KIDNEY TRANSPLANTATION Expression Levels of lncRNAs in the Patients with the Renal Transplant Rejection Mohsen Nafar1, Shiva Kalantari1, Sayyed Mohammad Hossein Ghaderian2, Mir Davood Omrani3, Hamid Fallah4, Shahram Arsang-Jang5, Tahereh Abbasi1, Shiva Samavat1, Nooshin Dalili1, Mohammad Taheri6*, Soudeh Ghafouri-Fard4** Purpose: Long non-coding RNAs (lncRNAs) include a vast portion of human transcripts. They exert regulatory roles in immune responses and participate in diverse biological functions. Recent studies indicated dysregulation of lncRNAs in the process of transplant rejection. In the current study, we aimed at identification of the expression of five lncRNAs (OIP5-AS1, FAS-AS1, TUG1, NEAT1 and PANDAR) in association with the process of transplant rejection. Material and Methods: We assessed expression of these lncRNAs in the peripheral blood of 61 kidney transplant receivers including 29 transplant rejected patients and 32 transplant non-rejected patients using real time PCR technique. Results: Expression of FAS-AS1 was significantly higher in rejected group compared to non-rejected group in males, however, differences between case and control groups were insignificant among females. For other lncR- NAs no significant differences were detected between two study groups. Quantile regression model showed that patients’ gender was an important parameter in determination of FAS-AS1 expression (Beta = - 9.46, t =- 2.82, P = 0.007) but not for other lncRNAs expressions. Significant pairwise correlations were detected between expression levels of lncRNAs in a disease related manner. Conclusion: Based on the higher expression of FAS-AS1 in patients with transplant rejection, this lncRNA might be associated with the pathogenesis of renal transplant rejection. Keywords: kidney transplant; rejection; lncRNA; OIP5-AS1; FAS-AS1; TUG1; NEAT1; PANDAR INTRODUCTION End stage renal disease (ESRD) is a catastrophic con-dition which has limited therapeutic options includ- ing renal transplantation(1). The fate of renal transplants is endangered by acute rejection which might happen in spite of application of immunosuppressive treatment and sophisticated surgical techniques(2). The depend- ence of diagnosis of acute rejection on the invasive method of renal biopsy has encouraged researchers to find suitable non-invasive methods for this purpose(3). Long non-coding RNAs (lncRNAs) as main regulators of immune response have been suggested to exert func- tional roles in the process of immune-mediated tissue rejection(3). These transcripts regulate expression of tar- get genes through different mechanisms and at different genomic, transcriptomic and post-transcriptomic levels. They have interaction domains for almost all funda- mental biological molecules including DNA, mRNAs, miRNAs, and proteins(4). Consequently, they participate 1Chronic Kidney Disease Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 2Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 3Urology and Nephrology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 4Department of Medical Genetics, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 5Clinical Research Development Center (CRDU), Qom University of Medical Sciences, Qom, Iran. 6Urogenital Stem Cell Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. *Correspondence: Urogenital Stem Cell Research Center, Shahid Beheshti University of Medical Sciences, Teh- ran, Iran. Tel & Fax: 00982123872572. E mail: mohammad_823@yahoo.com. **Correspondence: Department of Medical Genetics, Shahid Beheshti University of Medical Sciences, Tehran, Iran. E mail: s.ghafourifard@sbmu.ac.ir. Received July 2019 & Accepted November 2019 in regulation of nearly all aspects of life. Their inter- actions with Toll-like receptors result in modulation of expression of immune response genes(5). A previous study in patients with acute rejection and control sub- jects revealed that tens of lncRNAs are differentially expressed between groups(6). Besides, lncRNA mi- croarrays have shown distinctive expression profiles of acute rejection in renal transplant biopsies(7). Another study in animal models has shown the role of PRINS lncRNA in allograft rejection linking between persis- tent ischemia and transplant rejection(8). In the present study, we selected five lncRNAs to assess their expres- sion profile in the peripheral blood of renal transplant receivers including patients with acute rejection and those with normal glomerular filtration rate (GFR) and no sign of rejection. LncRNAs were selected based on their involvement in regulation of immune response or cell apoptosis. The lncRNA OPA-interacting protein 5 antisense transcript 1 (OIP5-AS1) participates in regu- Urology Journal/Vol 16 No. 6/ November-December2019/ pp. 572-577. [DOI: 10.22037/uj.v0i0.5456] Vol 16 No 06 November-December2019 573 lation of cell proliferation and apoptosis through inter- action with PTEN/PI3K/AKT pathway(9). This pathway has crucial roles in regulation of chemokine-induced recruitment of immune cells and their function. More- over, a certain isoform of PI3K controls development and activity of B and T cells. Notably, suppression of this pathway has decreased the strength of inflamma- tory responses in animal models(10). Fas –antisense 1 (FAS-AS1) is transcribed from antisense stand of Fas. Defects in Fas or Fas ligand (FasL) leads to systemic autoimmune responses in both human subjects and an- imals(11). Moreover, excessive release of autoantibodies have been reported following Fas defects in T and B lymphocytes or dendritic cells(12). Taurine up-regulated gene 1 (TUG1) participates in regulation of cell apopto- sis and inflammatory responses in diverse pathological conditions. Such functions are possibly exerted through modulation of activation of immune-related signaling pathways namely NF-κB and JAK/STAT (13). Nuclear paraspeckle assembly transcript 1 (NEAT1) binds to Splicing Factor Proline And Glutamine Rich and par- ticipates in modulation of the innate immune system and production of IL-8(14). The lncRNA Promoter Of CDKN1A Antisense DNA Damage Activated RNA (PANDAR) recruits polycomb repressive complexes and inhibits expression of senescence-enhancing genes (15). A recent study has reported up-regulation of this lncRNA in peripheral blood of multiple sclerosis (MS) patients(16). Consequently, the selected lncRNAs in the current project are possibly associated with immune re- sponse regulation and are hypothesized to participate in acute transplant rejection. PATIENTS AND METHODS Patients The current retrospective study was conducted on 29 transplant rejected patients (18 males and 11 females) and 32 transplant non-rejected patients (24 males and 8 females). Patients were admitted to Labbafi-Nejad hospital, Tehran, Iran during 2016-2018. Patients who received renal transplant during the mentioned period entered the study. Exclusion criteria were delayed graft function, urinary obstruction and urinary tract infection. Protocol biopsies of the renal allografts were performed based on the guidelines of the transplant center. Renal function was evaluated by creatinine clearance, protein excretion and renal ultrasound and angiography. Biop- sy was performed in cases with at least 25% creatinine rise during two consecutive measurements after rule out of drug toxicity and obstructions. Acute rejection was scored based on Banff criteria(17). Control subjects (non-rejected group) were matched to rejected group regarding sex and age parameters. These individuals either had no creatinine rise in the prior 3 months or protocol biopsy ruled out the transplant rejection. Pa- tients were under treatment with Tacrolimus, CellCept and Prednisolone with no significant inter/intra-group differences in treatment regimens. In antibody-mediat- ed rejection, T cell-mediated rejection and non-rejected groups, 10%, 28% and 50% of patients received trans- plants from live donors, respectively. The study protocol was approved by ethical commit- tee of Shahid Beheshti University of Medical Sciences (IR.SBMU.RETECH.REC.1398.193). Written consent forms were obtained from all study participants. Expression analysis Peripheral blood was obtained from the enrolled pa- tients at the day of biopsy in the same time and stored at -80 °C until additional investigations. Total RNA was isolated from all specimens using Hybrid-R Blood RNA (GeneAll Biotech, Korea). All steps were performed based on the protocol provided by the company. Sub- sequently, cDNA was produced from all samples using PrimeScript 1st strand cDNA Synthesis Kit (Clontech, Japan). Expressions of five lncRNAs were measured in real-time PCR system (Rotor Gene 6000, Corbett, Australia) using the HPRT1 gene as the reference gene. The RealQ Plus Master Mix (Ampliqon, Denmark) was used for amplification of lncRNAs. Primers and PCR conditions were the same as our recently published study(18). Statistical methods Mean values (± standard error of mean) of lncRNAs ex- Table 1. Demographic and clinical data of study participants. Groups Parameters Antibody-mediated rejected T cell-mediated rejected Non-rejected Age (mean ± standard error of mean) 40.7 (±15.2) 39.33 (±15.1) 35.6 (±16.1) Sex ratio (Female/ male) 9/ 13 1/ 6 8/ 24 Estimated GFR (eGFR) Before transplantation 8.76 ± 1.4 8.98 ± 1.32 7.96 ± 1.24 1 month after transplantation 41.9 ± 3.2 43.7 ± 3.4 55.39 ± 3.8 2 months after transplantation 47.195 ± 2.9 57.064 ± 3.9 64.119 ± 4.32 3 months after transplantation 40.97 ± 1.95 60.29 ± 4.2 60.935 ± 4.2 Figure 1. Relative expression of lncRNAs in study groups based on the gender of subjects (Black dots show the expression level in each patient, Red crosses show outlier values). LncRNAs and renal transplant rejection-Nafar et al. pressions were compared between study groups using Bayesian Regression model. The observation effects were regarded as random in the analysis model. The t student/Gaussian prior distribution was assumed for parameters with 8000 iteration and 1000 warm-up. The effects of possible confounding parameters were judged by Quantile regression model. The Box-Cox transfor- mation was used for normalization of the data. P-values were estimated from Frequentist method. Stan package in R 3.5.1 environment was used for statistical analysis. P < 0.05 was regarded as significant. RESULTS General data of patients Table 1 shows demographic and clinical data of pa- tients. In transplant rejected patients, graft biopsy re- vealed T cell mediated rejection and antibody-mediated rejection in 22 and 7 patients respectively. In non-re- jected patients, 12 patients had no creatinine rise where- as others had creatinine rise. In rejected group, mean (± standard error of mean) values of serum levels of creatinine were 3.14 ± 1.8 mg/dL and 2.04 ±1.74 mg/ dL prior and post-transplantation, respectively. In this group, one patient died and nephrectomy was done for one patient(19). Expression assays The results of expression analysis of lncRNAs in re- jected and non-rejected groups are shown in Figure 1. Expression of FAS-AS1 was significantly higher in re- jected group compared to non-rejected group in males, however, differences between case and control groups were insignificant among females. For other lncRNAs no significant differences were detected between two study groups. As shown in Table 2, expression of FAS- AS1 was different between rejected and non-rejected groups (relative expression difference = 4.0647, P val- ue = 0.005). However, gender-based analysis showed that the difference was due to the dissimilar expression levels in males, as females did not show any significant difference in this regard. Quantile regression model showed that patients’ gender was an important parameter in determination of FAS- AS1 expression (Beta = -9.46, t = -2.82, P = 0.007) but not for other lncRNAs expressions. Table 3 shows the results of Quantile regression for assessment of asso- ciation between expression ratio and independent var- iables. Subsequently, we assessed expression of lncRNAs be- tween four subgroups (T cell mediated rejection, anti- body-mediated rejection, stable GFR, non-rejected with creatinine rise). The results of Bayesian Regression model after adjustment of the effects of gender showed no significant difference in lncRNA expressions be- tween four study subgroups (Table 4). Correlations between expression levels of lncRNAs Finally, we assessed correlations between expression LncRNAs Groups Rejected Non-Rejected Relative Expression difference a SE P-value b 95% CrI OIP5-AS1 Total 29 32 -0.0936 0.07 0.623 [-0.22, 0.04] Male 25 19 -0.1268 0.07 0.581 [-0.27, 0.02] Female 7 10 -0.0102 0.17 0.765 [-0.35, 0.33] FAS-AS1 Total 29 32 4.0647 2.07 0.005 [0.01, 8.18] Male 25 19 4.0609 2 0.005 [0.19, 8] Female 7 10 -1.822 3.88 0.347 [-9.42, 5.95] TUG1 Total 29 32 -0.0964 0.08 0.310 [-0.24, 0.05] Male 25 19 -0.0664 0.08 0.282 [-0.22, 0.1] Female 7 10 -0.1718 0.19 0.744 [-0.55, 0.21] NEAT1 Total 29 32 -0.057 0.06 0.210 [-0.18, 0.07] Male 25 19 -0.0647 0.07 0.194 [-0.2, 0.07] Female 7 10 -0.0406 0.15 0.949 [-0.33, 0.26] PANDAR Total 29 32 -0.0462 0.06 0.527 [-0.17, 0.07] Male 25 19 -0.0496 0.08 0.572 [-0.2, 0.1] Female 7 10 -0.0344 0.11 0.917 [-0.26, 0.19] Table 2. The results of Bayesian Regression model to compare gene expression ratios between study groups with adjusting the effects of gender (a Expression difference: Rejected - Non-Rejected, b P-Value estimated from Frequentist method). LncRNA Variable Beta SE t P-Value 95 % CI for Beta OIP5-AS1 Group -0.05 0.10 -0.49 0.623 [-0.26, 0.16] Gender -0.09 0.15 -0.61 0.542 [-0.38, 0.2] Group*Gender -0.03 0.20 -0.15 0.884 [-0.42, 0.37] FAS-AS1 Group 5.14 1.76 2.91 0.005 [1.61, 8.68] Gender 3.36 2.48 1.35 0.181 [-1.61, 8.32] Group*Gender -9.46 3.36 -2.82 0.007 [-16.19, -2.74] TUG1 Group -0.13 0.13 -1.03 0.310 [-0.38, 0.12] Gender 0.02 0.18 0.11 0.909 [-0.33, 0.37] Group*Gender 0.04 0.24 0.18 0.859 [-0.44, 0.52] NEAT1 Group -0.11 0.09 -1.27 0.210 [-0.3, 0.07] Gender -0.09 0.13 -0.74 0.460 [-0.35, 0.16] Group*Gender 0.13 0.17 0.74 0.460 [-0.22, 0.47] PANDAR Group -0.05 0.07 -0.64 0.527 [-0.19, 0.1] Gender -0.12 0.10 -1.15 0.257 [-0.32, 0.09] Group*Gender 0.03 0.14 0.23 0.818 [-0.25, 0.31] Table 3. The results of Quantile regression for assessment of association between expression ratio and independent variables (Group: Rejected/ Non-rejected; Gender: Male/Female). LncRNAs and renal transplant rejection-Nafar et al. Kidney Transplantation 574 Vol 16 No 06 November-December2019 575 levels of lncRNAs in rejected and non-rejected groups (Figures 2 and 3). Significant inverse correlations were found between expression levels of OIP5-AS1 and FAS-AS1 as well as FAS-AS1 and NEAT1 in both study groups. Expression levels of FAS-AS1 and TUG1 were inversely correlated in transplant-rejected group. However, no significant correlation was found between expressions of them in the other group. Expression levels of FAS-AS1 and PANDAR were inversely cor- related in non-rejected group but not the other group. Expression levels of OIP5-AS1 and PANDAR were positively correlated in transplant-rejected patients but not the other group. Expressions of other pairs of lncR- NAs were correlated in both groups. DISCUSSION In the current study, we assessed expression of five ln- cRNAs in the peripheral blood of transplant receivers with and without transplant rejection. The role of ln- cRNAs in the process of transplant rejection has been assessed previously though high throughput or single gene expression analysis in biopsied samples(7,20). More- over, genome-wide assessment of lncRNA signatures in peripheral blood samples has shown that expression profile of two lncRNAs can be used as non-invasive di- agnostic biomarker for transplant rejection(3). Although the selected lncRNAs in the current study were previ- ously shown to be associated with regulation of immune response, our expression analysis showed dysregulation of only one of them in patients with transplant rejec- tion. Expression of FAS-AS1 was significantly higher in rejected group compared to non-rejected group in males, however, differences between case and control groups were insignificant among females. FAS-AS1 is transcribed from the antisense direction of intron 1 Table 4. The results of Bayesian Regression model for comparison of gene expression ratios between subgroups with adjusting the effects of gender (Reference category: Antibody-Mediated Rejection). LncRNA Group Relative Expression difference SE P-value 95% CrI OIP5-AS1 T-Cell Mediated Rejection -0.15 0.11 0.259 [-0.37, 0.07] Stable GFR 0.02 0.1 0.836 [-0.17, 0.21] Non-rejected with Creatinine Rise 0.08 0.08 0.750 [-0.08, 0.23] FAS-AS1 T-Cell Mediated Rejection -0.09 0.12 0.220 [-0.33, 0.16] Stable GFR 0.07 0.1 0.327 [-0.13, 0.28] Non-rejected with Creatinine Rise 0.08 0.09 0.283 [-0.11, 0.25] TUG1 T-Cell Mediated Rejection 5 2.94 0.540 [-0.83, 10.88] Stable GFR 0.34 2.53 0.602 [-4.56, 5.24] Non-rejected with Creatinine Rise -1.99 2.11 0.430 [-6.05, 2.17] NEAT1 T-Cell Mediated Rejection -0.14 0.1 0.159 [-0.33, 0.06] Stable GFR -0.03 0.08 0.989 [-0.19, 0.14] Non-rejected with Creatinine Rise 0.05 0.07 0.537 [-0.09, 0.18] PANDAR T-Cell Mediated Rejection -0.17 0.1 0.835 [-0.36, 0.03] Stable GFR 0.02 0.08 0.557 [-0.15, 0.19] Non-rejected with Creatinine Rise 0 0.07 0.760 [-0.14, 0.14] Figure 2. Correlation between expression levels of lncRNAs in transplant rejected individuals (Bivariate scatter plots with con- fidence ellipses below the diagonal, histograms on the diagonal, and Pearson correlations above the diagonal; Expression levels of lncRNAs are shown in X- and Y- axes; The expression value of an lncRNA (designated by points) determines the relative position of the symbol along the X-axis and the expression value of a second lncRNA determines the relative position of the symbol along the Y-axis.). Figure 3. Correlation between expression levels of lncRNAs in transplant non-rejected individuals (Bivariate scatter plots with confidence ellipses below the diagonal, histograms on the diago- nal, and Pearson correlations above the diagonal; Expression levels of lncRNAs are shown in X- and Y- axes; The expression value of an lncRNA (designated by points) determines the relative position of the symbol along the X-axis and the expression value of a sec- ond lncRNA determines the relative position of the symbol along the Y-axis.). LncRNAs and renal transplant rejection-Nafar et al. of the FAS gene(21). This lncRNA has a putative role in preservation of T lymphocytes from Fas-induced apoptosis(21). Consequently, higher expression of this lncRNA in peripheral blood of transplant-rejected pa- tients reflects higher activity of lymphocytes in these patients and is in accordance with pathogenic process of graft rejection. Previous studies have shown inverse correlation between levels of FAS-AS1 and soluble Fas (sFas)(22). sFas is regarded as an endogenous apoptosis suppressor that hinders the binding of Fas to Fas-L, pre- cludes monocyte-induced and T cell–induced endothe- lial cell apoptosis which participate in the process of rejection(23). The observed effect of gender on expres- sion of FAS-AS1 has also been reported previously. For instance, FAS-AS1 expression has been associated with schizophrenia in a subgroup of male subjects but not in female subjects(24). Moreover, a previous study has demonstrated an association between female sex hor- mones and the Fas/FasL system in reproductive tissues (25). When dividing patients into four subgroups, we could not detect any significant difference between expres- sions of mentioned lncRNAs between them. Such lack of difference might be due to the small sample size in each subgroup. So we recommend design of similar studies with larger sample sizes to appraise whether ex- pressions of these lncRNAs are involved in the patho- genesis of T cell mediated or antibody mediated trans- plant rejection. Finally, we appraised correlations between expression levels of lncRNAs in the study groups and found dis- tinct patterns of correlation in each group. From this data, it is possible to speculate that immune-related mechanisms during allograft rejection influence/ are in- fluenced by the interactive network between lncRNAs. Future studies are required to unravel the underlying mechanisms and clarify the cause-effect relationship. In brief, in the current study we have shown dysregula- tion of FAS-AS1 in male transplant receivers who expe- rienced acute rejection. Future studies in larger sample sizes are needed to evaluate the potential of this lncR- NA as peripheral biomarker for allograft rejection. The difference in expression level of this lncRNA between rejected and non-rejected groups might be applied as bi- omarker for stratifying patients if future studies in larg- er sample sizes verify the results of the current study. Our study has limitations regarding sample size and lack of functional assessment of underlying molecular mechanisms for participation of FAS-AS1 in transplant rejection. When dividing patients into subgroups, the size of sample in each subgroup was small, so the rela- tion between FAS-AS1 expression and rejection should be interpreted with caution. Another limitation of our study was lack of assessment of other lncRNAs with putative function in transplant rejection. Moreover, the retrospective nature of the study limits its potential to be translated into clinical application. CONCLUSIONS Based on the higher expression of FAS-AS1 in patients with transplant rejection, this lncRNA might be associ- ated with the pathogenesis of renal transplant rejection. ACKNOWLEDGEMENT This study was financially supported by Urology and Nephrology Research Center. CONFLICT of INTERESET The authors declare they have no conflict of interest. REFERENCES 1. Saidi RF, Broumand B. Current Challenges of Kidney Transplantation in Iran: Moving Beyond The "Iranian Model". Transplantation. 2018;102:1195-7. 2. Jalalzadeh M, Mousavinasab N, Peyrovi S, Ghadiani MH. The impact of acute rejection in kidney transplantation on long-term allograft and patient outcome. Nephrourol Mon. 2015;7:e24439. 3. Ge YZ, Xu T, Cao WJ, et al. A Molecular Signature of Two Long Non-Coding RNAs in Peripheral Blood Predicts Acute Renal Allograft Rejection. Cell Physiol Biochem. 2017;44:1213-23. 4. Fernandes JCR, Acuna SM, Aoki JI, Floeter- Winter LM, Muxel SM. Long Non-Coding RNAs in the Regulation of Gene Expression: Physiology and Disease. Noncoding RNA. 2019;5. 5. Carpenter S, Aiello D, Atianand MK, et al. 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