microRNA Target Sites Research Articles

De novo computational identification of stress-related sequence motifs and microRNA target sites in untranslated regions of a plant translatome

Gene regulation at the transcriptional and translational level leads to diversity in phenotypes and function in organisms. Regulatory DNA or RNA sequence motifs adjacent to the gene coding sequence act as binding sites for proteins that in turn enable or disable expression of the gene. Whereas the known DNA and RNA binding proteins range in the thousands, only a few motifs have been examined. In this study, we have predicted putative regulatory motifs in groups of untranslated regions from genes regulated at the translational level in Arabidopsis thaliana under normal and stressed conditions. The test group of sequences was divided into random subgroups and subjected to three de novo motif finding algorithms (Seeder, Weeder and MEME). In addition to identifying sequence motifs, using an in silico tool we have predicted microRNA target sites in the 3′ UTRs of the translationally regulated genes, as well as identified upstream open reading frames located in the 5′ UTRs. Our bioinformatics strategy and the knowledge generated contribute to understanding gene regulation during stress, and can be applied to disease and stress resistant plant development.

A microRNA family exerts maternal control on sex determination in C. elegans.

Gene expression in early animal embryogenesis is in large part controlled post-transcriptionally. Maternally contributed microRNAs may therefore play important roles in early development. We elucidated a major biological role of the nematode mir-35 family of maternally contributed essential microRNAs. We show that this microRNA family regulates the sex determination pathway at multiple levels, acting both upstream of and downstream from her-1 to prevent aberrantly activated male developmental programs in hermaphrodite embryos. Both of the predicted target genes that act downstream from the mir-35 family in this process, suppressor-26 (sup-26) and NHL (NCL-1, HT2A, and LIN-41 repeat) domain-containing-2 (nhl-2), encode RNA-binding proteins, thus delineating a previously unknown post-transcriptional regulatory subnetwork within the well-studied sex determination pathway of Caenorhabditis elegans Repression of nhl-2 by the mir-35 family is required for not only proper sex determination but also viability, showing that a single microRNA target site can be essential. Since sex determination in C. elegans requires zygotic gene expression to read the sex chromosome karyotype, early embryos must remain gender-naïve; our findings show that the mir-35 family microRNAs act in the early embryo to function as a developmental timer that preserves naïveté and prevents premature deleterious developmental decisions.

Association of Casp3 microRNA Target Site (1049216) SNP With the Risk and Progress of Cervical Squamous Cell Carcinoma.

In the present study, we investigated the relationship between the single-nucleotide polymorphism (SNP) of caspase-3 rs1049216 (C > T), a miRNA target site, and the risk and progression of cervical cancer. Using polymerase chain reaction-restriction fragment length polymorphism, we evaluated the genotype and distribution of caspase-3 rs1049216 in 515 patients with cervical squamous cell cancer and 415 controls. In additional experiments, we transfected luciferase reporter plasmids carrying T or C allele and/or miRNA mimics into the human cervical cell lines (HeLa and C-33A) to analyze its roles in the regulation of caspase-3 expression. By immunohistochemistry, the protein level of caspase-3 expression was examined in tumor tissues from 515 patients with cervical squamous cell cancer. We found that the TT genotype of caspase-3 rs1049216 conferred a significantly decreased risk of cervical cancer (adjusted odds ratio, 0.35; 95% confidence interval, 0.154-0.581) and may be associated with the progression of this cancer. Although the expression of caspase-3 in the TT genotype was higher than that in CC/CT genotype in peripheral blood mononuclear cells and tumor tissues. Additional luciferase analysis showed that the rs1049216 variant T allele was associated with significantly higher luciferase activity, compared with the C allele in the transfected cells, and when cotransfected with miRNAs, miRNA-181a could downregulate the luciferase activity in the cells that transfected the construct containing C allele, compared with T allele, which had not happened in the presence of other miRNAs selected. These data indicate that through upregulating the expression of caspase-3, the TT genotype of caspase-3 rs1049216 can be associated with not only the risk of cervical cancer but also the progression of this cancer.

Transcriptome Analysis of Leaves, Flowers and Fruits Perisperm of Coffea arabica L. Reveals the Differential Expression of Genes Involved in Raffinose Biosynthesis.

Coffea arabica L. is an important crop in several developing countries. Despite its economic importance, minimal transcriptome data are available for fruit tissues, especially during fruit development where several compounds related to coffee quality are produced. To understand the molecular aspects related to coffee fruit and grain development, we report a large-scale transcriptome analysis of leaf, flower and perisperm fruit tissue development. Illumina sequencing yielded 41,881,572 high-quality filtered reads. De novo assembly generated 65,364 unigenes with an average length of 1,264 bp. A total of 24,548 unigenes were annotated as protein coding genes, including 12,560 full-length sequences. In the annotation process, we identified nine candidate genes related to the biosynthesis of raffinose family oligossacarides (RFOs). These sugars confer osmoprotection and are accumulated during initial fruit development. Four genes from this pathway had their transcriptional pattern validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, we identified ~24,000 putative target sites for microRNAs (miRNAs) and 134 putative transcriptionally active transposable elements (TE) sequences in our dataset. This C. arabica transcriptomic atlas provides an important step for identifying candidate genes related to several coffee metabolic pathways, especially those related to fruit chemical composition and therefore beverage quality. Our results are the starting point for enhancing our knowledge about the coffee genes that are transcribed during the flowering and initial fruit development stages.

MiR-200a-3p promotes the proliferation of human esophageal cancer cells by post-transcriptionally regulating cytoplasmic collapsin response mediator protein-1.

The dysregulation of cytoplasmic collapsin response mediator protein1 (CRMP1) has been reported in lung cancer, medulloblastoma and esophageal cancer. However, the role of CRMP1 and its regulatory mechanisms in esophageal cancer remain unclear. In this study, we demonstrated that CRMP1 expression was downregulated in esophageal cancer tissues and that there were differences in its expression levels in different esophageal cancer cell lines. We found that CRMP1 overexpression inhibited the proliferation of esophageal cancer cells, whereas the silencing of CRMP1 promoted cell proliferation. We performed an analysis of potential microRNA(miRNA or miR) target sites using a commonly used prediction algorithm(TargetScan). The algorithm predicted that miR‑200a-3p targets the 3'untranslated region(3'UTR) of CRMP1. Further experiments confirmed this prediction. In addition, we found that miR‑200a-3p promoted the proliferation of esophageal cancer cells. Thus, our findings indicate that miR‑200a-3p promotes the proliferation of human esophageal cancer cells by post-transcriptionally regulating CRMP1.

In silico dissection of miRNA targetome polymorphisms and their role in regulating miRNA-mediated gene expression in esophageal cancer.

Esophageal cancer is the eighth most common cancer worldwide. Also middle-aged obese adults with higher body mass index during childhood have a greater risk to develop esophageal cancer. The contribution of microRNAs to esophageal cancer has been extensively studied and it became clear that these noncoding RNAs may play crucial roles in pathogenesis, diagnosis and prognosis of the disease. Increasing evidences have suggested that polymorphisms perturbing microRNA targetome (i.e., the compendium of all microRNA target sites) are associated with cancers including esophageal cancer. However, the extent to which such variants contribute to esophageal cancer is still unclear. In this study, we applied an in silico approach to systematically identify polymorphisms perturbing microRNA targetome in esophageal cancer and performed various analyses to predict the functional consequences of the occurrence of these variants. The computational results were integrated to provide a prioritized list of the most potentially disrupting esophageal cancer-implicated microRNA targetome polymorphisms along with the in silico insight into the mechanisms with which such variations may modulate microRNA-mediated regulation. The results of this study will be valuable for future functional experiments aimed at dissecting the roles of microRNA targetome polymorphisms in the onset and progression of esophageal cancer.

Abstract 1903: A genetic variation in microRNA target site of ETS2 is associated with clinical outcomes of paclitaxel-cisplatin chemotherapy in non-small cell lung cancer

Abstract Background: Recently, crosslinking, ligation, and sequencing of hybrids (CLASH) provided direct observation of transcriptome-wide miRNA-target pairs. The present study was performed to investigate the association of single nucleotide polymorphisms (SNPs) located in the miRNA target sites, which were experimentally verified by CLASH, with the clinical outcome of first line paclitaxel-cisplatin chemotherapy in advanced non-small cell lung cancer (NSCLC). Methods: Eighty SNPs in miRNA target sites of cancer related genes selected from 18,500 miRNA:target interactions in CLASH data were investigated in 379 advanced NSCLC patients using a sequenom mass spectrometry-based genotype assay. Quantitative reverse transcription-polymerase chain reaction and luciferase assay were conducted to examine functional relevance of potentially functional SNPs in miRNA target sites. Results: Of the 80 SNPs analyzed, 16 SNPs were significantly associated with the clinical outcome after chemotherapy. Among these, ANAPC1 rs3814026C>T, ETS2 rs461155A>G, SORBS1 rs7081076C>A and POLR2A rs2071504C>T were found to be significantly associated with both chemotherapy response and survival. Notably, the relative expression level of ETS2 was significantly associated with rs461155A>G genotypes in both tumor and paired normal lung tissues (Ptrend = 4 × 10-7,and3×10-4,respectively). Consistently, a decreased expression of the reporter gene for the G allele of rs461155 compared with the A allele was observed by luciferase assay. Conclusion: These findings suggest that the four SNPs, especially ETS2 rs461155A>G, could be used as biomarkers predicting the response and survival of NSCLC patients treated with first-line paclitaxel-cisplatin chemotherapy. Citation Format: SON JI WOONG, Shin Yup Lee, Chang Ho Kim, Jae Yong Park. A genetic variation in microRNA target site of ETS2 is associated with clinical outcomes of paclitaxel-cisplatin chemotherapy in non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1903.

TarPmiR: a new approach for microRNA target site prediction.

Motivation: The identification of microRNA (miRNA) target sites is fundamentally important for studying gene regulation. There are dozens of computational methods available for miRNA target site prediction. Despite their existence, we still cannot reliably identify miRNA target sites, partially due to our limited understanding of the characteristics of miRNA target sites. The recently published CLASH (crosslinking ligation and sequencing of hybrids) data provide an unprecedented opportunity to study the characteristics of miRNA target sites and improve miRNA target site prediction methods.Results: Applying four different machine learning approaches to the CLASH data, we identified seven new features of miRNA target sites. Combining these new features with those commonly used by existing miRNA target prediction algorithms, we developed an approach called TarPmiR for miRNA target site prediction. Testing on two human and one mouse non-CLASH datasets, we showed that TarPmiR predicted more than 74.2% of true miRNA target sites in each dataset. Compared with three existing approaches, we demonstrated that TarPmiR is superior to these existing approaches in terms of better recall and better precision.Availability and Implementation: The TarPmiR software is freely available at http://hulab.ucf.edu/research/projects/miRNA/TarPmiR/.Contacts: haihu@cs.ucf.edu or xiaoman@mail.ucf.eduSupplementary information: Supplementary data are available at Bioinformatics online.

The RNA Binding Protein IMP2 Preserves Glioblastoma Stem Cells by Preventing let-7 Target Gene Silencing.

Cancer stem cells (CSCs) can drive tumor growth, and their maintenance may rely on post-transcriptional regulation of gene expression, including that mediated by microRNAs (miRNAs). The let-7 miRNA family has been shown to induce differentiation by silencing stem cell programs. Let-7-mediated target gene suppression is prevented by LIN28A/B, which reduce let-7 biogenesis in normal embryonic and some cancer stem cells and ensure maintenance ofstemness. Here, we find that glioblastoma stem cells (GSCs) lack LIN28 and express both let-7 and their target genes, suggesting LIN28-independent protection from let-7 silencing. Using photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP), we show that insulin-like growth factor 2 mRNA-binding protein 2 (IMP2) binds to let-7 miRNA recognition elements (MREs) and prevents let-7 target gene silencing. Our observations define the RNA-binding repertoire of IMP2 and identify a mechanism whereby it supports GSC and neural stem cell specification.

ImiRP: a computational approach to microRNA target site mutation.

BackgroundMicroRNAs (miRNAs) are small ~22 nucleotide non-coding RNAs that function as post-transcriptional regulators of messenger RNA (mRNA) through base-pairing to 6–8 nucleotide long target sites, usually located within the mRNA 3’ untranslated region. A common approach to validate and probe microRNA-mRNA interactions is to mutate predicted target sites within the mRNA and determine whether it affects miRNA-mediated activity. The introduction of miRNA target site mutations, however, is potentially problematic as it may generate new, “illegitimate sites” target sites for other miRNAs, which may affect the experimental outcome. While it is possible to manually generate and check single miRNA target site mutations, this process can be time consuming, and becomes particularly onerous and error prone when multiple sites are to be mutated simultaneously. We have developed a modular Java-based system called ImiRP (Illegitimate miRNA Predictor) to solve this problem and to facilitate miRNA target site mutagenesis.ResultsThe ImiRP interface allows users to input a sequence of interest, specify the locations of multiple predicted target sites to mutate, and set parameters such as species, mutation strategy, and disallowed illegitimate target site types. As mutant sequences are generated, ImiRP utilizes the miRBase high confidence miRNA dataset to identify illegitimate target sites in each mutant sequence by comparing target site predictions between input and mutant sequences. ImiRP then assembles a final mutant sequence in which all specified target sites have been mutated.ConclusionsImiRP is a mutation generator program that enables selective disruption of specified miRNA target sites while ensuring predicted target sites for other miRNAs are not inadvertently created. ImiRP supports mutagenesis of single and multiple miRNA target sites within a given sequence, including sites that overlap. This software will be particularly useful for studies looking at microRNA cooperativity, where mutagenesis of multiple microRNA target sites may be desired. The software is available at imirp.org and is available open source for download through GitHub (https://github.com/imirp).Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-1057-y) contains supplementary material, which is available to authorized users.

MiR-143 inhibits oncogenic traits by degrading NUAK2 in glioblastoma.

Despite evidence that the crucial role of NUAK family, SNF1-like kinase,2[NUAK2; also known as sucrose non-fermenting1(SNF1)-like kinase(SNARK)], has been highlighted in cancer development and in tumor progression, to the best of our knowledge, there are no studies available to date on the role of NUAK2 in glioblastoma. Thus, in this study, in order to determine the role of NUAK2 in glioblastoma, we performed western blot analysis to detect its expression in glioma. The results demonstrated that NUAK2 expression was upregulated in glioma tissues and that its expression was associated with the advanced stages of the disease. Invitro, NUAK2 overexpression promoted the proliferation, migration and invasion of A172 glioblastoma cells, whereas the silencing of NUAK2 with a plasmid carrying shRNA targeting NUAK2 inhibited the proliferation of A172 glioblastoma cells. Moreover, NUAK2 regulated cancer stem cell(CSC)-related gene expression in the glioblastoma cells. We performed an analysis of potential microRNA(miR or miRNA) target sites using 3commonly used prediction algorithms, miRanda, TargetScan and PicTar. All 3algorithms predicted that miR‑143 targeted the 3'-untranslated region(3'UTR) of NUAK2. Subsequent experiments confirmed this prediction. Finally, we found that miR‑143 inhibited the proliferation, migration and invasion of the glioblastoma cells. Thus, the findings of the present study demonstrate that miR‑143 inhibits oncogenic traits by degrading NUAK2 in glioblastoma.

Suppression of SIK1 by miR-141 in human ovarian cancer cell lines and tissues.

Epithelial ovarian cancer(EOC), the sixth most common cancer in women worldwide, is the most commonly fatal gynecologic malignancy in developed countries. One of the main reasons for this is that relatively little was known about the molecular events responsible for the development of this highly aggressive disease. In the present study, we demonstrated that salt‑inducible kinase1(SIK1; which is also known as MSK/SIK/SNF1LK) was downregulated in ovarian cancer tissue samples. Using HEY ovarian cancer cells, we noted that SIK1 overexpression inhibited proliferation as well as cancer stem cell-associated traits. Silencing SIK1 promoted the proliferation of the EG ovarian cancer cell line. We performed an analysis of potential microRNAs(miRNAs or miRs) target sites using threecommonly used prediction algorithms: miRanda, TargetScan and PicTar. All three algorithms predicted that miR-141 targets the 3'UTR of SIK1. Subsequent experiments not only confirmed this prediction, but also showed that miR-141 was associated with the progression of this disease. Finally, we found that miR-141 promoted proliferation of EG cells, whereas silencing miR-141 restored SIK1 expression and inhibited the proliferation of the HEY cells. Elucidating the molecular mechanism of ovarian cancer not only enables us to further understand the pathogenesis and progression of the disease, but also provides new targets for effective therapies.

A genetic variation in microRNA target site of ETS2 is associated with clinical outcomes of paclitaxel-cisplatin chemotherapy in non-small cell lung cancer.

The present study was performed to investigate the association of single nucleotide polymorphisms (SNPs) located in the miRNA target sites with the clinical outcomes of first line paclitaxel-cisplatin chemotherapy in advanced NSCLC. Eighty SNPs in miRNA binding sites of cancer related genes selected from 18,500 miRNA:target bindings in crosslinking, ligation, and sequencing of hybrids (CLASH) data were investigated in 379 advanced NSCLC patients using a sequenom mass spectrometry-based genotype assay. qRT-PCR and luciferase assay were conducted to examine functional relevance of potentially functional SNPs in miRNA binding sites. Of the 80 SNPs analyzed, 16 SNPs were significantly associated with the clinical outcomes after chemotherapy. Among these, ANAPC1 rs3814026C>T, ETS2 rs461155A>G, SORBS1 rs7081076C>A and POLR2A rs2071504C>T could predict both chemotherapy response and survival. Notably, ETS2 rs461155A>G was significantly associated with decreased ETS2 mRNA expression in both tumor and paired normal lung tissues (Ptrend = 4 × 10−7, and 3 × 10−4, respectively). Consistently, a decreased expression of the reporter gene for the G allele of rs461155 compared with the A allele was observed by luciferase assay. These findings suggest that the four SNPs, especially ETS2 rs461155A>G, could be used as biomarkers predicting the clinical outcomes of NSCLC patients treated with first-line paclitaxel-cisplatin chemotherapy.

Generation and analysis of an improved Foxg1-IRES-Cre driver mouse line

Foxg1 expression is highly restricted to the telencephalon and other head structures in the early embryo. This expression pattern has been exploited to generate conditional knockout mice, based on a widely used Foxg1-Cre knock-in line (Foxg1tm1(cre)Skm), in which the Foxg1 coding region was replaced by the Cre gene. The utility of this line, however, is severely hampered for two reasons: (1) Foxg1-Cre mice display ectopic and unpredictable Cre activity, and (2) Foxg1 haploinsufficiency can produce neurodevelopmental phenotypes. To overcome these issues, we have generated a new Foxg1-IRES-Cre knock-in mouse line, in which an IRES-Cre cassette was inserted in the 3′UTR of Foxg1 locus, thus preserving the endogenous Foxg1 coding region and un-translated gene regulatory sequences in the 3′UTR, including recently discovered microRNA target sites. We further demonstrate that the new Foxg1-IRES-Cre line displays consistent Cre activity patterns that recapitulated the endogenous Foxg1 expression at embryonic and postnatal stages without causing defects in cortical development. We conclude that the new Foxg1-IRES-Cre mouse line is a unique and advanced tool for studying genes involved in the development of the telencephalon and other Foxg1-expressing regions starting from early embryonic stages.

Dynamic Regulation of Tandem 3' Untranslated Regions in Zebrafish Spleen Cells during Immune Response.

Alternative polyadenylation (APA) has been found to be involved in tumorigenesis, development, and cell differentiation, as well as in the activation of several subsets of immune cells in vitro. Whether APA takes place in immune responses in vivo is largely unknown. We profiled the variation in tandem 3' untranslated regions (UTRs) in pathogen-challenged zebrafish and identified hundreds of APA genes with ∼ 10% being immune response genes. The detected immune response APA genes were enriched in TLR signaling, apoptosis, and JAK-STAT signaling pathways. A greater number of microRNA target sites and AU-rich elements were found in the extended 3' UTRs than in the common 3' UTRs of these APA genes. Further analysis suggested that microRNA and AU-rich element-mediated posttranscriptional regulation plays an important role in modulating the expression of APA genes. These results indicate that APA is extensively involved in immune responses in vivo, and it may be a potential new paradigm for immune regulation.

MicroRNA-regulated viral vectors for gene therapy.

Safe and effective gene therapy approaches require targeted tissue-specific transfer of a therapeutic transgene. Besides traditional approaches, such as transcriptional and transductional targeting, microRNA-dependent post-transcriptional suppression of transgene expression has been emerging as powerful new technology to increase the specificity of vector-mediated transgene expression. MicroRNAs are small non-coding RNAs and often expressed in a tissue-, lineage-, activation- or differentiation-specific pattern. They typically regulate gene expression by binding to imperfectly complementary sequences in the 3' untranslated region (UTR) of the mRNA. To control exogenous transgene expression, tandem repeats of artificial microRNA target sites are usually incorporated into the 3' UTR of the transgene expression cassette, leading to subsequent degradation of transgene mRNA in cells expressing the corresponding microRNA. This targeting strategy, first shown for lentiviral vectors in antigen presenting cells, has now been used for tissue-specific expression of vector-encoded therapeutic transgenes, to reduce immune response against the transgene, to control virus tropism for oncolytic virotherapy, to increase safety of live attenuated virus vaccines and to identify and select cell subsets for pluripotent stem cell therapies, respectively. This review provides an introduction into the technical mechanism underlying microRNA-regulation, highlights new developments in this field and gives an overview of applications of microRNA-regulated viral vectors for cardiac, suicide gene cancer and hematopoietic stem cell therapy, as well as for treatment of neurological and eye diseases.

Double-strand break repair and colorectal cancer: gene variants within 3' UTRs and microRNAs binding as modulators of cancer risk and clinical outcome.

Genetic variations in 3′ untranslated regions of target genes may affect microRNA binding, resulting in differential protein expression. microRNAs regulate DNA repair, and single-nucleotide polymorphisms in miRNA binding sites (miRSNPs) may account for interindividual differences in the DNA repair capacity. Our hypothesis is that miRSNPs in relevant DNA repair genes may ultimately affect cancer susceptibility and impact prognosis.In the present study, we analysed the association of polymorphisms in predicted microRNA target sites of double-strand breaks (DSBs) repair genes with colorectal cancer (CRC) risk and clinical outcome. Twenty-one miRSNPs in non-homologous end-joining and homologous recombination pathways were assessed in 1111 cases and 1469 controls. The variant CC genotype of rs2155209 in MRE11A was strongly associated with decreased cancer risk when compared with the other genotypes (OR 0.54, 95% CI 0.38–0.76, p = 0.0004). A reduced expression of the reporter gene was observed for the C allele of this polymorphism by in vitro assay, suggesting a more efficient interaction with potentially binding miRNAs. In colon cancer patients, the rs2155209 CC genotype was associated with shorter survival while the TT genotype of RAD52 rs11226 with longer survival when both compared with their respective more frequent genotypes (HR 1.63, 95% CI 1.06-2.51, p = 0.03 HR 0.60, 95% CI 0.41–0.89, p = 0.01, respectively).miRSNPs in DSB repair genes involved in the maintenance of genomic stability may have a role on CRC susceptibility and clinical outcome.

MiR-203 promotes proliferation, migration and invasion by degrading SIK1 in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is among the most lethal human cancers and it is insensitive to many chemotherapeutic drugs. The molecular basis of pancreatic cancer remains to be elucidated. Investigations into the molecular mechanism involved in the development and progression as well as drug resistance of the disease may be useful to understand the pathogenesis and progression of the disease and offer new targets for effective therapies. In the present study, we showed that salt-inducible kinase 1 (SIK1) was downregulated and loss of SIK1 was associated with gemcitabine resistance in pancreatic cancer. In pancreatic cancer cells, SIK1 inhibited proliferation, migration and invasion. An analysis of potential microRNA target sites was performed using the prediction algorithms, miRanda, TargetScan and PicTar. The three algorithms predicted that miR-203 is capable of targeting 3'UTR of SIK1. Subsequent experiments confirmed the prediction. In addition, the results showed that miR-203 promotes proliferation, migration and invasion in pancreatic cancer cells, whereas the restoration of SIK1 abrogated the regulation of pre-miR‑203-mediated proliferation, migration and invasion.

Cohort of estrogen-induced microRNAs regulate adrenomedullin expression.

Estrogen regulates the expression of many genes and has been correlated with differences in cardiac contraction; however, the underlying mechanisms remain poorly defined. Adrenomedullin (Adm = gene; AM = protein) is a multifunctional peptide with inotropic actions. Previous studies have demonstrated that estrogen enhances the expression of Adm, suggesting a relationship between AM and estrogen in cardiac contraction during physiological and pathological states. In this study, female mice in a mouse model of genetic Adm overexpression, abbreviated as Adm(hi/hi), were found to express 60 times more Adm in the heart than wild-type littermates, compared with the three-fold elevation of Adm previously reported in Adm(hi/hi) male hearts. Thus, this study sought to further investigate any functional consequences of increased cardiac Adm expression and begin exploring the mechanisms that regulate Adm expression in an estrogen-dependent fashion. This study revealed that heart function is enhanced in Adm(hi/hi) females, which along with Adm expression levels, was reversed following ovariectomization. Since the Adm(hi/hi) line was generated by the displacement of the 3' untranslated region (UTR), the native 3'UTR was examined for estrogen-induced microRNAs target sites to potentially explain the aberrant overexpression observed in Adm(hi/hi) female hearts. Using a bioinformatic approach, it was determined that the mouse Adm 3'UTR contains many target sites for previously characterized estrogen-induced microRNAs. This study also determined that the novel microRNA, miR-879, is another estrogen-induced microRNA that interacts with the 3'UTR of Adm to destabilize the mRNA. Together, these studies revealed that estrogen-induced microRNAs are important for balancing cardiac Adm expression in females.

Detection of canonical A-to-G editing events at 3' UTRs and microRNA target sites in human lungs using next-generation sequencing.

RNA editing is a post-transcriptional modification of RNA. The majority of these changes result from adenosine deaminase acting on RNA (ADARs) catalyzing the conversion of adenosine residues to inosine in double-stranded RNAs (dsRNAs). Massively parallel sequencing has enabled the identification of RNA editing sites in human transcriptomes. In this study, we sequenced DNA and RNA from human lungs and identified RNA editing sites with high confidence via a computational pipeline utilizing stringent analysis thresholds. We identified a total of 3,447 editing sites that overlapped in three human lung samples, and with 50% of these sites having canonical A-to-G base changes. Approximately 27% of the edited sites overlapped with Alu repeats, and showed A-to-G clustering (>3 clusters in 100 bp). The majority of edited sites mapped to either 3' untranslated regions (UTRs) or introns close to splice sites; whereas, only few sites were in exons resulting in non-synonymous amino acid changes. Interestingly, we identified 652 A-to-G editing events in the 3' UTR of 205 target genes that mapped to 932 potential miRNA target binding sites. Several of these miRNA edited sites were validated in silico. Additionally, we validated several A-to-G edited sites by Sanger sequencing. Altogether, our study suggests a role for RNA editing in miRNA-mediated gene regulation and splicing in human lungs. In this study, we have generated a RNA editome of human lung tissue that can be compared with other RNA editomes across different lung tissues to delineate a role for RNA editing in normal and diseased states.