ABSTRACT
Despite continuous improvement of immunosuppression, small bowel transplantation (SBT) is plagued by a high incidence of acute cellular rejection (ACR) that is frequently intractable. Therefore, there is a need to uncover novel insights that will lead to strategies to achieve better control of ACR. We hypothesized that particular miRNAs provide critical regulation of the intragraft immune response. The aim of our study was to identify miRNAs involved in intestinal ACR. We examined 26 small intestinal mucosal biopsies (AR/NR group; 15/11) obtained from recipients after SBT or multivisceral transplantation. We investigated the expression of 384 mature human miRNAs and 280 mRNAs associated with immune, inflammation and apoptosis processes. We identified differentially expressed 28 miRNAs and 58 mRNAs that characterized intestinal ACR. We found a strong positive correlation between the intragraft expression levels of three miRNAs (miR-142-3p, miR-886-3p and miR-132) and 17 mRNAs including CTLA4 and GZMB. We visualized these miRNAs within cells expressing CD3 and CD14 proteins in explanted intestinal allografts with severe ACR. Our data suggested that miRNAs have a critical role in the activation of infiltrating cells during intestinal ACR. These differences in miRNA expression patterns can be used to identify novel biomarkers and therapeutic targets for immunosuppressive agents.
Subject(s)
Gene Expression Regulation , Graft Rejection/genetics , Intestinal Mucosa/pathology , Intestine, Small/transplantation , MicroRNAs/genetics , Acute Disease , Adolescent , Adult , Aged , Biopsy , Child , Child, Preschool , Female , Fixatives/pharmacology , Formaldehyde/pharmacology , Gene Expression Profiling , Graft Rejection/metabolism , Graft Rejection/pathology , Humans , In Situ Hybridization, Fluorescence , Infant , Infant, Newborn , Intestinal Mucosa/metabolism , Intestine, Small/metabolism , Intestine, Small/pathology , Male , MicroRNAs/biosynthesis , Middle Aged , Paraffin Embedding , Real-Time Polymerase Chain Reaction , Transplantation, Homologous , Young AdultABSTRACT
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.
Subject(s)
Gene Expression Profiling , Intestinal Mucosa/pathology , Intestine, Small/transplantation , MicroRNAs/genetics , Biopsy , Down-Regulation , Graft Rejection/diagnosis , Graft Rejection/pathology , Humans , MicroRNAs/isolation & purification , Polymerase Chain Reaction/methods , RNA/genetics , RNA/isolation & purification , Transplantation, Homologous , Up-RegulationABSTRACT
It is well known that aberrations in the nuclear matrix contribute to the development of cancer, but many aspects of this process remain unknown. The mammalian sperm nuclear matrix serves as a distinctive model of DNA loop domain organization by the nuclear matrix since the integrity of the DNA structure can be measured by the ability of the paternal chromosomes to participate in embryogenesis. The structure of the nuclear matrix is known to be important for normal cellular functions such as transcriptional regulation and DNA replication. Even small aberrations in DNA structural organization in the sperm cell could have disastrous consequences for the embryo if they were essential for function. Recent work from our laboratory suggests that sperm nuclei with disrupted nuclear matrix structures but intact DNA cannot participate fully in embryogenesis, suggesting that the structural organization of DNA may provide important, heritable information that is necessary for development. We term the DNA sequence together with its three-dimensional organization the "genomic code." We suggest that the sperm nucleus is an ideal model for understanding the principles of the involvement of the three-dimensional structure of DNA in normal cellular function. Finally, the implications for cancer about what we can learn using sperm DNA as a model about the "genomic code" are discussed. J. Cell. Biochem. Suppl. 35:23-26, 2000.
