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Gene Expression Profiling of MicroRNAs in Small-Bowel Transplantation Paraffin-Embedded Mucosal Biopsy Tissue
Gene Expression Profiling of MicroRNAs in Small-Bowel Transplantation Paraffin-Embedded Mucosal Biopsy Tissue B. Sotolongo, T. Asaoka, E. Island, M. Carreno, V. Delacruz, D. Cova, C. Russo, P. Tryphonopoulos, J. Moon, D. Weppler, A. Tzakis, and P. Ruiz ABSTRACT Background. The molecular mechanisms and regulation of immune-mediated rejection of organ allografts remains unclear. Recent studies have reported that small non-coding RNAs, microRNAs (miRNAs) play a critical role in the immune system via modulation of transcription and translation. Purpose. We hypothesized that particular miRNAs provide regulation of an ensuing intragraft immune effector response. The aim of our study was to detect miRNAs involved in acute cellular rejection (AR) in human small intestinal allografts. Materials. We examined 12 small intestinal mucosal biopsies (AR, 7 cases, all grade 2 or 3) and non-rejecting (NR) allografts (5 cases, all grade 0) obtained from recipients after small bowel or multivisceral transplantation. RNA was isolated from the formalin-fixed paraffin-embedded (FFPE) biopsy samples and transcribed to cDNA. After preamplification we utilized a PCR based TaqMan Low Density Array (TLDA) containing 365 mature human miRNAs. Relative quantification was done based on pooled normal intestine using a comparative Ct method. Results. We identified 62 miRNA upregulated genes in small bowels with ACR, and 35 were downregulated. Forty-two miRNA genes were upregulated in non-ACR small bowel biopsy samples (grade IND), and 45 were downregulated. The relative fold change ratio of ACR to non-ACR was calculated, and 50 upregulated and 8 downregulated miRNAs were detected as significant. Several interesting miRNAs will be evaluated further from this preliminary study. Our data suggests that intragraft miRNAs are potentially involved in the activation of a host alloimmune response to donor. These miRNAs may serve as targets for appropriate intervention and may be useful to monitor the allograft status. ISTOLOGIC ANALYSIS of mucosal biopsy tissue and clinical assessments have been the norm for measuring graft function and rejection in small-bowel transplantation. To date, few noninvasive biomarkers have been used reliably to assess graft function. MicroRNAs (miRNAs) are small singlestranded RNA molecules 21 to 23 nucleotides long, cleaved from larger hairpin precursor transcripts. MicroRNAs are conserved noncoding RNAs that are encoded by genes but are not transcribed into protein; they regulate gene expression by cleaving or repressing the translation of their messenger RNA (mRNA) targets to cause mRNA degradation or inhibition.1 In this regard, miRNA has emerged as having different roles in cellular functions such as cellular proliferation, cell differentiation, cell mobility, and cell
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death.2 MicroRNAs could become biomarkers correlative to human disease states by either upregulation or downregulation of gene expression patterns.2,3 In the present
From the Departments of Surgery (B.S., T.A., E.I., M.C., V.D., D.C., P.T., J.M., D.W., A.T., P.R.) and Pathology (P.R.), University of Miami Leonard M. Miller School of Medicine, Miami, Florida, and the Microbiology Unit, Laboratory Department, Bambino Gesu Children’s Hospital, Rome, Italy (C.R.). Address reprint requests to Philip Ruiz, MD, PhD, Department of Surgery, University of Miami School of Medicine, Rosensteil Medical Sciences Building, Transplant Laboratories, 1600 NW 10th Ave, Suite 8150, Miami, FL 33136. E-mail: pruiz@med. miami.edu © 2010 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 42, 62– 65 (2010)
MICRO-RNA IN MUCOSAL BIOPSY TISSUE
study, we used the gene expression profiling arrays for a panel of known miRNAs to monitor gene expression patterns in formalin-fixed paraffin-embedded (FFPE) biopsy tissues. We evaluated several FFPE samples from smallbowel transplant biopsy specimens that either demonstrated acute cellular rejection (ACR; moderate to severe, grades 2 or 3) or were indeterminate for rejection (grade IND), all in comparison with pooled normal bowel by using real-time polymerase chain reaction (PCR) gene expression profiling using a Taqman miRNA gene array. Liang et al1 characterized miRNA expression profiles in normal human tissues and showed clear clustering of gene
63
expression by tissue types in fresh tissue; however, that study did not investigate FFPE tissue. Szafranska et al3 demonstrated that accurate molecular characterization and expression profiling could be performed on FFPE and frozen tissue in assessment of miRNAs. Many studies have been conducted on the nature of miRNA genes in their role in cancer as tumor suppressors and as oncogenes,2,4 as in the case of miRNA 106b-25 cluster (106b, 92 and 25) found in gastric carcinomas; miRNA 21, which is highly expressed in pancreatic cancer; and miRNA 145 downregulated in colon cancer.4 Saito et al5 described the role of miRNAs 15b, 16, 34a, 143, and 145 as tumor suppressors, and
Fig 1. Upregulated (A) and downregulated (B) microRNA genes in FFPE from mucosal biopsy specimens exhibit ACR/ non-ACR relative fold change.
64
miRNA genes 17–92 cluster, 21, 106a, 106b-25 cluster, and 155 in gastrointestinal cancers. The role of miRNA genes in immune regulation is just beginning to emerge, with little information in the area of alloimmunity. Recent studies have shown that miRNAs 125b, 146, 155, and 223 have a role in the innate immune system and that miRNAs 17–92, 150, 155, and 181 in B and T cells.6 A recent study by Kohlhaas et al7 found that Foxp3 targets miRNA 155 and contributes to the development of regulatory T cells. Hou et al8 have shown that mice macrophages after vesicular stomatitis viral challenge exhibit upregulation of miRNA genes 146a, 155, 7a, 574-5p, and 125a, which demonstrates the influence of viruses on miRNA genes. Sui et al9 found that 20 miRNA genes differentially expressed upregulation and downregulation in comparison with normal control samples in kidney allograft rejection transplant samples. Among the miRNA genes in that study that were differentially expressed were upregulated miRNA genes 125a, 320, 381, 602, 629, and 658, and downregulated miRNA genes 324a, 611, 654, 330, 524,
SOTOLONGO, ASAOKA, ISLAND ET AL
17-3p, 483, 663, 516-5p, 326, 197, and 346.9 In another renal transplant biopsy study conducted by Anglicheau et al,10 acute rejection allograft status was indicative of overexpression of miRNAs 142-5p, 155, and 223. MATERIALS AND METHODS Real-time PCR gene expression profiling was performed on FFPE samples from bowel transplant recipients and from RNA isolated from a control pool composed of 25 samples of normal small-bowel FFPE biopsies. The RNA for the experimental groups was collected from small-bowel biopsy samples from the following 2 groups: moderate to severe ACR (grade 2 or 3; 7 samples) and indeterminate for rejection (grade IND; 5 non-ACR samples). The FFPE samples (ⱕ35 mg) were deparaffinized with xylene and ethanol washes. The samples were then treated with protease to remove all proteins. Next, the total RNA was isolated without depletion of miRNAs (which can occur with most RNA isolation kits) using a total nucleic acid isolation kit for FFPE (Recoverall; Applied Biosystems, Inc, Foster City, California). A glass filter method that included a deoxyribonuclease treatment was used to purify the total RNA with this kit. The RNA was then transcribed
Fig 2. Differential expression pattern of 98 microRNAs in intestinal transplant biopsy specimens comparing non-ACR and ACR upregulated and downregulated microRNAs using Treeview PCR software to cluster genes.
MICRO-RNA IN MUCOSAL BIOPSY TISSUE into complementary DNA using a MicRNA reverse transcription kit (Applied Biosystems, Inc). The cDNA was preamplified with a pool of miRNA primers to amplify target miRNA sequences. Real-time PCR was performed using, on average, 100 ng of cDNA per port loaded onto a Taqman MiRNA assay (Early Access microfluidic card, version 1.0; Applied Biosystems, Inc) and run on a 7900 HT Fast real-time PCR system (Applied Biosystems, Inc). The miRNA array has 384 miRNAs. Each array was ensured for gene specificity against both transcript and genome public databases such as BLAST (Basic Local Alignment Search Tool; National Center for Biotechnology. Information, US National Library of Medicine, Bethesda, Maryland). The Taqman MiRNA array was analyzed using relative quantitation software (Sequence Detection Systems, version 2.3; Applied Biosystems, Inc) using the ⌬⌬ Ct method. All samples were normalized to a miRNA mammalian endogenous control gene, U6. A pool of 25 normal small bowel FFPE biopsy tissues was used as the calibrator. Treeview PC-based software was used for heat map image analysis of the gene expression pattern.
RESULTS
Our experimental biopsy specimens were grouped into 2 groups: indeterminate for rejection (non-ACR) and moderate to severe rejection (ACR). Compared with pooled normal FFPE control tissues, the experimental samples showed both upregulation and downregulation of several miRNAs. The miRNA genes with a fold change (relative quantitation) to the normal pool with ratios of ACR and non-ACR median 2.0 or greater (upregulated) or 0.5 or less (downregulated) were recognized as significant. The patterns of upregulated and downregulated genes were differentially expressed for the non-ACR samples vs ACR samples. Sixty-two miRNA genes were upregulated in small bowels with ACR, and 35 were downregulated (data not shown). Forty-two miRNA genes were upregulated in nonACR small bowel biopsy samples (grade IND), and 45 were downregulated (data not shown). The relative fold change ratio of ACR to non-ACR was calculated, and 50 upregulated and 8 downregulated miRNAs were detected as significant (Fig 1). Treeview PCR software was used for a gene cluster of miRNA genes and detected 98 miRNAs in non-ACR and ACR sample genes with a P value of ⱕ.5 (significant). This image showed a differential gene clustering expression pattern of 98 miRNAs in non-ACR vs ACR samples (Fig 2). Among the most interesting miRNAs to be evaluated in the future from the many that were observed in this microarray were the downregulated miRNA genes 15b, 28-5p, RNU48, 106b, 192, 197, 200c, and 221. Among the most promising upregulated miRNA genes were 125a, 142, 155, and 629, which also appear upregulated in kidney acute rejection.4,9 DISCUSSION
Our initial experiments using FFPE mucosal small bowel biopsy specimens demonstrate that miRNA genes can be
65
successfully measured. Our data show that the histologic degree of acute rejection is associated with various patterns of upregulation or downregulation of miRNA genes. Moreover, the transplantation biopsy specimens exhibited differential profiles of upregulation and downregulation for ACR vs non-ACR samples as compared with control miRNA from a pool of tissue from 25 normal bowels. While these studies are preliminary, they are promising in that they suggest a possible role for various miRNAs in potentially regulating or potentiating an alloimmune response. Should one or more of these miRNAs be demonstrated as consistently associated with the onset and progression of acute rejection, there are many potential directions that can be pursued including the establishment of possible biomarkers, targets for intervention such as anti-miR miRNA inhibitors,11 and studies of upstream (eg, pre-miR miRNA precursors) and downstream genes in the miRNA pathways. Taqman miRNA arrays may also be used to evaluate changes between various treatment regimens in bowel transplantation and whether certain miRNA gene expression patterns can be linked to rejection vs graft survival. ACKNOWLEDGEMENT Dayami Hernandez provided histologic support; Irvana EloundouAvomo, technical support; and Cristina Hersh, clerical expertise.
REFERENCES 1. Liang Y, Ridzon D, Wong L, et al: Characterization of microRNA expression profiles in normal human tissues. BMC Genomics 8:166, 2007 2. Garzon R, Calin GA, Croce CM: MicroRNAs in cancer. Ann Rev Med 60:167, 2009 3. Szafranska A, Davison T, Shingara J, et al: Accurate molecular characterization of formalin-fixed, paraffin-imbedded tissues by microRNA expression profiling. J Mol Diagn 10:415, 2008 4. Visone R, Petrocca F, Croce CM: Micro-RNAs in gastrointestinal and liver disease. Gastroenterology 135:1866, 2008 5. Saito Y, Suzuki H, Hibi T: The role of microRNAs in gastrointestinal cancers. J Gastroenterol 44:18022, 2009 6. Lu L, Liston A: MicroRNA in the immune system, microRNA as an immune system. Immunology 27:291, 2008 7. Kohlhaas S, Garden O, Scudamore C, et al: Cutting edge: the Foxp3 target mir-155 contributes to the development of regulatory T-cells. J Immunol 182:2578, 2009 8. Hou J, Wang P, Lin L, et al: MicroRNA-146a feedback inhibits RIG-I-dependent type I IFN production in macrophages by targeting TRAF6, IRAK1, and IRAK2. J Immunol 183:2150, 2009 9. Sui W, Dai Y, Huang Y, et al: Microarray analysis of microRNA expression in acute rejection after renal transplantation. Transplant Immunol 19:81, 2008 10. Anglicheau D, Sharma VK, Ding R, et al: MicroRNA expression profiles predictive of human renal allograft status. Proc Natl Acad Sci USA 106:5330, 2009 11. Brown BD, Naldini L: Exploiting and antagonizing microRNA regulation for therapeutic and experimental applications. Nature 10:578, 2009
H
0041-1345/10/$–see front matter doi:10.1016/j.transproceed.2009.12.018 62
death.2 MicroRNAs could become biomarkers correlative to human disease states by either upregulation or downregulation of gene expression patterns.2,3 In the present
From the Departments of Surgery (B.S., T.A., E.I., M.C., V.D., D.C., P.T., J.M., D.W., A.T., P.R.) and Pathology (P.R.), University of Miami Leonard M. Miller School of Medicine, Miami, Florida, and the Microbiology Unit, Laboratory Department, Bambino Gesu Children’s Hospital, Rome, Italy (C.R.). Address reprint requests to Philip Ruiz, MD, PhD, Department of Surgery, University of Miami School of Medicine, Rosensteil Medical Sciences Building, Transplant Laboratories, 1600 NW 10th Ave, Suite 8150, Miami, FL 33136. E-mail: pruiz@med. miami.edu © 2010 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 42, 62– 65 (2010)
MICRO-RNA IN MUCOSAL BIOPSY TISSUE
study, we used the gene expression profiling arrays for a panel of known miRNAs to monitor gene expression patterns in formalin-fixed paraffin-embedded (FFPE) biopsy tissues. We evaluated several FFPE samples from smallbowel transplant biopsy specimens that either demonstrated acute cellular rejection (ACR; moderate to severe, grades 2 or 3) or were indeterminate for rejection (grade IND), all in comparison with pooled normal bowel by using real-time polymerase chain reaction (PCR) gene expression profiling using a Taqman miRNA gene array. Liang et al1 characterized miRNA expression profiles in normal human tissues and showed clear clustering of gene
63
expression by tissue types in fresh tissue; however, that study did not investigate FFPE tissue. Szafranska et al3 demonstrated that accurate molecular characterization and expression profiling could be performed on FFPE and frozen tissue in assessment of miRNAs. Many studies have been conducted on the nature of miRNA genes in their role in cancer as tumor suppressors and as oncogenes,2,4 as in the case of miRNA 106b-25 cluster (106b, 92 and 25) found in gastric carcinomas; miRNA 21, which is highly expressed in pancreatic cancer; and miRNA 145 downregulated in colon cancer.4 Saito et al5 described the role of miRNAs 15b, 16, 34a, 143, and 145 as tumor suppressors, and
Fig 1. Upregulated (A) and downregulated (B) microRNA genes in FFPE from mucosal biopsy specimens exhibit ACR/ non-ACR relative fold change.
64
miRNA genes 17–92 cluster, 21, 106a, 106b-25 cluster, and 155 in gastrointestinal cancers. The role of miRNA genes in immune regulation is just beginning to emerge, with little information in the area of alloimmunity. Recent studies have shown that miRNAs 125b, 146, 155, and 223 have a role in the innate immune system and that miRNAs 17–92, 150, 155, and 181 in B and T cells.6 A recent study by Kohlhaas et al7 found that Foxp3 targets miRNA 155 and contributes to the development of regulatory T cells. Hou et al8 have shown that mice macrophages after vesicular stomatitis viral challenge exhibit upregulation of miRNA genes 146a, 155, 7a, 574-5p, and 125a, which demonstrates the influence of viruses on miRNA genes. Sui et al9 found that 20 miRNA genes differentially expressed upregulation and downregulation in comparison with normal control samples in kidney allograft rejection transplant samples. Among the miRNA genes in that study that were differentially expressed were upregulated miRNA genes 125a, 320, 381, 602, 629, and 658, and downregulated miRNA genes 324a, 611, 654, 330, 524,
SOTOLONGO, ASAOKA, ISLAND ET AL
17-3p, 483, 663, 516-5p, 326, 197, and 346.9 In another renal transplant biopsy study conducted by Anglicheau et al,10 acute rejection allograft status was indicative of overexpression of miRNAs 142-5p, 155, and 223. MATERIALS AND METHODS Real-time PCR gene expression profiling was performed on FFPE samples from bowel transplant recipients and from RNA isolated from a control pool composed of 25 samples of normal small-bowel FFPE biopsies. The RNA for the experimental groups was collected from small-bowel biopsy samples from the following 2 groups: moderate to severe ACR (grade 2 or 3; 7 samples) and indeterminate for rejection (grade IND; 5 non-ACR samples). The FFPE samples (ⱕ35 mg) were deparaffinized with xylene and ethanol washes. The samples were then treated with protease to remove all proteins. Next, the total RNA was isolated without depletion of miRNAs (which can occur with most RNA isolation kits) using a total nucleic acid isolation kit for FFPE (Recoverall; Applied Biosystems, Inc, Foster City, California). A glass filter method that included a deoxyribonuclease treatment was used to purify the total RNA with this kit. The RNA was then transcribed
Fig 2. Differential expression pattern of 98 microRNAs in intestinal transplant biopsy specimens comparing non-ACR and ACR upregulated and downregulated microRNAs using Treeview PCR software to cluster genes.
MICRO-RNA IN MUCOSAL BIOPSY TISSUE into complementary DNA using a MicRNA reverse transcription kit (Applied Biosystems, Inc). The cDNA was preamplified with a pool of miRNA primers to amplify target miRNA sequences. Real-time PCR was performed using, on average, 100 ng of cDNA per port loaded onto a Taqman MiRNA assay (Early Access microfluidic card, version 1.0; Applied Biosystems, Inc) and run on a 7900 HT Fast real-time PCR system (Applied Biosystems, Inc). The miRNA array has 384 miRNAs. Each array was ensured for gene specificity against both transcript and genome public databases such as BLAST (Basic Local Alignment Search Tool; National Center for Biotechnology. Information, US National Library of Medicine, Bethesda, Maryland). The Taqman MiRNA array was analyzed using relative quantitation software (Sequence Detection Systems, version 2.3; Applied Biosystems, Inc) using the ⌬⌬ Ct method. All samples were normalized to a miRNA mammalian endogenous control gene, U6. A pool of 25 normal small bowel FFPE biopsy tissues was used as the calibrator. Treeview PC-based software was used for heat map image analysis of the gene expression pattern.
RESULTS
Our experimental biopsy specimens were grouped into 2 groups: indeterminate for rejection (non-ACR) and moderate to severe rejection (ACR). Compared with pooled normal FFPE control tissues, the experimental samples showed both upregulation and downregulation of several miRNAs. The miRNA genes with a fold change (relative quantitation) to the normal pool with ratios of ACR and non-ACR median 2.0 or greater (upregulated) or 0.5 or less (downregulated) were recognized as significant. The patterns of upregulated and downregulated genes were differentially expressed for the non-ACR samples vs ACR samples. Sixty-two miRNA genes were upregulated in small bowels with ACR, and 35 were downregulated (data not shown). Forty-two miRNA genes were upregulated in nonACR small bowel biopsy samples (grade IND), and 45 were downregulated (data not shown). The relative fold change ratio of ACR to non-ACR was calculated, and 50 upregulated and 8 downregulated miRNAs were detected as significant (Fig 1). Treeview PCR software was used for a gene cluster of miRNA genes and detected 98 miRNAs in non-ACR and ACR sample genes with a P value of ⱕ.5 (significant). This image showed a differential gene clustering expression pattern of 98 miRNAs in non-ACR vs ACR samples (Fig 2). Among the most interesting miRNAs to be evaluated in the future from the many that were observed in this microarray were the downregulated miRNA genes 15b, 28-5p, RNU48, 106b, 192, 197, 200c, and 221. Among the most promising upregulated miRNA genes were 125a, 142, 155, and 629, which also appear upregulated in kidney acute rejection.4,9 DISCUSSION
Our initial experiments using FFPE mucosal small bowel biopsy specimens demonstrate that miRNA genes can be
65
successfully measured. Our data show that the histologic degree of acute rejection is associated with various patterns of upregulation or downregulation of miRNA genes. Moreover, the transplantation biopsy specimens exhibited differential profiles of upregulation and downregulation for ACR vs non-ACR samples as compared with control miRNA from a pool of tissue from 25 normal bowels. While these studies are preliminary, they are promising in that they suggest a possible role for various miRNAs in potentially regulating or potentiating an alloimmune response. Should one or more of these miRNAs be demonstrated as consistently associated with the onset and progression of acute rejection, there are many potential directions that can be pursued including the establishment of possible biomarkers, targets for intervention such as anti-miR miRNA inhibitors,11 and studies of upstream (eg, pre-miR miRNA precursors) and downstream genes in the miRNA pathways. Taqman miRNA arrays may also be used to evaluate changes between various treatment regimens in bowel transplantation and whether certain miRNA gene expression patterns can be linked to rejection vs graft survival. ACKNOWLEDGEMENT Dayami Hernandez provided histologic support; Irvana EloundouAvomo, technical support; and Cristina Hersh, clerical expertise.
REFERENCES 1. Liang Y, Ridzon D, Wong L, et al: Characterization of microRNA expression profiles in normal human tissues. BMC Genomics 8:166, 2007 2. Garzon R, Calin GA, Croce CM: MicroRNAs in cancer. Ann Rev Med 60:167, 2009 3. Szafranska A, Davison T, Shingara J, et al: Accurate molecular characterization of formalin-fixed, paraffin-imbedded tissues by microRNA expression profiling. J Mol Diagn 10:415, 2008 4. Visone R, Petrocca F, Croce CM: Micro-RNAs in gastrointestinal and liver disease. Gastroenterology 135:1866, 2008 5. Saito Y, Suzuki H, Hibi T: The role of microRNAs in gastrointestinal cancers. J Gastroenterol 44:18022, 2009 6. Lu L, Liston A: MicroRNA in the immune system, microRNA as an immune system. Immunology 27:291, 2008 7. Kohlhaas S, Garden O, Scudamore C, et al: Cutting edge: the Foxp3 target mir-155 contributes to the development of regulatory T-cells. J Immunol 182:2578, 2009 8. Hou J, Wang P, Lin L, et al: MicroRNA-146a feedback inhibits RIG-I-dependent type I IFN production in macrophages by targeting TRAF6, IRAK1, and IRAK2. J Immunol 183:2150, 2009 9. Sui W, Dai Y, Huang Y, et al: Microarray analysis of microRNA expression in acute rejection after renal transplantation. Transplant Immunol 19:81, 2008 10. Anglicheau D, Sharma VK, Ding R, et al: MicroRNA expression profiles predictive of human renal allograft status. Proc Natl Acad Sci USA 106:5330, 2009 11. Brown BD, Naldini L: Exploiting and antagonizing microRNA regulation for therapeutic and experimental applications. Nature 10:578, 2009