Exploring lncRNAs associated with human pancreatic islet cell death induced by transfer of adoptive lymphocytes in a humanized mouse model.

Background: Long noncoding RNA (lncRNA)-mediated posttranscriptional and epigenetic landscapes of gene regulation are associated with numerous human diseases. However, the regulatory mechanisms governing human ß-cell function and survival remain unknown. Owing to technical and ethical constraints, studying the direct role of lncRNAs in ß-cell function and survival in humans in vivo is difficult. Therefore, we utilized humanized mice with human islets to investigate lncRNA expression using whole transcriptome shotgun sequencing. Our study aimed to characterize lncRNAs that may be crucial for human islet cell function and survival. Methods: Human ß-cell death was induced in humanized mice engrafted with functional human islets. Using these humanized mice harboring human islets with induced ß-cell death, we investigated lncRNA expression through whole transcriptome shotgun sequencing. Additionally, we systematically identified, characterized, and explored the regulatory functions of lncRNAs that are potentially important for human pancreatic islet cell function and survival. Results: Human islet cell death was induced in humanized mice engrafted with functional human islets. RNA sequencing analysis of isolated human islets, islet grafts from humanized mice with and without induced cell death, revealed aberrant expression of a distinct set of lncRNAs that are associated with the deregulated mRNAs important for cellular processes and molecular pathways related to ß-cell function and survival. A total of 10 lncRNA isoforms (SCYL1-1:22, POLG2-1:1, CTRB1-1:1, SRPK1-1:1, GTF3C5-1:1, PPY-1:1, CTRB1-1:5, CPA5-1:1, BCAR1-2:1, and CTRB1-1:4) were identified as highly enriched and specific to human islets. These lncRNAs were deregulated in human islets from donors with different BMIs and with type 2 diabetes (T2D), as well as in cultured human islets with glucose stimulation and induced cell death induced by cytokines. Aberrant expression of these lncRNAs was detected in the exosomes from the medium used to culture islets with cytokines. Conclusion: Islet-enriched and specific human lncRNAs are deregulated in human islet grafts and cultured human islets with induced cell death. These lncRNAs may be crucial for human ß-cell function and survival and could have an impact on identifying biomarkers for ß-cell loss and discovering novel therapeutic targets to enhance ß-cell function and survival.

Circulating miRNA-150-5p is associated with immune-mediated early ß-cell loss in a humanized mouse model.

BACKGROUND: Aberrant microRNA (miRNA) expression levels are associated with various graft rejections. We used our humanized mouse model with transplanted human islets to identify miRNAs in islet grafts related to xenograft rejection and circulating miRNAs associated with xenograft rejection-mediated ß-cell loss. METHODS: Diabetic immunodeficient NOD.scid mice were transplanted with human islets and subsequently achieved stable normoglycemia. Lymphocytes from NOD mice were then adoptively transferred to the humanized mice to induce human ß-cell destruction. Islet graft and plasma were collected immediately once blood glucose reached >200 mg/dL. miRNAs in the islet grafts and in the plasma with or without adoptive lymphocyte transfer (ALT) were measured using NanoString nCounter® miRNA Expression Assay and qPCR. RESULTS: A set of immune-related miRNAs was significantly increased in human islet grafts of ALT-treated mice compared to control mice. Of these miRNAs, miR-150-5p was significantly increased in the circulation of ALT-treated mice at tissue collection and the increase was a result of immune activation rather than simply the presence of lymphocytes in circulation. Furthermore, miR-150-5p was significantly increased in human islet graft and circulation prior to the development of hyperglycemia in the ALT-treated mice. CONCLUSIONS: Our data demonstrated that immune-related miRNAs are associated with human islet xenograft rejection in mice. miR-150-5p is increased in human islet graft and in the circulation during islet xenograft rejection and ß-cell destruction prior to hyperglycemia and may be an early biomarker for islet xenograft rejection.

MicroRNA Expression Profile in Bovine Granulosa Cells of Preovulatory Dominant and Subordinate Follicles during the Late Follicular Phase of the Estrous Cycle.

In bovine, ovarian follicles grow in a wave-like fashion with commonly 2 or 3 follicular waves emerging per estrous cycle. The dominant follicle of the follicular wave which coincides with the LH-surge becomes ovulatory, leaving the subordinate follicles to undergo atresia. These physiological processes are controlled by timely and spatially expressed genes and gene products, which in turn are regulated by post-transcriptional regulators. MicroRNAs, a class of short non-coding RNA molecules, are one of the important posttranscriptional regulators of genes associated with various cellular processes. Here we investigated the expression pattern of miRNAs in granulosa cells of bovine preovulatory dominant and subordinate follicles during the late follicular phase of bovine estrous cycle using Illumina miRNA deep sequencing. In addition to 11 putative novel miRNAs, a total of 315 and 323 known miRNAs were detected in preovulatory dominant and subordinate follicles, respectively. Moreover, in comparison with the subordinate follicles, a total of 64 miRNAs were found to be differentially expressed in preovulatory dominant follicles, of which 34 miRNAs including the miR-132 and miR-183 clusters were significantly enriched, and 30 miRNAs including the miR-17-92 cluster, bta-miR-409a and bta-miR-378 were significantly down regulated in preovulatory dominant follicles. In-silico pathway analysis revealed that canonical pathways related to oncogenesis, cell adhesion, cell proliferation, apoptosis and metabolism were significantly enriched by the predicted target genes of differentially expressed miRNAs. Furthermore, Luciferase reporter assay analysis showed that one of the differentially regulated miRNAs, the miR-183 cluster miRNAs, were validated to target the 3'-UTR of FOXO1 gene. Moreover FOXO1 was highly enriched in granulosa cells of subordinate follicles in comparison with the preovulatory dominant follicles demonstrating reciprocal expression pattern with miR-183 cluster miRNAs. In conclusion, the presence of distinct sets of miRNAs in granulosa cells of preovulatory dominant and subordinate follicles supports the potential role of miRNAs in post-transcriptional regulation of genes involved in bovine follicular development during the late follicular phase of the estrous cycle.

Polycystic ovarian syndrome is accompanied by repression of gene signatures associated with biosynthesis and metabolism of steroids, cholesterol and lipids.

BACKGROUND: Polycystic ovarian syndrome (PCOS) is a spectrum of heterogeneous disorders of reproduction and metabolism in women with potential systemic sequel such as diabetes and obesity. Although, PCOS is believed to be caused by genetic abnormalities, the genetic background that can be associated with PCOS phenotypes remains unclear due to the complexity of the trait. In this study, we used a rat model which exhibits reproductive and metabolic abnormalities similar to the human PCOS to unravel the molecular mechanisms underlining this complex syndrome. METHODS: Female Sprague-Dawley rats were randomly assigned to DHT and control (CTL) groups. Rats in the DHT group were implanted with a silicone capsule continuous-releasing 83 µg 5α-dihydrotestosterone (DHT) per day for 12 weeks to mimic the hyperandrogenic state in women with PCOS. The animals were euthanized at 15 weeks of age and the pairs of ovaries were excised and the ovarian cortex tissues were used for gene expression analysis. Total RNA was from the ovarian cortex was amplified, labeled and hybridized to the Affymetrix GeneChip® Rat Genome 230 2.0 Array. A linear model system for microarray data analysis was used to identify genes affected in DHT treated rat ovaries and the molecular pathway of those genes were analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis tool. RESULTS: A total of 573 gene transcripts, including CPA1, CDH1, INSL3, AMH, ALDH1B1, INHBA, CYP17A1, RBP4, GAS6, GAS7 and GATA4, were activated while 430 others including HSD17B7, HSD3B6, STAR, HMGCS1, HMGCR, CYP51, CYP11A1 and CYP19A1 were repressed in DHT-treated ovaries. Functional annotation of the dysregulated genes revealed that biosynthesis and metabolism of steroids, cholesterol and lipids to be the most top functions enriched by the repressed genes. However, cell differentiation/proliferation, transcriptional regulation, neurogenesis, cell adhesion and blood vessel development processes were enriched by activated genes. CONCLUSION: The dysregulation of genes associated with biosynthesis and metabolism of steroids, cholesterol and lipids, cell differentiation/proliferation in DHT- treated ovaries could be a molecular clue for abnormal steroidogenesis, estrous cycle irregularity, abnormal folliculogenesis, anovulation and lipid metabolism in PCOS patients.

Sulforaphane epigenetically regulates innate immune responses of porcine monocyte-derived dendritic cells induced with lipopolysaccharide.

Histone acetylation, regulated by histone deacetylases (HDACs) is a key epigenetic mechanism controlling gene expressions. Although dendritic cells (DCs) are playing pivotal roles in host immune responses, the effect of epigenetic modulation of DCs immune responses remains unknown. Sulforaphane (SFN) as a HDAC inhibitor has anti-inflammatory properties, which is used to investigate the epigenetic regulation of LPS-induced immune gene and HDAC family gene expressions in porcine monocyte-derived dendritic cells (moDCs). SFN was found to inhibit the lipopolysaccharide LPS induced HDAC6, HDAC10 and DNA methyltransferase (DNMT3a) gene expression, whereas up-regulated the expression of DNMT1 gene. Additionally, SFN was observed to inhibit the global HDAC activity, and suppressed moDCs differentiation from immature to mature DCs through down-regulating the CD40, CD80 and CD86 expression and led further to enhanced phagocytosis of moDCs. The SFN pre-treated of moDCs directly altered the LPS-induced TLR4 and MD2 gene expression and dynamically regulated the TLR4-induced activity of transcription factor NF-κB and TBP. SFN showed a protective role in LPS induced cell apoptosis through suppressing the IRF6 and TGF-ß1 production. SFN impaired the pro-inflammatory cytokine TNF-α and IL-1ß secretion into the cell culture supernatants that were induced in moDCs by LPS stimulation, whereas SFN increased the cellular-resident TNF-α accumulation. This study demonstrates that through the epigenetic mechanism the HDAC inhibitor SFN could modulate the LPS induced innate immune responses of porcine moDCs.

Massive deregulation of miRNAs from nuclear reprogramming errors during trophoblast differentiation for placentogenesis in cloned pregnancy.

BACKGROUND: Low efficiency of Somatic Cell Nuclear Transfer (NT) has been widely addressed with high incidence of placental abnormalities due to genetic and epigenetic modifications. MiRNAs are shown to be major regulators of such modifications. The present study has been carried out to identify the expression patterns of 377 miRNAs, their functional associations and mechanism of regulation in bovine placentas derived from artificial insemination (AI), in vitro production (IVP) and NT pregnancies. RESULTS: This study reveals a massive deregulation of miRNAs as chromosomal cluster or miRNA families without sex-linkage in NT and in-vitro derived IVP placentas. Cell specific localization miRNAs in blastocysts and expression profiling of embryos and placentas at different developmental stages identified that the major deregulation of miRNAs exhibited in placentas at day 50 of pregnancies is found to be less dependent on global DNA methylation, rather than on aberrant miRNA biogenesis molecules. Among them, aberrant AGO2 expression due to hypermethylation of its promoter was evident. Along with other factors, aberrant AGO2 expression was observed to be associated with multiple defects in trophoblast differentiation through deregulation of miRNAs mediated mechanisms. CONCLUSION: These aberrant miRNA activities might be associated with genetic and epigenetic modifications in abnormal placentogenesis due to maldifferentiation of early trophoblast cell lineage in NT and IVP pregnancies. This study provides the first insight into genome wide miRNA expression, their role in regulation of trophoblast differentiation as well as abnormal placental development in Somatic Cell Nuclear Transfer pregnancies to pave the way to improve the efficiency of cloning by nuclear transfer.

Altered expression of miRNAs in a dihydrotestosterone-induced rat PCOS model.

BACKGROUND: The polycystic ovary syndrome (PCOS) is a complex and heterogeneous endocrine condition characterized by hyperandrogenism, hyperinsulinemia, insulin resistance and chronic anovulation. Regulation and interaction of a multitude of genes required for follicular development are found to be altered in PCOS. MicroRNAs (miRNAs) mediate posttranscriptional gene regulation by binding to the 3´ untranslated region of mRNAs to either inhibit or enhance translation. However, the extent and regulation of miRNA expression in PCOS is poorly understood and the current study is the first to describe altered expression of miRNAs in PCOS. METHODS: A chronically androgenized [5α-dihydrotestosterone (DHT)-treated] rat model which recapitulates many of the phenotypes of human PCOS, and miRNA PCR array was used to investigate the expression of 349 miRNAs in DHT treated rat ovaries. The ovarian expression of several selected miRNAs was also analyzed by in situ localization experiment. RESULTS: DHT-treated rats exhibit increased body weight, disrupted estrus cyclicity, decreased insulin sensitivity and decreased ovarian weight, with the latter phenomenon readily rescued by gonadotropin treatment in vivo. In general, 24% of the 349 miRNAs investigated were found to be differentially expressed between DHT-treated and control rats. Most of the differentially expressed miRNAs were found to be predominantly localized in the theca cells of the follicles. In silico analysis of the potential target genes of dysregulated miRNAs revealed their possible involvement in various pathways in the regulation of ovarian function. CONCLUSION: Our current findings suggest that miRNAs are differentially regulated in hyperandrogenism, a condition possibly involved in the dysregulation of steroid hormone receptors and intra-ovarian factors, and that miRNAs may be involved in the etiology of PCOS.

Identification and characterization of miRNAs expressed in the bovine ovary.

BACKGROUND: MicroRNAs are the major class of gene-regulating molecules playing diverse roles through sequence complementarity to target mRNAs at post-transcriptional level. Tightly regulated expression and interaction of a multitude of genes for ovarian folliculogenesis could be regulated by these miRNAs. Identification of them is the first step towards understanding miRNA-guided gene regulation in different biological functions. Despite increasing efforts in miRNAs identification across various species and diverse tissue types, little is known about bovine ovarian miRNAs. Here, we report the identification and characterization of miRNAs expressed in the bovine ovary through cloning, expression analysis and target prediction. RESULTS: The miRNA library (5'-independent ligation cloning method), which was constructed from bovine ovary in this study, revealed cloning of 50 known and 24 novel miRNAs. Among all identified miRNAs, 38 were found to be new for bovine and were derived from 43 distinct loci showing characteristic secondary structure. While 22 miRNAs precursor loci were found to be well conserved in more than one species, 16 were found to be bovine specific. Most of the miRNAs were cloned multiple times, in which let-7a, let-7b, let-7c, miR-21, miR-23b, miR-24, miR-27a, miR-126 and miR-143 were cloned 10, 28, 13, 4, 11, 7, 6, 4 and 11 times, respectively. Expression analysis of all new and some annotated miRNAs in different intra-ovarian structures and in other multiple tissues showed that some were present ubiquitously while others were differentially expressed among different tissue types. Bta-miR-29a was localized in the follicular cells at different developmental stages in the cyclic ovary. Bio-informatics prediction, screening and Gene Ontology analysis of miRNAs targets identified several biological processes and pathways underlying the ovarian function. CONCLUSION: Results of this study suggest the presence of miRNAs in the bovine ovary, thereby elucidate their potential role in regulating diverse molecular and physiological pathways underlying the ovarian functionality. This information will give insights into bovine ovarian miRNAs, which can be further characterized for their role in follicular development and female fertility as well.