Commentary
microRNA in Transplantation: Small in Name Only Brian J. Nankivell, MD, MSc, PhD, FRACP1 WHAT ARE microRNA? The discovery of noncoding RNAs has revolutionized biology and medicine. No longer considered as ordinary cellular housekeepers, these important functional molecules control chromatin architecture, transcription, RNA splicing, editing, translation, and turnover. MicroRNAs (miRNAs) are one class of short RNA molecules, usually 18 to 22 nucleotides in length, which are not translated into protein (“noncoding”) but instead, bind to messenger RNA (mRNA) by basepairing to complementary sequences which down-regulates protein expression. Cytosolic miRNAs specifically induce cleavage (“silencing”) or destabilization of mRNA, or inhibit ribosomal protein translation (“translational repression”). The end result is reduced functional mRNA and protein translation. Originally discovered in the nematode, Caenorhabditis elegans, miRNAs are highly conserved across species, and abundantly expressed in almost all mammalian cells. Knockout of Dicer, a critical cytosolic RNase III enzyme needed for processing to miRNA, is embryonically lethal. Biogenesis of miRNAs is under tight temporal and spatial control, and dysregulation occurs in disease. The human genome encodes over 1000 miRNAs, which target and regulate approximately 60% of the cell's mRNAs. Other cytosolic long noncoding RNAs regulate mRNA stability, and some can act as microRNA sponges. These regulatory networks refine the molecular response, optimizing the fine control of cellular differentiation and function. Recent evidence suggests that miRNA have a role in acute rejection of transplant organs.1 In this issue of Transplantation, Vitalone et al2 have evaluated the interplay between miRNA and mRNA expression in kidney allografts from pediatric recipients with acute T cell– mediated rejection. Samples from indication biopsies were compared with stable surveillance biopsies. From a previously analyzed sample set (n = 70 biopsies), 1035 mRNAs were differentially expressed by acute rejection using DNA microarrays and identified as potential targets of 19 miRNAs using a customized software database (GO-Elite, http://www. genmapp.org/go_elite), which identifies minimal nonredundant sets of biological ontology terms or pathways. These candidate miRNAs from “in silico” prediction were confirmed by microfluidic quantitative polymerase chain reaction in a separate sample set (n = 97): narrowing down the number of significantly altered miRNAs to just 9 (UP: miR142-3p, Received 30 March 2015. Accepted 9 April 2015. 1
Department of Renal Medicine, Westmead Hospital, Sydney, Australia.
Correspondence: Brian J. Nankivell, MD, MSc, PhD, FRACP, Department of Renal Medicine Westmead Hospital, Westmead, 2145, NSW, Sydney, Australia. (Brian. [email protected]). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0041-1337/9909-1754 DOI: 10.1097/TP.0000000000000807
1754
www.transplantjournal.com
miR342-3p, and miR25) and (DOWN: miR181a, miR192, miR204, miR215, miR10b*, and miR615-3p). Only predicted miRNAs were measured, and a broader miRNA genomic study was not undertaken. These altered miRNA were then backlinked to potential mRNA targets using predictive software and to allograft histopathology. Perturbed miRNAs correlated with the intensity of interstitial inflammation and tubulitis (suggesting an infiltrating cell origin) and with Banff tubular atrophy grades (as a response to injury). Predicted mRNA targets of miRNA are present in tubules using public database information. Pathway level analyses of each altered miRNA predicted a diverse range of biological processes (such as TOR, Notch, HIDAC2 and Hedgehog signalling pathways, gap junction, and synaptic vesicle). A common enriched pathway was TGFß signalling. Enrichment analysis of mRNA that were altered in acute rejection, identified lymphocytes, and other immune cells. The dominant predicted upstream transcriptional regulator of altered miRNA/mRNA was FOXP3, which associated with miR25, miR181a, miR192, miR215, and miR342-3p.
UNDERSTANDING THE BIOLOGY OF REJECTION This work adds to a growing literature that acute allograft rejection initiates substantial alterations both in mRNA and their corresponding miRNA targets. Anglicheau et al3 found 3 unique miRNAs (miR142-5p, miR155, and miR223) in T cell–mediated rejection, corresponding with circulating mononuclear cells. Wilflingseder et al1 found distinct miRNA signatures mediating specific biological pathways in allograft dysfunction. Acute rejection was enriched for immune response pathways, and differed from ischemic injury, which displayed angiogenesis, proliferation, and apoptosis pathways. Sui et al4 found 5 transcription factors activated in acute rejection, which correlated with 12 miRNAs and 32 long noncoding RNAs, and identified an acute rejection signature. In allograft biopsies with interstitial fibrosis, Scian et al5 found 3 differentially expressed miRNAs (miR142-3p, miR204, and miR211) that were also present in paired urine samples. Ben-Dov et al6 found 4 miRNAs upregulated in interstitial fibrosis (miR21, miR142-5p, miR142-3p, and miR506) and 2 suppressed (miR30b and miR30c). So although concordance between studies appears imperfect, there is commonality across organs with comparable phenotypes. Each miRNAs may pair to hundreds of different mRNA molecules, and a given target mRNA might be regulated by several miRNAs. This difficult yet critical step in miRNA biology—the prediction of target mRNA genes, is undertaken by bioinformatic programs that link experimental databases. Multiple putative target genes are then generated. Shortlisted biologically significant targets then need experimental verification: a tedious, subjective, and challenging process. Even then, the effect of miRNA binding on protein Transplantation
■
September 2015
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
■
Volume 99
■
Number 9
Commentary
© 2015 Wolters Kluwer
production depends on location, number, and binding strength of the mRNA targets. Some miRNA tissue pertubations found in rejection will originate from infiltrating cells, which correlated with interstitial inflammation and tubulitis by histopathology. Other studies of acutely rejecting allografts contain high abundance of miRNAs typically expressed in mononuclear cells, correlating with T- and B-cell markers.3 The origin of dysregulated miR146a identified by tissue microarrays from rejecting rodent liver transplants was localized to inflammatory cells infiltrating portal tracts by in situ hybridization.7 Other miRNA perturbations result from disrupted allograft tissue. Negative correlations between the miRNA and their matched mRNA targets suggest mRNA message degradation; however, other correlations were absent or positive and other explanations are required. Some differentially expressed miRNA may result from altered regulation of the miRNAs themselves within injured cells.3 The functional proof that any miRNA is important includes miRNA mimics and antisense oligonucleotides (which overexpress or inhibit a particular miRNA's function), and transgenic animal models which can conditionally overexpress or knockdown an miRNA of interest. NONINVASIVE DIAGNOSIS OF ACUTE REJECTION The miRNA have been mooted a potential biomarkers for the noninvasive diagnosis of transplant rejection and ischemic transplant injury (e.g., miR182-5p).1 Circulating miRNAs are released after cell death or sublethal injury as larger shedding microvesicles (100 nm-1 μm), or actively secreted smaller exosomes (30-120 nm diameter) derived from the endolysosomal compartment. They can carry genetic information between cells which facilitate cellular crosstalk: with uptake of miRNA by recipient cells altering transcription.8 One practical advantage of miRNA over mRNA is their remarkable stability. They can withstand repetitive freezing and thawing cycles, resist degradation by RNAses, and survive in blood, urine, or paraffin tissue blocks. Laboratory challenges to measure miRNAs include the slow turnaround time for the processes of RNA isolation, reverse transcription, quantitative PCR analysis, and interpretation of results, as well as technical issues, such as normalization to adjust for variability in RNA isolation and low concentrations. Clinical
1755
difficulties include discrimination of allograft rejection from other pathologies with comparable inflammatory phenotypes, such as bacterial or BK virus infection. Oghumu et al9 reported 25 miRNAs that differentiated acute pyelonephritis from acute cellular rejection. Although injury from rejection or ischemia were different from stable control samples, diagnostic overlap of common inflammatory and cytokine signaling pathways limit clinical utility.1,10 Potential noninvasive biomarkers derived in the bioinformatics laboratory need further unbiased testing in realistic clinical scenarios.1 CONCLUSIONS Overall, the assessment and understanding of miRNA together with their mRNA targets offer a more nuanced and holistic understanding of cell biology compared to isolated transcriptomics of mRNA levels to infer protein expression. However, much more work is needed. REFERENCES 1. Wilflingseder J, Reindl-Schwaighofer R, Sunzenauer J, et al. MicroRNAs in kidney transplantation. Nephrol Dial Transplant. 2014. 2. Vitalone M, Sigdel TK, Salomonis N, et al. Transcriptional perturbations in graft rejection. Transplantation. 2015;99(9):1882–1893. 3. Anglicheau D, Sharma VK, Ding R, et al. MicroRNA expression profiles predictive of human renal allograft status. Proc Natl Acad Sci U S A. 2009;106:5330–5335. 4. Sui W, Lin H, Peng W, et al. Molecular dysfunctions in acute rejection after renal transplantation revealed by integrated analysis of transcription factor, microRNA and long noncoding RNA. Genomics. 2013;102:310–322. 5. Scian MJ, Maluf DG, David KG, et al. MicroRNA profiles in allograft tissues and paired urines associate with chronic allograft dysfunction with IF/TA. Am J Transplant. 2011;11:2110–2122. 6. Ben-Dov IZ, Muthukumar T, Morozov P, et al. MicroRNA sequence profiles of human kidney allografts with or without tubulointerstitial fibrosis. Transplantation. 2012;94:1086–1094. 7. Hu J, Wang Z, Tan CJ, et al. Plasma microRNA, a potential biomarker for acute rejection after liver transplantation. Transplantation. 2013;95: 991–999. 8. Fleissner F, Goerzig Y, Haverich A, et al. Microvesicles as novel biomarkers and therapeutic targets in transplantation medicine. Am J Transplant. 2012;12:289–297. 9. Oghumu S, Bracewell A, Nori U, et al. Acute pyelonephritis in renal allografts: a new role for microRNAs? Transplantation. 2014;97:559–568. 10. Wilflingseder J, Regele H, Perco P, et al. miRNA profiling discriminates types of rejection and injury in human renal allografts. Transplantation. 2013;95:835–841.
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
microRNA in Transplantation: Small in Name Only Brian J. Nankivell, MD, MSc, PhD, FRACP1 WHAT ARE microRNA? The discovery of noncoding RNAs has revolutionized biology and medicine. No longer considered as ordinary cellular housekeepers, these important functional molecules control chromatin architecture, transcription, RNA splicing, editing, translation, and turnover. MicroRNAs (miRNAs) are one class of short RNA molecules, usually 18 to 22 nucleotides in length, which are not translated into protein (“noncoding”) but instead, bind to messenger RNA (mRNA) by basepairing to complementary sequences which down-regulates protein expression. Cytosolic miRNAs specifically induce cleavage (“silencing”) or destabilization of mRNA, or inhibit ribosomal protein translation (“translational repression”). The end result is reduced functional mRNA and protein translation. Originally discovered in the nematode, Caenorhabditis elegans, miRNAs are highly conserved across species, and abundantly expressed in almost all mammalian cells. Knockout of Dicer, a critical cytosolic RNase III enzyme needed for processing to miRNA, is embryonically lethal. Biogenesis of miRNAs is under tight temporal and spatial control, and dysregulation occurs in disease. The human genome encodes over 1000 miRNAs, which target and regulate approximately 60% of the cell's mRNAs. Other cytosolic long noncoding RNAs regulate mRNA stability, and some can act as microRNA sponges. These regulatory networks refine the molecular response, optimizing the fine control of cellular differentiation and function. Recent evidence suggests that miRNA have a role in acute rejection of transplant organs.1 In this issue of Transplantation, Vitalone et al2 have evaluated the interplay between miRNA and mRNA expression in kidney allografts from pediatric recipients with acute T cell– mediated rejection. Samples from indication biopsies were compared with stable surveillance biopsies. From a previously analyzed sample set (n = 70 biopsies), 1035 mRNAs were differentially expressed by acute rejection using DNA microarrays and identified as potential targets of 19 miRNAs using a customized software database (GO-Elite, http://www. genmapp.org/go_elite), which identifies minimal nonredundant sets of biological ontology terms or pathways. These candidate miRNAs from “in silico” prediction were confirmed by microfluidic quantitative polymerase chain reaction in a separate sample set (n = 97): narrowing down the number of significantly altered miRNAs to just 9 (UP: miR142-3p, Received 30 March 2015. Accepted 9 April 2015. 1
Department of Renal Medicine, Westmead Hospital, Sydney, Australia.
Correspondence: Brian J. Nankivell, MD, MSc, PhD, FRACP, Department of Renal Medicine Westmead Hospital, Westmead, 2145, NSW, Sydney, Australia. (Brian. [email protected]). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0041-1337/9909-1754 DOI: 10.1097/TP.0000000000000807
1754
www.transplantjournal.com
miR342-3p, and miR25) and (DOWN: miR181a, miR192, miR204, miR215, miR10b*, and miR615-3p). Only predicted miRNAs were measured, and a broader miRNA genomic study was not undertaken. These altered miRNA were then backlinked to potential mRNA targets using predictive software and to allograft histopathology. Perturbed miRNAs correlated with the intensity of interstitial inflammation and tubulitis (suggesting an infiltrating cell origin) and with Banff tubular atrophy grades (as a response to injury). Predicted mRNA targets of miRNA are present in tubules using public database information. Pathway level analyses of each altered miRNA predicted a diverse range of biological processes (such as TOR, Notch, HIDAC2 and Hedgehog signalling pathways, gap junction, and synaptic vesicle). A common enriched pathway was TGFß signalling. Enrichment analysis of mRNA that were altered in acute rejection, identified lymphocytes, and other immune cells. The dominant predicted upstream transcriptional regulator of altered miRNA/mRNA was FOXP3, which associated with miR25, miR181a, miR192, miR215, and miR342-3p.
UNDERSTANDING THE BIOLOGY OF REJECTION This work adds to a growing literature that acute allograft rejection initiates substantial alterations both in mRNA and their corresponding miRNA targets. Anglicheau et al3 found 3 unique miRNAs (miR142-5p, miR155, and miR223) in T cell–mediated rejection, corresponding with circulating mononuclear cells. Wilflingseder et al1 found distinct miRNA signatures mediating specific biological pathways in allograft dysfunction. Acute rejection was enriched for immune response pathways, and differed from ischemic injury, which displayed angiogenesis, proliferation, and apoptosis pathways. Sui et al4 found 5 transcription factors activated in acute rejection, which correlated with 12 miRNAs and 32 long noncoding RNAs, and identified an acute rejection signature. In allograft biopsies with interstitial fibrosis, Scian et al5 found 3 differentially expressed miRNAs (miR142-3p, miR204, and miR211) that were also present in paired urine samples. Ben-Dov et al6 found 4 miRNAs upregulated in interstitial fibrosis (miR21, miR142-5p, miR142-3p, and miR506) and 2 suppressed (miR30b and miR30c). So although concordance between studies appears imperfect, there is commonality across organs with comparable phenotypes. Each miRNAs may pair to hundreds of different mRNA molecules, and a given target mRNA might be regulated by several miRNAs. This difficult yet critical step in miRNA biology—the prediction of target mRNA genes, is undertaken by bioinformatic programs that link experimental databases. Multiple putative target genes are then generated. Shortlisted biologically significant targets then need experimental verification: a tedious, subjective, and challenging process. Even then, the effect of miRNA binding on protein Transplantation
■
September 2015
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
■
Volume 99
■
Number 9
Commentary
© 2015 Wolters Kluwer
production depends on location, number, and binding strength of the mRNA targets. Some miRNA tissue pertubations found in rejection will originate from infiltrating cells, which correlated with interstitial inflammation and tubulitis by histopathology. Other studies of acutely rejecting allografts contain high abundance of miRNAs typically expressed in mononuclear cells, correlating with T- and B-cell markers.3 The origin of dysregulated miR146a identified by tissue microarrays from rejecting rodent liver transplants was localized to inflammatory cells infiltrating portal tracts by in situ hybridization.7 Other miRNA perturbations result from disrupted allograft tissue. Negative correlations between the miRNA and their matched mRNA targets suggest mRNA message degradation; however, other correlations were absent or positive and other explanations are required. Some differentially expressed miRNA may result from altered regulation of the miRNAs themselves within injured cells.3 The functional proof that any miRNA is important includes miRNA mimics and antisense oligonucleotides (which overexpress or inhibit a particular miRNA's function), and transgenic animal models which can conditionally overexpress or knockdown an miRNA of interest. NONINVASIVE DIAGNOSIS OF ACUTE REJECTION The miRNA have been mooted a potential biomarkers for the noninvasive diagnosis of transplant rejection and ischemic transplant injury (e.g., miR182-5p).1 Circulating miRNAs are released after cell death or sublethal injury as larger shedding microvesicles (100 nm-1 μm), or actively secreted smaller exosomes (30-120 nm diameter) derived from the endolysosomal compartment. They can carry genetic information between cells which facilitate cellular crosstalk: with uptake of miRNA by recipient cells altering transcription.8 One practical advantage of miRNA over mRNA is their remarkable stability. They can withstand repetitive freezing and thawing cycles, resist degradation by RNAses, and survive in blood, urine, or paraffin tissue blocks. Laboratory challenges to measure miRNAs include the slow turnaround time for the processes of RNA isolation, reverse transcription, quantitative PCR analysis, and interpretation of results, as well as technical issues, such as normalization to adjust for variability in RNA isolation and low concentrations. Clinical
1755
difficulties include discrimination of allograft rejection from other pathologies with comparable inflammatory phenotypes, such as bacterial or BK virus infection. Oghumu et al9 reported 25 miRNAs that differentiated acute pyelonephritis from acute cellular rejection. Although injury from rejection or ischemia were different from stable control samples, diagnostic overlap of common inflammatory and cytokine signaling pathways limit clinical utility.1,10 Potential noninvasive biomarkers derived in the bioinformatics laboratory need further unbiased testing in realistic clinical scenarios.1 CONCLUSIONS Overall, the assessment and understanding of miRNA together with their mRNA targets offer a more nuanced and holistic understanding of cell biology compared to isolated transcriptomics of mRNA levels to infer protein expression. However, much more work is needed. REFERENCES 1. Wilflingseder J, Reindl-Schwaighofer R, Sunzenauer J, et al. MicroRNAs in kidney transplantation. Nephrol Dial Transplant. 2014. 2. Vitalone M, Sigdel TK, Salomonis N, et al. Transcriptional perturbations in graft rejection. Transplantation. 2015;99(9):1882–1893. 3. Anglicheau D, Sharma VK, Ding R, et al. MicroRNA expression profiles predictive of human renal allograft status. Proc Natl Acad Sci U S A. 2009;106:5330–5335. 4. Sui W, Lin H, Peng W, et al. Molecular dysfunctions in acute rejection after renal transplantation revealed by integrated analysis of transcription factor, microRNA and long noncoding RNA. Genomics. 2013;102:310–322. 5. Scian MJ, Maluf DG, David KG, et al. MicroRNA profiles in allograft tissues and paired urines associate with chronic allograft dysfunction with IF/TA. Am J Transplant. 2011;11:2110–2122. 6. Ben-Dov IZ, Muthukumar T, Morozov P, et al. MicroRNA sequence profiles of human kidney allografts with or without tubulointerstitial fibrosis. Transplantation. 2012;94:1086–1094. 7. Hu J, Wang Z, Tan CJ, et al. Plasma microRNA, a potential biomarker for acute rejection after liver transplantation. Transplantation. 2013;95: 991–999. 8. Fleissner F, Goerzig Y, Haverich A, et al. Microvesicles as novel biomarkers and therapeutic targets in transplantation medicine. Am J Transplant. 2012;12:289–297. 9. Oghumu S, Bracewell A, Nori U, et al. Acute pyelonephritis in renal allografts: a new role for microRNAs? Transplantation. 2014;97:559–568. 10. Wilflingseder J, Regele H, Perco P, et al. miRNA profiling discriminates types of rejection and injury in human renal allografts. Transplantation. 2013;95:835–841.
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
