A novel DSN‐based fluorescence assay for MicroRNA‐133a detection and its application for LVH diagnosis in maintenance hemodialysis patients

Abstract Left ventricular hypertrophy (LVH) is the most powerful predictor of cardiovascular mortality in maintenance hemodialysis (MHD) patients. Circulating microRNA‐133a (miR‐133a) was reported to be a potential biomarker for LVH in MHD patients. The aim of this experiment is to establish a novel DSN (duplex‐specific‐nuclease)‐based fluorescence assay for the ultrasensitive detection of miR‐133a and investigate its application for LVH diagnosis in MHD patients. The results indicate DSN enzyme combined with ultrathin metallic MoS2 nanosheets presents high sensitivity, specificity, and low fluorescence background for miR‐133a detection. Then, circulating miR‐133a levels in plasma from 40 MHD patients and 20 healthy controls are analyzed by such assay. The levels of miR‐133a are down‐regulated in MHD patients with LVH compared to MHD patients without LVH and healthy controls, and the ROC (receiver operating characteristic) curve shows strong separation between MHD with LVH patients and MHD without LVH patients. Furthermore, the liner regression analysis shows negative correlation of miR‐133a level and interventricular septum thickness (IVS) as well as left ventricular mass index (LVMI), the indicators of LVH. Therefore, our findings reveal DSN‐based fluorescence assay for miR‐133a is suitable for LVH diagnosis in MHD patients.

133a levels in plasma from 40 MHD patients and 20 healthy controls are analyzed by such assay. The levels of miR-133a are down-regulated in MHD patients with LVH compared to MHD patients without LVH and healthy controls, and the ROC (receiver operating characteristic) curve shows strong separation between MHD with LVH patients and MHD without LVH patients. Furthermore, the liner regression analysis shows negative correlation of miR-133a level and interventricular septum thickness (IVS) as well as left ventricular mass index (LVMI), the indicators of LVH. Therefore, our findings reveal DSN-based fluorescence assay for miR-133a is suitable for LVH diagnosis in MHD patients.

K E Y W O R D S
DSN-based fluorescence assay, left ventricular hypertrophy, maintenance hemodialysis,

MicroRNA-133a
ECG is somewhat limited by its poor sensitivity in dialysis patients. [7][8][9] Echocardiography represents a valuable method for the detection of LVH due to its wide availability and its relatively low cost; however, the main limitation is represented by the low spatial resolution and reproducibility. 10 Although new markers of cardiac risk such as cardiac hormone brain natriuretic peptide (BNP), troponin T (cTnT), and troponin I (cTnI) draw extensive attention, the detection of these biomarkers is still scarcely applied for clinical practice in dialysis patients. 11 MicroRNAs (miRNAs) are endogenous small non-coding RNAs (18-25 nucleotides) that regulate gene expression at the post-transcriptional level. 12 MiRNAs have been shown to play important roles in multiple biological processes and aberrant expressions of which in tissues and cells can promote various diseases. 13,14 Thus, miRNAs in circulation have been proposed as being useful in diagnostics as biomarkers for diseases and different types of disease.
Existed evidence has shown that miRNAs are key modulators of cardiovascular function, and several of them have been confirmed to be useful biomarkers for cardiovascular diseases. 15,16 MiR-133a with high expression in cardiac and skeletal muscle is dysregulated during heart hypertrophy and failure. Several studies have reported circulating miR-133a is changed in patients with cardiac dysfunction.
Study from Wen et al have revealed that miR-133a is an effective biomarker for prediction of cardiac hypertrophy in MHD patients.
The conventional used method for microRNA detection is qRT-PCR (quantitative real-time polymerase chain reaction); however, multiple sample processing steps and unstable characters limit its application in clinical. DSN (duplex-specific-nuclease)-based fluorescence assay has emerged as a versatile component in bioanalytical strategy for miRNA detection owing to high sensitivity, specificity, short assay time, and low contamination risk. DSN shows a strong preference for digesting DNA strands in double-stranded DNA or in DNA-RNA hybrid duplexes. Additionally, it discriminates between fully matched and slightly mismatched short duplexes. 17 In the present study, a novel DSN-based fluorescence assay is established for miR-133a detection and its application for LVH diagnosis in MHD patients is further evaluated.  Table 1. Blood samples are immediately collected using EDTA (ethylene diamine tetraacetic acid) tubes. All blood samples are centrifuged at 3500 g for 10 minutes within 4 hours of collection for the isolation of plasma. Then, plasma is transferred to RNase-free tubes and stored at −80℃.

| DSN-based fluorescence assay
Two-steps reaction steps are conducted in this assay. In the first-step reaction, sample from plasma (10 μL) is added to reaction mixture

| Statistical analysis
Biostatistical analyses are conducted by the SPSS 16.0 software package (IL, USA). All experiments are repeated three times, and data are presented as means ± SD. Difference between groups is analyzed with independent two-sample t test or Mann-Whitney U test. A P < .05 is accepted as statistically significant.

| Clinical analysis of the study population
A total of 60 subjects including 40 ESRD patients with MHD treatment and 20 healthy controls without kidney and cardiac diseases are studied. Clinical characteristics of the included subjects are presented in Table 1. According to the LVMI value, forty MHD patients are divided into two groups with or without LVH. There is no significant difference between two groups with common clinical characteristics except for CRP (C-reactive protein) and LVMI.

| Establishment of DSN-based fluorescence assay for miR-133a
To determine the circulating miR-133a levels, a novel fluorescence assay combined DSN enzyme with ultrathin metallic MoS 2 nanosheets is designed, which is improved according to the previous study. 19 The basis of such assay is shown as Figure 1, upon the addition of target miR-133a, the ssDNA probe with FAM-labeled hybridizes to the miR-133a to form a heteroduplex of DNA/RNA.
The cleavage of probe from DNA/RNA duplex occurs when DSN is added, which triggers another hybridization, cleavage and release, and leads to a large amplification. After reaction, the product incubates with ultrathin metallic MoS 2 nanosheets and the cleaved short FAM-labeled oligonucleotide fragments cannot be absorbed and remains a strong signal. Then, the sensitivity, linear dynamic range, and specificity are analyzed. As shown in Figure 2, the limit of detection (LOD) is approximately 1 pM according to the standard curve, and the signal from miR-133a is stronger than that of miR-133b, indicating high sensitivity and specificity of such assay.

| Evaluation of DSN-based fluorescence assay for LVH diagnosis
MiR-133a was previously reported to be a potential biomarker for cardiac hypertrophy in MHD patients, and in this study, the diagnostic value of miR-133a detection based on fluorescence assay in such disease is analyzed. As shown in Figure 3A  potential biomarker for cardiac hypertrophy in MHD patients. 25 In this study, a novel DSN-based fluorescence assay is established for miR-133a detection and applied in the diagnosis of LVH in MHD patients.
High sensitivity efficiency makes qRT-PCR to be the widely used method for microRNA detection, while multiple sample processing limits its application in diagnosis of heart failure. 26 Methods based on fluorescence are recently developed for rapid detecting microRNAs; however, low sensitivity and specificity limit these methods. 27