MicroRNAs Differential Expression In Rejection After Renal Transplantation

Renal transplantation is the most ideal and oecumenical treatment of choice for end-stage renal disease (ESRD) patients. But transplantation rejection is still a strong risk factor for recipients of renal grafts. At present, the diagnosis of renal transplantation rejection can only be made by renal biopsy, which is costly, inconvenient and carries risks of complication. Clinical management of renal transplant patients would be improved if new rapid, noninvasive and reliable methods for detecting biomarkers of rejection were available. Despite a lot of work have been done, until now investigators have not been able to use the results of studies for early biomarkers form urine and peripheral blood of the patients in clinically.MicroRNAs (miRNAs) are noncoding RNA molecules of 21 to 24 nt that regulate the expression of target genes in a post-transcriptional manner. Evidence indicates that miRNAs play essential roles in embryogenesis, cell differentiation and pathogenesis of human diseases. Novel biomarkers are readily, accessible and predict the fate of the transplant early and could help to further illustrate the mechanism of rejection. Our study Objective was to compare the levels of miRNAs expression in renal biopsies of acute rejection and chronic rejection after renal transplantation between the normal samples and tried to reveal the mechanism of renal transplantation through predicting the target genes of the differentially expressed miRNAs.Methods1. Collected three biopsies of patients with acute rejection after renal transplantation as acute rejection group, four biopsies of patients with chronic rejection after renal transplantation biopsies as chronic rejection group, three renal cortexes of tissue eumorphism patients as normal control group.2. Total RNA was extracted using trizol according to the manufacturer's instructions and assessed RNA yield and quality by UV absorbance and denaturing agarose gel electrophoresis. 3. The total RNA samples were labeling and concentrating and hybridization with miRNA array using miRCURYTM Array microarray kit.4. Scaned the slide using the Genepix 4000B. The 635nm laser was used. The images were saved as TIF files. Analyzed the data in Genepix Pro 6.0 and saved the results as EXCEL files.5. Validated the results of miRNA array through Realtime-PCR.6. TargetScan software predicted the target genes of the differentially expressed miRNAs. Searched the molecules associated with the renal transplantation rejection among those target genes.Results1. The quality of RNA was satisfied the demand of miRNA microarray experiment.2. In acute rejection after renal transplantation, there were 21 miRNAs significant differential expression of which 8 up-regulated and 13 down-regulated. 14 significant differential expression miRNAs could be inquired about the information as sequence and genetic locus.3. In chronic rejection after renal transplantation, there were 63 miRNAs significant differential expression of which 35 up-regulated and 40 down-regulated. 45 significant differential expression miRNAs could be inquired about the information as sequence and genetic locus.4. Hsa-miR-320 and hsa-miR-324-3p in acute rejection and hsa-miR-637, hsa-miR-648, hsa-miR-516-5p in chronic rejection were selected for relative quantification by Realtime-PCR. It showed that the results were conformable to the result of array.5. Predicted the target genes of the differentially expressed miRNAs by TargetScan software. It was showed that 2617 target genes of 13 differentially expressed miRNAs were predicted in acute rejection.7177 target genes of 32 differentially expressed miRNAs were predicted in chronic rejection, of which 6 miRNAs were the same as acute rejection. The target genes of 18 miRNAs which predicted in acute rejection and chronic rejection were directly connected with human disease such as tumor and Hematologic disease. The other differentially expressed miRNAs play a part in regulation of human physiological functions.Conclusions1. Microarray chip is the effective approach for the high-throughput analysis of miRNAs expression profile in rejection of renal transplantation.2. There were 20 miRNAs significant differential expression in acute rejection after renal transplantation and 63 miRNAs significant differential expression in chronic rejection after renal transplantation by Microarray chip. MiRNAs were significant differential expression in rejection of renal transplantation.3. 5 MiRNAs were selected for relative quantification by Realtime-PCR.All results was conformable to the result of array which confirmed the reliability of Microarray chip results. Realtime-PCR was an effective approach for detecting expression level of the sing miRNA.4. The target genes of the differentially expressed miRNAs through TargetScan software predicting included many oncogene and Hematologic disease gene. Those played a great part in regulation of human physiological functions. MiRNAs may be the new biomarkers for transplantation rejection.The study of differentially expressed miRNAs could help to further investigate the mechanism underlying the development of rejection of renal transplantation.