, miRNA levels were slightly altered, and unsaturated fatty acid-containing lysophosphatidylcholine concentration increased after hypoxia and reoxygenation injury; this suggested that the changes in exosomal components associated with ischemia-reperfusion injury activates inflammation, including the NF-κB pathway. This study elucidated the multiple changes that co-occur in exosomes after ischemic stimulation and partially clarified the mechanism underlying exosome-mediated inflammation after intestinal ischemic recanalization.

An overview of biochemical technologies for the cancer biomarker miR-21 detection.

In recent years, the incidence of cancer has continuously increased, in which various miRNAs have been proposed as biomarkers for the early screening of cancer patients. As a consequence, the development of accurate methods for miRNA quantification has become a major research challenge worldwide. As one of the first discovered oncogenic miRNAs, microRNA-21 (miR-21) has been highlighted for its critical role in cancers. This review describes the main techniques currently available for miR-21 detection, compares the differences of the methods and the amplification strategies,and provides an overview of the state of knowledge in the field.

Non-Coding RNA Investigations in Cutaneous Melanoma: A Step forward in Discovering Novel Biomarkers

Non-Coding RNA Investigations in Cutaneous Melanoma: A Step forward in Discovering Novel Biomarkers

The miR-20a/miR-92b Profile Is Associated with Circulating γδ T-Cell Perturbations in Mild Psoriasis.

Psoriasis vulgaris (PV) is an autoinflammatory dermatosis of unknown etiology. Current evidence suggests a pathogenic role of γδT cells, but the growing complexity of this population has made the offending subset difficult to pinpoint. The work on γδTCRint and γδTCRhi subsets, which express intermediate and high levels of γδTCR at their surface, respectively, is particularly scarce, leaving their inner workings in PV essentially unresolved. We have shown here that the γδTCRint/γδTCRhi cell composition and their transcriptom are related to the differential miRNA expression by performing a targeted miRNA and mRNA quantification (RT-qPCR) in multiplexed, flow-sorted γδ blood T cells from healthy controls (n = 14) and patients with PV (n = 13). A significant loss of miR-20a in bulk γδT cells (~fourfold decrease, PV vs. controls) largely mirrored increasing Vδ1-Vδ2- and γδintVδ1-Vδ2- cell densities in the bloodstream, culminating in a relative excess of γδintVδ1-Vδ2- cells for PV. Transcripts encoding DNA-binding factors (ZBTB16), cytokine receptors (IL18R1), and cell adhesion molecules (SELPLG) were depleted in the process, closely tracking miR-20a availability in bulk γδ T-cell RNA. Compared to controls, PV was also associated with enhanced miR-92b expression (~13-fold) in bulk γδT cells that lacked association with the γδT cell composition. The miR-29a and let-7c expressions remained unaltered in case-control comparisons. Overall, our data expand the current landscape of the peripheral γδT cell composition, underlining changes in its mRNA/miRNA transcriptional circuits that may inform PV pathogenesis.

A platform for high-resolution immune liquid biopsy analysis to predict response in patients with renal cell carcinoma treated with nivolumab or cabozantinib: Preliminary data from I-RENE trial (Meet-URO 8 study).

712 Background: Nivolumab, an immune checkpoint inhibitor of PD-1, demonstrated a significant OS benefit in patients with metastatic renal cell carcinoma (mRCC), in progression after a previous line of therapy with anti-VEGFR agents. However, the features of effective immune response and predictive biomarkers of clinical benefit to PD-1 blockade have not yet been recognized. Methods: I-RENE is a prospective translational multicenter Italian study of a real-life mRCC patients treated with nivolumab or cabozantinib after failure of therapy with anti-VEGFR agents. 82 patients were enrolled from December 2018 to August 2022 (nivo 60, cabo 22), with blood samples obtained at baseline and at different time points in both treatment groups. An extended concept of "immune liquid biopsy" is being applied to the study, consisting in the phenotypic and transcriptional profile of lymphoid and myeloid subsets, immune-related miRNA quantification, cyto/chemo-kinome and RNAseq of extracellular vesicle. Results: Multiparametric flow cytometry, performed to monitor the blood frequency and different myeloid and lymphoid cells, show that monocyte subsets (classical, intermediate and non-classical CD14+ cells), monocytic myeloid derived suppressor cells (MDSC, such as CD14+HLA-DRneg and CD14+PD-L1+) and polymorphonucleate (PMN)-MDSC, remain either stable or increase during treatment; concomitantly, CD8+PD-1+ T cells (detected by anti-nivolumab IgG4) increment frequency, acquire the effector CD45RA-CCR7+ phenotype and express the proliferating marker Ki67. Patients receiving cabozantinib display instead a remarkable decrease of all myeloid cell subsets, paired by the boost of cytotoxic CD3-CD16+CD56dim NK cells and more marginally of CD8+PD1+ cells, Preliminary correlations indicate that clinical benefit of nivolumab seems to cluster with the lack of CD14+ cells and M-MDSC increase and blood frequency of total, and the boost in CD8+CD45RA-CCR7+Ki67+ effector T cells. In contrast, the cabozantinib-induced immune modulation occurring in patients treated with does not associate with clinical response Conclusions: This first set of data indicate blood as promising source of dynamic biomarkers for the development of algorithms predicting response to PD-1 blockade. Furthermore, the results so far collected suggest that the potent immunomodulation induced by cabozantinib on the immunosuppressive myeloid compartment may not lead to any tumor control in the absence of specific immune stimulation by checkpoint inhibitors. This study was supported by the Italian Ministry of health (RF-2016_02363001). Clinical trials.gov: NCT04891055 Clinical trial information: NCT04891055 .

MiR-148b as a Potential Biomarker for IgA Nephropathy

Background: IgA nephropathy (IgAN) is one of the most common glomerular diseases worldwide. Approximately 25 percent of IgAN patients reach the kidney failure stage within twenty years of diagnosis. The histopathological examination of kidney biopsy is needed to confirm the diagnosis of IgAN. microRNA (miRNA) is a small RNA that plays an important role at the post-transcriptional level by downregulating mRNAs (messenger RNA). We tried to establish a miRNA-based biomarker for IgAN. Methods: We recruited 30 IgAN patients and 15 healthy controls as study participants after taking their informed written consent. A real-time PCR-based method was used for the absolute quantification of miRNAs. A logistic regression method and receiver operating characteristic analysis were performed to find the diagnostic and prognostic accuracy of miR-148b and let-7b for IgAN in histopathological MEST-C scores. Results: miR-148b and let-7b levels were higher in IgAN patients compared to the healthy controls. miR-148b was positively correlated with glomerular filtration rate (GFR) and negatively correlated with segmental glomerulosclerosis, tubular atrophy/interstitial fibrosis (T), and blood pressure (BP). The sensitivity, specificity, and area under the curve (AUC) of the receiver operating characteristic (ROC) for miR-148b against T were 0.87, 0.77, and 0.85, respectively. The threshold value of the miR-148b copy number was 8479 to differentiate the severe condition of IgAN. Conclusion: miR-148b can be used as a potential biomarker for IgAN.