Liang Xu, Xiu-Xiu Chen, Jian Xu, Hui-Ling Gan, Hong-Tao Jiang

Department of organ transplantation, Second Affiliated Hospital, Hainan Medical University, Haikou 570100, China

Keywords:Kidney transplantation Acute T cell mediated rejection miRNA

ABSTRACT Objective: To explore the expression profile of miRNA in peripheral blood of patients with acute T cell mediated rejection after kidney transplantation. Method: 4 patients with acute T cell mediated rejection(TCMR) after kidney transplantation were collected as experimental group. Meanwhile,4 patients under stable condition after kidney transplantation were collected as control group. The elbow blood was taken from two groups. The next generation sequencing was used to study miRNA libraries. Then, they were analyzed of variance by R language software. Results: 14 miRNAs were differently expressed in the experimental group and control group, including 5 up-regulated miRNAs (P＜0.05) and 9 down-regulated miRNAs(P＜0.05). Conclusions: Peripheral blood miRNAs expression variations might be ideal biomarkers and reveal mechanisms for acute TCMR.

1. Introduction

How to identify acute rejection (acute rejection, AR) after kidney transplantation early, and to give timely and optimal treatment in the absence of graft damage or minor damage is considered the key[1] to improve graft survival.Therefore, how to detect AR early before AR causes irreversible transplant kidney injury is a severe clinical difficulty we face.Studies have shown that microRNA RNA (miRNA) is involved in regulating transplanted renal AR,which occurs not only before clinical symptoms, but also before pathological changes [2,3].Acute TCMR is a common clinical type of AR, accounting for about 90% [4].In this study, miRNA in acute TCMR was selected by second-generation sequencing and explored the possible mechanism, in order to find miRNA in peripheral blood and closely related to AR as new biological markers for early diagnosis of acute TCMR, and provide new and stronger basis for early diagnosis and treatment of acute TCMR after kidney transplantation.

2. Materials and Methods

2.1 General information

Eight patients undergoing kidney allotransplantation from August 1,2019 to December 31,31,2020 were selected, including 6 male and 2 female, 29-53 years old, average age 41.2 years old,population reactive antibody and lymphotoxicity test were negative,and all were treated with tacromous and mesocoated Maccohenol plus methylprednisolone after surgery.Four cases were each in the experimental group and the control group.In the experimental group, the transplanted kidney biopsy puncture confirmed TCMR,and the control group were the recipients with good recovery and renal function was stable after kidney transplantation in the same period.There were no significant differences in age, sex, or BMI between the experimental and control groups (P＞ 0.05), and the two groups were comparable.After the diagnosis of TCMR, hormone or rabbit anti-human thymic immunoglobulin was used, and the blood creatinine recovered to the level before TCMR, and no obvious abnormalities were found in the review of the transplanted kidney color ultrasound.The study was approved by the hospital ethics committee and all recipients participating in the study signed informed consent.

2.2 Methods

For study subjects, 30ml of elbow vein blood was loaded into three collecting vessels containing heparin sodium anticoagulant,and PBMC was obtained by centrifugation at 2000 r/min.Total RNA of samples was extracted with TRIzol reagent, RNA quality control criteria: ① 260 / 280 ratio, 260 / 230 ratio 1.8; 100ng total② RNA; ③ RNA integrity test 28S / 18S＞ 1.5, RNA Integrity Number (RIN)＞ 7. We sent the total RNA stored with dry ice to Shanghai Biotech by cold chain, which used the original data of second-generation sequencing to obtain the samples, and then conducted differential analysis with R language software.To obtain significant different genes, we set the screening condition as:pvalue 0.05 and difference multiple 2.

2.3 Statistical methods

For samples without biological replicates, read count (read length number) data were normalized with TNM and then for differential analysis with the R language edgeR package.For samples with biological replicates, we analyzed them using the R language DESeq2 package.To obtain significant different genes, we set the screening condition as: pvalue 0.05 and difference multiple 2.Meanwhile, differential gene MA maps and differential miRNA target genes were tionally annotated using R software.

2.4 GO enrichment analysis

GO enrichment analysis of differential miRNA target genes in the experimental and control groups was performed using the clusterProfiler and igraph packages of R language, and finally, the enrichment results were visualized.

3. Results

3.1 Screening of the differential miRNA

Analyzing the original data using the R language DESeq2 package, we found 71 upregulated genes and 123 downregulated genes in the experimental and control group sample data and drew MA plots (Figure 1).The horizontal and vertical coordinates are the log2 (CPM) values for the two groups of samples.Each dot in the figure represents a gene, the closer to the origin the lower the expression, red dots indicate up-regulated genes, green dots indicate downregulated genes, black indicates non-differential genes, upregulated and downregulated are vertical axis samples relative to horizontal axis samples.then, We list the first 14 significantly differentially expressed miRNA in the experimental and control groups, Five miRNA were expressed (miR-122-5p, miR-124-5p,miR-1273g-5p, miR-125a-5 p, miR-miR-miR-101-101-3p, miR-1185-5p miR-1 250-5p, miR-127-3p, miR-1299) (Table 1).

Figure 1 The MA plot of the differential miRNA

3.2 Working characteristic curve of differential miRNA subjects (ROC curve)

Differential miRNA ROC curves were drawn using the pROC package in the R language (Figure 2).Where the cross-ordinate indicates sensitivity and specificity, respectively.Thus, we calculated the area under the ROC curve (AUC) as 0.88, which indicates the good prediction accuracy of the data of this study.

Figure 2 The ROC curves for the differential miRNA

3.3 GO functional enrichment analysis

From the experimental and control data, we obtained 49 GO entries,and after the functional enrichment analysis of differential miRNA target genes, we obtained 14 significantly different GO entries (P＜0.5).Six entries participate in the function of cell composition,including cytoplasm, cytoskeleton, synapse, extracellular matrix of proteins, two entries participate in molecular function, embodied in protein connection and guanine nucleotide exchange factors,

six entries are involved in biological processes, including DNA transcription regulation, positive regulation of GTP enzymes,formation and phosphorylation of various organelles.We drew the bar chart of target gene GO annotation classification (Figure 3) and the significantly enriched function-gene interaction network map of differential miRNA target genes (Figure 4).

Table 1 Comparison of miRNA expression profiles between the experimental and control groups

Figure 3 Functional enrichment analysis of the top 10 GO positions

Figure 4 Significantly enriched function-gene network map

4. Discussion

Since the first successful implementation of homogeneous living kidney transplantation in humans in 1954, current renal transplantation techniques still fail to replace [6].Although the quality of life and survival time of kidney transplant recipients are continuously improved thanks to the progressive kidney transplant technology and the application of various new immunosuppressive agents[7,8], transplant AR remains the main risk factor for the longterm survival of grafts.At present, the clinical diagnosis of AR is mainly conducted through clinical manifestations (including fever,increased blood pressure, reduced urine volume, transplant kidney enlargement and pain) and laboratory examination (the magnitude of blood creatinine increase).Similarly, color ultrasound has indirectly diagnosed AR through some non-specific changes (graft morphology and flow resistance index).Therefore, the gold standard for the diagnosis of AR at this stage is still tissue biopsy, but its accuracy is still affected by the pathologists' diagnostic level, sampling error and other [10].Given that the above diagnostic methods are diagnosed based on the discovery of structural and functional impairment of transplanted kidney, new, reliable, non-invasive biomarkers are urgently needed for early diagnosis of AR before not changing transplanted kidney structure and function, so as to minimize the damage of AR to transplanted kidney[11].

Recently, miRNA has gradually become a hot spot to explore the early diagnosis of AR due to its stability, organ-tissue specificity,and involvement in graft kidney rejection[12,13,14].The miRNA is a class of non-coding single-stranded RNA small molecules consisting of 19 - 23 nucleotides.The miRNA regulates gene expression at the post-transcriptional level and suppresses translation and grades mRNA by base complementary pairing with the 3 ′ -UTR and 5 ′ -UTR regions of the target mRNA, thus reducing translation efficiency or blocking mRNA involved in gene regulation and expression of[15].On the one hand, more and more studies show that about 60% of human transcriptome is regulated; on the other hand, with the recent development of high-throughput sequencing technology and bioinformatics methods, miRNA research, including regulatory targets and related functions to predict [16].Heegaard[17]noted that miRNA can be found in the peripheral blood several months before the clinical diagnosis of the disease, and is also confirmed to be differentially expressed among different diseases,including AR[18], after kidney transplantation.Anglicheau et al[19] analyzed the expression profile of acute rejection after kidney transplantation and found that upregulated miR-142-5p, miR-155 and miR-223 were closely associated with CD3 and CD20 mRNA,suggesting that these miRNA may cause immune cell infiltration in the graft.Using transplanted kidney biopsy, miR-10b was found to be down-regulated in acutely rejected biopsy tissues, and to inhibit cell activity and accelerated apoptotic[20] through targeted regulation of BCL2L11.Peng et al[21] found that the high expression of miR-650 in the transplanted kidney was associated with acute rejection.Further study found that miR-650 targeted the high expression of caspase-8, thus inhibiting cell proliferation, inducing apoptosis and inhibiting IL-13 release by increasing INF-release, thus increasing the chemotaxis of macrophages.Some scholars used multivariate logistic regression analysis to believe that the five miRNAs (miR-15b, miR-16, miR103a, miR106a and miR-107) detected from whole blood cells could accurately distinguish between the recipients of TCMR (BanffII-III) and the[22] of other types of injury.However, this team studied the same miRNA and failed to get the same results as[23] in PBMC, indicating that the biomarker identification results of some samples can not be simply extended to the whole population, so more studies are needed to explain the mechanisms related to the difference in miRNA expression between different samples.For the TCMR receptor, the expression of miR-99a and miR-100 upregulated[24].A previous study was performed only to find that the expression of miR-99a in TCMR biopsy tissues decreased [19].We hypothesized that the differential expression of miR-99a in tissues and serum may be due to the smaller number of immune cells associated with rejection in the peripheral blood than in the graft.Lorenzen et al[25] found that urinary levels of miR-210 were significantly reduced during TCMR, while decreased miR-210 levels correlated with a decrease in allograft function one year after transplantation.Therefore, he suggested that miR-210 is a potent biomarker for TCMR.In addition, he also confirmed that the expression of miRNA-210 was reduced in the urine of TCMR patients, but miRNA-155-5p expressed better TCMR than miRNA-210 (sensitivity 85%, specificity 86%, AUC=0.875; P=0.046)[26].In China, high-throughput miRNA GeneChip detection technology was used to find the high[27] expression of miR-142-5p, miR-144-5p and miR-130a-3p in the plasma of AR recipients.

Compared with previous gene chips can only detect specific miRNA, however, the whole genome sequencing besides the advantages of high sensitivity and high throughput.This would yield a more complete miRNA expression profile.At the same time, we analyzed the raw data and found that the differential miRNA mainly affected DNA transcriptional regulation, positively regulating GTP enzyme, guanine nucleotide exchange factor, extracellular matrix of protein, protein connection and other related functions.In addition,it has been shown that miRNA-1250-5p is the miRNA located in the second intron of the host gene cell apoptosis-associated tyrosine kinase (AATK), and AATK is located at 17q25.3[28].Zhang et al[29]found the presence of CpG islands in the promoter region of the miRNA-1250-5p host gene AATK, thus, the low expression of miRNA-1250-5p was achieved by methylation of the promoter region of the host gene AATK.Recently, in recent years, more studies have suggested that DNA methylation is involved in the development of AR and the development of [30].Similarly, whether miRNA-1250-5p involves DNA methylation in the occurrence and development of TCMR deserves our further investigation.

In this study, second-generation sequencing confirmed differences in peripheral PBMC miRNA expression profiles of TCMR patients after kidney transplantation, and a total of 14 significantly differentially expressed miRNA were detected, with 5 miRNA upregulated(P＜0.05). This suggests that these differentially expressed miRNA are most likely involved in the occurrence and development of TCMR.It is further predicted that miRNA-1250-5p is regulated by DNA methylation, and then involved in the occurrence and development of TCMR.Therefore, this study provides a theoretical basis for the development, diagnosis and treatment of TCMR by screening the molecular markers of TCMR after kidney transplantation through further classification of AR.

Author’s contribution The study design was the first author and the corresponding author.The first author wrote the paper and the corresponding author reviewed the manuscript. All authors participated in the specimen and data collection, and the authors contributed equally and involved no relevant conflicts of interest.