Differential expression of microRNAs in renal ...

Transplant Immunology 33 (2015) 1–6

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Differential expression of microRNAs in renal transplant patients with acute T-cell mediated rejection☆ Ehsan Soltaninejad a,b, Mohammad Hossein Nicknam b,c, Mohsen Nafar d, Pedram Ahmadpoor d, Fatemeh Pourrezagholi d, Mohammad Hossein Sharbafi b, Morteza Hosseinzadeh b, Farshad Foroughi c, Mir Saeed Yekaninejad e, Tayyeb Bahrami c, Ehsan Sharif-Paghaleh b, Aliakbar Amirzargar b,c,⁎ a

Department of Immunology, School of Medicine, Birjand University of Medical Sciences, Birjand, Iran Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran c Molecular Immunology Research Center, Tehran University of Medical Sciences, Tehran, Iran d Chronic Kidney Disease Research Center and Department of Nephrology, Shahid Labbafinejad Medical Center and Shahid Beheshti University of Medical Sciences, Tehran, Iran e Department of Epidemiology and Biostatistics, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran b

a r t i c l e

i n f o

Article history: Received 6 December 2014 Received in revised form 12 May 2015 Accepted 13 May 2015 Available online 20 May 2015 Keywords: Acute T-cell mediated rejection Biomarker MicroRNA Renal transplantation

a b s t r a c t Background: MicroRNAs (miRNAs) regulate most of encoding genes and protein. In this study, we aimed to investigate the expression levels of miR-142-5p, miR-142-3p, miR-155 and miR-223 in paired biopsy and peripheral blood mononuclear cell (PBMC) samples of renal allograft recipients with acute T-cell mediated rejection (ATCMR), compared with normal allografts (NA). Methods: In this study, the expression levels of individual miRNAs were determined in biopsy and PBMC samples of 17 recipients with ATCMR and 18 recipients with NA. Results: Our results showed that the intragraft expression levels of all studied miRNAs were significantly higher in ATCMR than NA. However, regarding the PBMC samples, miR-142-3p and miR-223 were significantly increased in ATCMR than NA. Receiver operating characteristic (ROC) analysis showed that miR-142-5p, miR-142-3p, miR-155 and miR-223 in biopsy samples and miR-142-3p and miR-223 in PBMC samples could discriminate ATCMR from NA recipients. Conclusion: It has been reported that high intragraft expressions of miRNAs have a profound role in the pathogenesis of ATCMR process. Our results showed that high expression of all the studied miRNAs in biopsies and miR-142-3p and miR-223 in PBMC samples could be used as suggestive diagnostic tools to discriminate ATCMR patients from NA. © 2015 Elsevier B.V. All rights reserved.

1. Introduction

Abbreviations: miRNA, MicroRNA; PBMC, Peripheral blood mononuclear cell; ATCMR, Acute T-cell mediated rejection; AR, Acute rejection; NA, Normal allograft; ROC, Receiver operating characteristic; ESRD, End-stage renal disease; CAF, Chronic allograft failure; FC, Fold change; IQR, Inter quartile range; AUC, Area under the curve; CI, Confidence interval. ☆ Authorship: Ehsan Soltaninejad participated in sample collection, performing the experiments and writing the paper draft. Mohammad Hossein Nicknam participated in designing the study and editing the manuscript. Mohammad Hossein Sharbafi participated in acquisition of clinical data and performing the experiments. Mohsen Nafar and Pedram Ahmadpoor were the responsible nephrologist of this study and participated in acquisition of clinical data and interpreting the data. Morteza Hosseinzadeh, Farshad Foroughi and Tayeb Bahrami participated in sample collection. Mir Saeed Yekaninejad participated in statistical analyzing the data. Ehsan Sharif-Paghaleh participated in editing the paper. Aliakbar Amirzargar is the main project manager, and participated in designing the study and editing the manuscript. ⁎ Corresponding author at: Immunogenetics Lab, Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran. Tel.: +98 21 8895 3009; fax: +98 21 6642 2337. E-mail addresses: [email protected], [email protected] (A. Amirzargar).

http://dx.doi.org/10.1016/j.trim.2015.05.002 0966-3274/© 2015 Elsevier B.V. All rights reserved.

Solid organ transplantation is the most effective treatment for end-stage organ failure. However, the rejection of transplanted organs remains as a main factor which impacts on the organ transplant effectiveness [1]. Renal transplantation, compared with dialysis, improved health-related quality of life of end-stage renal disease (ESRD) patients and it is associated with a high cost-effectiveness ratio [2]. However, acute rejection (AR) of renal allografts is an immune response that may occur at any time after renal transplantation [3–7]. Furthermore, AR is the most important risk factor leading to chronic allograft nephropathy in renal allograft recipients [8], that remains the major cause of graft loss [9]. Currently, tissue biopsy is considered as a gold standard for diagnosis of allograft rejection [10]; however, it is highly invasive and requires risky procedures along with variation in reports of renal biopsies between pathologists [11,12]. The lack of non-invasive, accurate and specific diagnostic assays for graft rejection is a major difficulty in the management of renal transplant recipients [13,14]. Therefore, development of

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sensitive and accurate biomarkers in the graft as well as non-invasive immunological biomarkers accessible in blood and other biological fluids corresponding to graft rejection is necessary. MicroRNAs (miRNAs) are evolutionary conserved non-coding RNA molecules, small (≈ 19–25 nucleotides in length) that regulate gene expression by affecting the translational repression or by mRNA degradation [15]. miRNAs functions by binding through the 3′ untranslated region (3′UTR) of target mRNA. Also, it has been shown that miRNAs play important roles in many biological processes such as development [16], cell proliferation, differentiation, apoptosis, fat metabolism and oncogenesis [17]. Moreover, MiRNAs are stably expressed in biologically body fluids such as urine, saliva, serum, plasma and other body fluids. Furthermore, expression levels of miRNAs are correlated with human diseases such as various types of kidney diseases, cancers, stroke, heart diseases or in physiological states such as pregnancy [18–22]. This suggests that miRNAs could be used for non-invasive diagnosis and monitoring of a variety of diseases. In this study, we focused on miR-142-5p, miR-142-3p, miR-155 and miR-223 because of their specificity to hematopoietic lineage [23, 24]; meanwhile there are some evidences on over-expression of these miRNAs in biopsy/PBMC samples of AR patients [24,25]. To our knowledge, the expression analysis of miR-142-3p and miR-155 in PBMC samples of ATCMR recipients and NA has not been performed yet. Moreover, this study is the first study that evaluated expression of these miRNAs simultaneously in biopsy and PBMC samples of renal transplant patients.

Table 1 Demographic and clinical characteristics of renal allograft recipients.

Number of patients Male (n; %) Female (n; %) Age (years; min–max) Types of allografts Deceased Living Types of donors Related donor Unrelated donor Recipient CMV (pos) CMV disease Donor CMV (pos) Donor HCV (pos) Donor HBS Ag (pos) Donor HBC Ab (pos) Donor HIV (pos) Blood creatinine level (mg/dl) Date of biopsy (months post-transplant)

Normal allograft

Acute T-cell mediated rejection

18 11 (61.1%) 7 (38.9%) 55 (34-67)

17 10 (58.8%) 7 (41.2%) 47 (21-61)

1 17

3 14

1 17 1 0 3 0 0 1 0 1.24 ± 0.21 13.66 ± 4.21

0 17 2 0 2 0 0 1 0 5.18 ± 1.49 11.65 ± 3.18

solution and then transferred to 4 °C overnight then stored at −80 °C until RNA extraction. 3.2. RNA isolation and cDNA synthesis

2. Objective

3. Materials and methods

Total RNA samples were isolated from renal biopsies or PBMCs using the mirVana miRNA isolation kit according to the manufacturer's instructions (Ambion). The concentration and quantity of total RNA were measured at 260 nm and 280 nm (A260/280) using a NanoDrop 2000 spectrophotometer (NanoDrop Technologies). Total RNA (10 ng) from ATCMR and NA subjects were reverse transcribed into cDNA using specific primers and reagents provided by TaqMan® MicroRNA Assays (Applied Biosystems, Foster City, CA, USA) and Taqman® MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) respectively.

3.1. Patients and sample collection

3.3. Quantitative real-time PCR

The study was approved by the Tehran University of Medical Sciences ethical committee; each patient signed written informed consent. In this cross sectional study, the patients were recruited from Shahid Labbafinejad Medical Center. Biopsy and PBMC samples were obtained from renal transplant patients with ATCMR (n = 17) and NA (n = 18) without histopathological evidence of rejection as controls. The exclusion criteria were BK virus infection, calcineurin inhibitor nephrotoxicity, urinary tract obstruction and patients with ≥2nd transplantation. The inclusion criteria were patients undergoing their first transplantation and signing the inform consent. Renal allograft biopsies were classified according to the 2009 update Banff classification criteria [26]. In our study, all 17 AR subjects developed a T-cell mediated rejection (12 IA and 5 IB) in category 4 based on the Banff diagnostic category [26]. The pathologists were blinded in their evaluation of the biopsies to determine the presence of rejection. Additional demographic and clinical characteristic information of the patients are shown in Table 1. Renal allograft biopsy samples were immediately placed in RNAlater solution (Ambion) according to the manufacturer's instructions. In summary, biopsy samples were stored at 4 °C overnight to allow penetration of the solution into tissues and subsequently stored at − 80 °C until RNA extraction. Peripheral whole blood on EDTA were collected at the time of obtaining biopsy samples from the same patients with ATCMR or NA. PBMCs were isolated from whole blood of ATCMR and NA recipients using standard Ficoll density-gradient centrifugation. To protect the RNA from degradation, PBMCs were kept in RNAlater

Real-time quantitative PCR was performed to measure the expression levels of microRNAs with the TaqMan® MicroRNA Assays (Applied Biosystems, Foster City, CA, USA), using probes for miR-142-5p (assay ID: 002248), miR-142-3p (assay ID: 000464), miR-155 (assay ID: 002623), miR-223 (assay ID: 002295), RNU44 (Assay ID: 001006) on a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The data were normalized using RNU44 as endogenous control.

The aim of this study was to investigate, whether ATCMR is associated with changes in miRNA expression levels of miR-142-5p, miR-142-3p, miR-155 and miR-223 within biopsies and paired PBMC samples. If so, whether the expression levels of these miRNAs in biopsy and PBMC samples of ATCMR could be used as diagnostic and predictive biomarkers for ATCMR.

3.4. Data analysis Threshold cycle number was used to calculate the relative expression between samples. We used the ΔΔCt (cycle threshold) method in which relative expression = 2−ΔΔCt, where ΔΔCt = (ΔCt of ATCMR) − (ΔCt of NA). Non-parametric Mann–Whitney test was used for comparing miRNA expression between acute T-cell mediated rejection and normal allograft and between T-cell mediated grade IA and IB rejection groups. Non parametric Spearman correlation was used for calculating the correlation between miRNAs in biopsies and PBMCs for normal allograft and acute T-cell mediated rejection groups. Receiver operating characteristic (ROC) analysis was applied to find the best cut off values of miRNAs for diagnosing the acute T-cell mediated rejection from non-rejected patients. Also multivariate ROC curve analysis was used to find the discriminative value of all studied miRNAs for discriminating acute T-cell mediated rejection from non-rejected

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patients. The Epi and pROC packages in R software were used for multivariate ROC curve. All tests were two sided and a P b 0.05 was considered as statistically significant and highly significant when P b 0.01.

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Table 2 Expression levels of individual miRNA measured in biopsy and PBMC samples. Normal allograft FCa median (IQRb)

Acute T-cell mediated rejection FC median (IQR)

P-value

0.971 (0.801–1.779) 1.123 (0.398–2.760) 1.103 (0.657–2.072) 0.961 (0.675–1.359) 1.100 (0.884–1.630) 0.820 (0.660–1.870) 1.105 (0.948–1.654) 1.049 (0.694–1.565)

6.958 (6.635–7.867) 4.906 (3.249–8.183) 5.013 (4.171–6.505) 3.115 (2.443–4.794) 1.554 (1.367–1.734) 2.161 (1.396–3.231) 1.658 (1.467–1.992) 2.441 (1.527–3.706)

b0.0001 b0.0001 b0.0001 0.001 0.112 0.023 0.059 0.003

4. Results 4.1. Identification of microRNA expression levels in biopsy samples of renal transplant recipients Using TaqMan® MicroRNA Assays, the expression of miR-142-5p, miR-142-3p, miR-155 and miR-223 was examined in biopsies from 17 ATCMR compared with 18 NA recipients. Biopsy samples were classified into two groups; ATCMR and NA, based on the histopathological reports according to the 2009 update Banff classification criteria [26]. In our study miR-142-5p (FC = 6.95, p b 0.0001), miR-142-3p (FC = 4.90, p b 0.0001), miR-155 (FC = 5.01, p b 0.0001), and miR-223 (FC = 3.11, p = 0.001) were expressed at significantly higher levels in the biopsy samples of ATCMR compared to NA biopsies (Fig. 1 and Table 2). Furthermore, there was no significant difference in the miRNAs expression levels between the acute T-cell mediated rejection grades (IA and IB) in the biopsies scored according to Banff grade (p N 0.30). The ROC curve analysis indicated that miR-142-5p, miR-142-3p, miR-155, and miR-223 could reliably distinguish ATCMR from NA by assessment of their expression level in biopsy samples (Table 3). Also, our study showed that miR-142-5p had the highest AUC (area under curve) of 1.00, CI95% = [1.00 to 1.00]; p b 0.0001, together with the highest sensitivity (100%) and highest specificity (100%). This was followed by miR-155 (AUC = 0.99, CI95% = [0.96 to 1.00]; p b 0.0001) with 100% sensitivity and 92% specificity, and miR142-3p (AUC = 0.97, CI95% = [0.90 to 1.00]; p b 0.0001) with 90% sensitivity and 100% specificity. The miR-223 (AUC = 0.94, CI95% = [0.84 to 1.00]; p = 0.001) had 90% sensitivity and 80% specificity. These sensitivity and specificity scores were obtained in biopsy samples (Table 3). All miRNAs were combined in a multivariate ROC curve model. Discrimination value was maximum in biopsy samples when miRNAs were combined (Sensitivity and Specificity = 100%) the independent effect of each of the miRNAs was also estimated in multivariate ROC curve model. miR-142-5p has the highest contribution for detection of acute T-cell mediated rejection in the biopsies (Fig. 1). 4.2. Identification of microRNA expression levels in PBMC samples of renal transplant recipients The expression levels of miR-142-5p, miR-142-3p, miR-155, and miR-223 were investigated in PBMC samples isolated from peripheral blood of patients undergoing biopsy procedure using TaqMan® MicroRNA Assays. Our results indicated that miR-223

Biopsy

PBMCs

a b

miR-142-5p miR-142-3p miR-155 miR-223 miR-142-5p miR-142-3p miR-155 miR-223

Fold change. Interquartile range.

(FC = 2.44, P = 0.002) and miR-142-3p (FC = 2.16, P = 0.023) were significantly upregulated in PBMC samples from ATCMR patients. Although fold changes of 1.59 and 1.82 were calculated for miR-142-5p and miR-155 respectively; but, miR-155 was borderline significant (p = 0.059) and miR-142-5p did not reach statistical significance (p = 0.112), (Fig. 2 and Table 2). Similarly, there was no significant difference in the miRNAs expression levels in the PBMCs between the biopsy proven acute T-cell mediated rejection subjects with the different grades (IA and IB) scored based on Banff grade (p N 0.30). The ROC curve analysis displayed well discrimination between two groups with expression measurements of miR-223 (AUC = 0.89, CI95% = [0.75 to 1.00]; p = 0.003) that had the highest sensitivity (100%) and highest specificity (76%) and miR-142-3p (AUC = 0.80, CI95% = [0.59 to 1.00]; p = 0.023) with 100% sensitivity and 65% specificity respectively in PBMC samples. Moreover, PBMC levels of miR-155 could have diagnostic importance, but with a lower level of accuracy (AUC = 0.75, CI95% = [0.52 to 0.98]; p = 0.059) while miR-142-5p was unable to discriminate ATCMR group from NA (Table 3). Multivariate ROC curve model with all miRNAs also had high discriminating value in PBMC samples (Sensitivity = 90%, Specificity = 85%) and miR-223 was the most important miRNA for detecting ATCMR in the PBMCs (Fig. 2).

Fig. 1. miRNA expression in the graft. A) miR-142-5p expression was significantly increased (p b 0.0001) in acute T-cell mediated rejection (ATCMR) biopsies from normal allografts (NA). B) miR-142-3p expression was significantly increased (p b 0.0001). C) miR-155 expression was significantly increased (p b 0.0001). D) miR-223 expression was significantly increased (p = 0.001). The data were normalized using RNU44 as the endogenous control and the relative expression was calculated using ΔΔCt method.

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Table 3 ROC curve analysis of real-time qPCR data. Sample

miRNAs

Cutoff point

Sensitivity %

Specificity %

AUCa (95% CIb)

P-value

Biopsy

miR-142-5p miR-142-3p miR-155 miR-223 miR-142-5p miR-142-3p miR-155 miR-223

3.397 2.953 2.366 1.442 1.364 0.955 1.246 1.273

100 90 100 90 80 100 100 100

100 100 92 80 70 65 62 76

1.00 (1.00–1.00) 0.97 (0.90–1.00) 0.99 (0.96–1.00) 0.94 (0.84–1.00) 0.71 (0.46–0.96) 0.80 (0.59–1.00) 0.75 (0.52–0.98) 0.89 (0.75–1.00)

b0.0001 b0.0001 b0.0001 0.001 0.112 0.023 0.059 0.003

PBMCs

a b

Area under the curve. Confidence interval.

4.3. Correlation between expression levels of individual miRNAs in the biopsy with those in the blood Calculation of Spearman correlation of miRNAs between biopsy and PBMC samples showed that there was a mild to high positive correlation for miR-142-5p and miR-155 in normal allografts (r = 0.733, p = 0.016); (r = 0.345, p = 0.328) and acute T-cell mediated rejection groups (r = 0.139, p = 0.701); (r = 0.394, p = 0.260) respectively. However, correlations for miR-142-3p and miR-223 between biopsy and PBMC were negative in normal allografts (r = −0.20, p = 0.580); (r = −0.430, p = 0.214) and acute T-cell mediated rejection groups (r = −0.455, p = 0.187); (r = −0.248, p = 0.489) respectively.

5. Discussion Acute rejection is an immune response that has a profound effect on renal transplantation outcome [1,3]. Currently, evaluation of graft function mainly relies on histological evidence of the biopsy taken, when physiological parameters indicate probable renal dysfunction [12,13].

MicroRNAs (miRNAs) play an important role in many biological processes, while they could regulate the expression of genes implicated in immune response [20,27,28]. Furthermore, some studies showed that miR-142, miR-155, miR-223, miR-150 and miR-144 are highly specific for hematopoietic cell lineage [21]. A differential expression of miRNAs has been shown in patients with acute rejection [23,25,29,30], interstitial fibrosis [31,32], operationally tolerant patients [33], and chronic antibody mediated rejection [24]. To our knowledge, until now miR142-3p and miR-155 expression analysis in the blood of patients with acute T-cell mediated rejection has not been performed yet. Moreover, this is the first study that evaluated expression levels of miRNAs simultaneously in biopsy and PBMC samples of the same renal transplant recipients with acute T-cell mediated rejection. Sui et al. found a differentially expressed miRNA profile in acute rejection of renal allograft and reported 8 miRNAs that were upregulated and 12 miRNA that were downregulated [30]. Anglicheau and his

Fig. 2. miRNA expression in PBMC samples. A) miR-142-5p expression was not significantly increased (p = 0.112) in PBMC samples from acute T-cell mediated rejection (ATCMR) patients compared with normal allografts (NA). B) miR-142-3p was significantly increased (p = 0.023) in PBMC samples. C) miR-155 was increased borderline significant (p = 0.059) in PBMC samples of acute T-cell mediated rejection. D) miR-223 was significantly increased (p = 0.003) and had the highest expression levels in PBMC samples of acute T-cell mediated rejection from normal allografts.

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colleagues have demonstrated different miRNA expression in biopsy during acute rejection. Furthermore, they analyzed the association of altered miRNAs with intragraft levels of mRNA CD3/CD20 (immune related) and NKCC-2/USAG-1 (tubule related). They have revealed the up-regulated miR-142-5p, miR-155 and miR-223 were highly associated with the intragraft levels of mRNA for CD3 and CD20. Anglicheau et al. used both living and deceased donor and normal allograft tissues in their study [23]. Whereas, Sui et al. studied miRNA expression levels in a very small population and used normal kidney tissues (not normal allograft tissues) as controls. Moreover, these two studies might use different array panels in their evaluations. MicroRNAs selected for this study have been reported differentially expressed in acute rejection and they play key roles in immune responses. miR-142 was expressed in higher levels in naive T cells than in differentiated Th1 and Th2 cells [34–36]. Our result revealed elevated levels of miR-142-5p and miR-142-3p in biopsy samples of ATCMR patient's results. Also, miR-142-3p was significantly overexpressed in PBMC samples of ATCMR biopsies. Scian et al. have identified differential expression of miR-142-3p in biopsies and urinary cells of renal allografts with evidence of interstitial fibrosis and tubular atrophy [32]. miR-155 resides within an exon of non-coding gene bic (B-cell integration cluster) that overexpressed after stimulation of antigen receptors in lymphocytes or through TLR (Toll-like receptor) stimulation in B-cells, dendritic cells and macrophages [37]. Therefore, miR-155 plays an important role in regulating immune responses. Furthermore, this miRNA regulates the activation-induced cytidine deaminase [38,39] which is necessary to induce somatic hypermutation in immunoglobulin class switching of B cells [40,41]. In this regard, our study showed that miR-155 was higher in biopsy and PBMC samples of patients with ATCMR compared with NA recipients which suggested that miR-155 may be implicated in immunological prospects of acute T-cell mediated rejection. miR-223 has been found strongly overexpressed in granulocytes, platelets and monocytes [42]. Liu and his colleagues revealed higher expression of this miRNA in PBMC samples of AR patients [25]. In our study, miR-223 was higher in biopsy samples as well as PBMC samples of patients with ATCMR compared with NA. Our results confirmed high expression levels of this miRNA in PBMC samples of ATCMR patients and this miRNA may have important roles in acute T-cell mediated rejection mechanisms. ROC curve analysis was performed to determine the predictive value of miRNAs in biopsy and PBMC samples. We found that ATCMR can be diagnosed very accurately with high level of precision using the intragraft expression level of miR-142-5p (100% sensitivity and 100% specificity, p b 0.0001), miR-155 (100% sensitivity and 92% specificity, p b 0.0001), miR-142-3p (90% sensitivity and 100% specificity, p b 0.0001), and miR-223 (90% sensitivity and 80% specificity, p = 0.001) (Tables 2, 3). While miR-223 (100% sensitivity and 76% specificity, p = 0.003) and miR-142-3p (100% sensitivity and 65% specificity, p = 0.23) could be able to predict ATCMR using expression levels of these miRNAs in PBMC samples. Calculation of Spearman correlation of MicroRNAs between biopsy and PBMC samples showed that there were mild to high positive correlation for miR-142-5p and miR-155 in normal allograft and acute T-cell mediated rejection groups (r = 0.13 to 0.73). However, correlations for miR-142-3p and miR-223 between biopsy and PBMC were negative (r = −0.20 to −0.45). Using this analysis it is suggested that miR1425p and miR-155 in PBMC could be predictive for the pathological process in allograft. In conclusion, cell infiltration might be the cause of the high expression of miR-142-5p, miR-142-3p, miR-155 and miR-223 in allografts, since during acute rejection numerous cell types including B cells and T cells and dendritic cells infiltrate into allografts [23]. However, in the PBMCs, the cause of large overlap between normal allograft and ATCMR might be the fewer amount of specific immune cells in the circulation than the cells infiltrated into graft which are involved in

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graft rejection. This study confirmed detection and differential expression of miR-142-5p, miR-142-3p, miR-155 and miR-223 in biopsy and PBMC samples of patients diagnosed with ATCMR. Since the early diagnosis of AR in renal transplant recipients is very important in renal transplant recipients' status, we validated that the expression levels of miR-142-5p, miR-142-3p, miR-155 and miR-223 in biopsies could be used as a diagnostic and predictive biomarkers for acute T-cell mediated rejection of renal allografts. Furthermore, miR-223 and miR-142-3p in PBMC samples could be used as non-invasive diagnostic and predictive biomarkers of ATCMR. As the miRNA expression in PBMCs showed a low specificity to diagnose ATCMR, we would rather suggest using the combined miRNAs profile to differentiate between AR and NA instead of the use of single miRNA expression levels. These findings may improve our understanding about acute T-cell mediated rejection mechanisms. Further studies on larger sample size are necessary to determine the expression levels of miRNAs in renal transplant recipients in order to identify good predictive/diagnostic biomarkers in scheduled time-lines after renal transplantation. Disclosure The authors of this manuscript have no conflict of interest. Acknowledgments This study (MSc student thesis) was supported by a grant from Tehran University of Medical Sciences, research deputy, grant number 21625, Tehran, Iran. References [1] D.O. Taylor, L.B. Edwards, M.M. Boucek, E.P. Trulock, D.A. Waltz, B.M. Keck, et al., Registry of the International Society for Heart and Lung Transplantation: twentythird official adult heart transplantation report—2006, J Heart Lung Transplant 25 (2006) 869–879. [2] A. Laupacis, P. Keown, N. Pus, H. Krueger, B. Ferguson, C. Wong, et al., A study of the quality of life and cost-utility of renal transplantation, Kidney Int 50 (1996) 235–242. [3] K. Solez, R.B. Colvin, L.C. Racusen, M. Haas, B. Sis, M. Mengel, et al., Banff 07 classification of renal allograft pathology: updates and future directions, Am J Transplant 8 (2008) 753–760. [4] B. Nazari, A. Amirzargar, B. Nikbin, M. Nafar, P. Ahmadpour, B. Einollahi, et al., Comparison of the Th1, IFN-γ secreting cells and FoxP3 expression between patients with stable graft function and acute rejection post kidney transplantation, Iran J Allergy Asthma Immunol 12 (3) (Jul 13 2013) 262–268. [5] F. Mohammadi, M.H. Niknam, M. Nafar, B. Einollahi, B. Nazari, M. Lessanpezeshki, et al., Dynamic changes of IFN-γ-producing cells, TGF-β and their predictive value in early outcomes of renal transplantation, Int J Organ Transplant Med 4 (2) (2013) 77–85. [6] M.A. Amirzargar, A. Amirzargar, A. Basiri, M. Hajilooi, G. Roshanaei, G. Rajabi, et al., Early post-transplant immune monitoring can predict long-term kidney graft survival: soluble CD30 levels, anti-HLA antibodies and IgA-anti-Fab autoantibodies, Hum Immunol 75 (1) (Jan 2014) 47–58. [7] H. Nikoueinejad, A. Amirzargar, A. Sarrafnejad, B. Einollahi, M. Nafar, P. Ahmadpour, et al., Dynamic changes of regulatory T cell and dendritic cell subsets in stable kidney transplant patients: a prospective analysis, Iran J Kidney Dis 8 (2) (Mar 2014) 130–138. [8] H.U. Meier-Kriesche, A.O. Ojo, J.A. Hanson, D.M. Cibrik, J.D. Punch, A.B. Leichtman, et al., Increased impact of acute rejection on chronic allograft failure in recent era, Transplantation 70 (2000) 1098–1100. [9] J.R. Chapman, P.J. O'Connell, B.J. Nankivell, Chronic renal allograft dysfunction, J Am Soc Nephrol 16 (2005) 3015–3026. [10] I. Ahmad, Biopsy of the transplanted kidney, Semin Interv Radiol 21 (2004) 275–281. [11] P.N. Furness, N. Taub, International variation in the interpretation of renal transplant biopsies: report of the CERTPAP Project, Kidney Int 60 (2001) 1998–2012. [12] J.M. Sorof, R.K. Vartanian, J.L. Olson, S.J. Tomlanovich, F.G. Vincenti, W.J. Amend, Histopathological concordance of paired renal allograft biopsy cores. Effect on the diagnosis and management of acute rejection, Transplantation 60 (1995) 1215–1219. [13] E.D. Poggio, D.S. Batty, S.M. Flechner, Evaluation of renal function in transplantation, Transplantation 84 (2007) 131–136. [14] J.D. Schold, B. Kaplan, The elephant in the room: failings of current clinical endpoints in kidney transplantation, Am J Transplant 10 (2010) 1163–1166. [15] A.-B. Shyu, M.F. Wilkinson, A. van Hoof, Messenger RNA regulation: to translate or to degrade, EMBO J 27 (2008) 471–481. [16] G. Stefani, F.J. Slack, Small non-coding RNAs in animal development, Nat Rev Mol Cell Biol 9 (2008) 219–230.

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