Putative miRNA Research Articles

Abstract A22: Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis in Wilms tumors

Abstract Wilms tumor is the most common pediatric kidney cancer and the fourth most common childhood cancer overall. It is treated with a combination of surgery, chemotherapy, and radiation, and while most children are cured, survival remains poor in those with advanced-stage disease. Known driver mutations (in WT1, WTX, and CTNNB1) account for only one-third of Wilms tumor cases. To better define the genomic landscape of Wilms tumor, we performed whole-exome sequencing of 44 Wilms tumors. We also sequenced matched germline DNA by whole-exome sequencing (15 cases) or targeted Sanger sequencing (29 cases) to identify somatic mutations. Through this approach, we identified recurrent somatic mutations in MYCN, DROSHA, and DICER1, which were mutually exclusive with known Wilms tumor mutations in WT1 and CTNNB1. The ribonucleases DROSHA and DICER1 perform two key steps in the biogenesis of microRNAs (miRNAs), which are ∼22-nt non-coding RNAs that regulate the stability and translation of target mRNAs. DROSHA and DICER1 mutations occurred at or near conserved metal-binding residues in their ribonuclease (RNase) IIIB domains. In vitro processing assays modeling these mutations showed that they partially or completely disrupted the ribonuclease activity of DROSHA and DICER1. In addition, next-generation sequencing of small RNAs in these tumors revealed that the DICER1 mutations preferentially impaired processing of miRNAs derived from the 5′ arm of pre-miRNA hairpins, including the let-7 tumor suppressor miRNA family. Processing of 5′-derived miRNAs is specifically dependent on DICER1 RNase IIIB activity. Although DROSHA-mutant tumors did not exhibit skewed expression of 5′ vs. 3′-derived miRNAs, expression of let-7 and other putative tumor suppressor miRNAs was reduced in these tumors as well. While germline and somatic mutations in DICER1 have been reported in some cancers, this is the first report of recurrent DROSHA mutations in any human tumor type. Moreover, these results suggest that impairment of miRNA processing by somatic mutation of DROSHA and DICER1 defines a novel subclass of Wilms tumor. Citation Format: Dinesh Rakheja, Kenneth S. Chen, Yangjian Liu, Abhay A. Shukla, Sara Hildebrand, Vanessa Schmid, Xiaoyong Sun, Xin Feng, Tsung-Cheng Chang, Shama Khokhar, Nitin J. Karandikar, James S. Malter, Joshua T. Mendell, James F. Amatruda. Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis in Wilms tumors. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A22.

Abstract 4371: Tumor suppressive microRNA-218 inhibits cancer cell migration and invasion via targeting LASP1 in prostate cancer

Abstract BACKGROUND: Prostate cancer (PCa) is the most frequently diagnosed cancer and the second leading cause of cancer death among men in developed countries. Disease progression and the development of hormone refractory disease remain major cause of cancer-related death.Understanding the molecular mechanisms of PCa progression and metastatic pathways using currently available genomic approaches would help to improve therapies for and prevention of the disease. Our recent study of microRNA (miRNA) expression signature of PCa has revealed that microRNA-218 (miR-218) was significantly downregulated in cancer tissues, suggesting that miR-218 may be a putative tumor-suppressive miRNA in PCa. The aim of this study was to investigate the functional significance of miR-218 in PCa and to identify novel miR-218-regulated cancer pathways and target genes involved in PCa oncogenesis and metastasis.METHODS: The expression levels of miR-218 were validated using real-time RT-PCR using PCa clinical specimens and cell lines (PC3 and DU145). Cell proliferation, migration and invasion assays were performed to investigate the functional significance of miR-218 and its target gene, LIM and SH3 protein 1 (LASP1) in PCa cell lines.Cells were transfected with mature miR-218 or si-LASP1 by reverse transfection methods. Genome-wide gene expression data and in silico analysis were used to identify the molecular pathways and putative targets regulated by miR-218.The luciferase reporter assay was applied to identify the actual binding site of miR-218 in target gene. The expression of LASP1 was investigated by immunohistochemistry.RESULTS: The expression levels of miR-218 were significantly downregulated in PCa tissues and PCa cell lines. Restoration of miR-218 in PCa cell lines revealed that miR-218 significantly inhibited cancer cell migration and invasion.Gene expression studies and luciferase reporter assays showed that LASP1 was directly regulated by miR-218. Silencing LASP1 resulted in significant inhibition of cell migration and invasion in PCa cells.The expression of LASP1 was upregulated in cancer tissues as demonstrated byimmunohistochemical staining.CONCLUSIONS: The miR-218 was frequently reduced in PCa clinical specimens and appeared to function as a tumor suppressor through regulation of oncogenic LASP1. The identification of novel molecularpathways and targets regulated by the tumor suppressive miRNAs may lead to a better understanding of PCa progression and metastasis, and the development of new therapeutic strategies to treat this disease. Citation Format: Rika Nishikawa, Yusuke Goto, Takashi Kinoshita, Shinichi Sakamoto, Takeshi Chiyomaru, Hideki Enokida, Satoko Kojima, Masayuki Nakagawa, Yukio Naya, Tomohiko Ichikawa, Naohiko Seki. Tumor suppressive microRNA-218 inhibits cancer cell migration and invasion via targeting LASP1 in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4371. doi:10.1158/1538-7445.AM2014-4371

Abstract 4350: Tumor-suppressive microRNA-29a inhibits cancer cell migration and invasion via targeting HSP47 in cervical squamous cell carcinoma

Abstract BACKGROUNDS: Cervical cancer is the second most common cause of cancer death in women worldwide, and approximately 500,000 new cases of cervical cancer are diagnosed each year, with 280,000 deaths. Cervical squamous cell carcinoma (SCC) is the most frequent type of cervical cancer and the most important risk factor for cervical-SCC is persistent human papilloma virus (HPV) infection. However, the molecular mechanisms of cervical SCC initiation, development, and metastasis have not yet been fully elucidated. Our recent studies of microRNA (miRNA) expression signatures have indicated that miRNA-29a (miR-29a) was significantly downregulated in several types of human cancers, suggesting that miR-29a may be a putative tumor-suppressive miRNA in human cancers. The aim of this study was to investigate the functional significance of miR-29a in cervical squamous cell carcinoma (SCC) and to identify novel miR-29a-regulated cancer pathways and target genes involved in cervical SCC oncogenesis and metastasis. METHODS: The expression levels of miR-29a were validated using real-time RT-PCR using cervical SCC clinical specimens and cell lines (CaSKi, HeLa, ME180, and YUMOTO). Cell proliferation, migration and invasion assays were performed to investigate the functional significance of miR-29a and its target gene, heat-shock protein 47 (HSP47) in cell lines. Cells were transfected with mature miR-29a or si-HSP47 by reverse transfection methods. Genome-wide gene expression data and in silico analysis were used to identify the molecular pathways and putative targets regulated by miR-29a. The luciferase reporter assay was applied to identify the actual binding site of miR-29a in target gene. The expression of HSP47 was investigated by immunohistochemistry. RESULTS: Restoration of miR-29a in cervical cancer cell lines revealed that this miRNA significantly inhibited cancer cell migration and invasion. Gene expression data and in silico analysis demonstrated that HSP47 was a potential target of miR-29a regulation. Luciferase reporter assays showed that miR-29a directly regulated HSP47. Moreover, silencing of the HSP47 gene significantly inhibited cell migration and invasion in cancer cells, and the expression of HSP47 was upregulated in cancer tissues and cervical intraepithelial neoplasia (CIN), as demonstrated by immunostaining. CONCLUSIONS: Downregulation of miR-29a was a frequent event in cervical SCC, and miR-29a acted as a tumor suppressor by directly targeting HSP47. Recognition of tumor-suppressive miRNA-regulated molecular targets provides new insights into the potential mechanisms of cervical SCC oncogenesis and metastasis and suggests novel therapeutic strategies for treatment of this disease. Citation Format: Noriko Yamamoto, Takashi Kinoshita, Nijiro Nohata, Akira Mitsuhashi, Hirokazu Usui, Hirofumi Yoshino, Toshihiko Itesako, Hideki Enokida, Masayuki Nakagawa, Makio Shozu, Naohiko Seki. Tumor-suppressive microRNA-29a inhibits cancer cell migration and invasion via targeting HSP47 in cervical squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4350. doi:10.1158/1538-7445.AM2014-4350

Abstract LB-204: Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis in Wilms tumors

Abstract Wilms tumor is the most common pediatric kidney cancer and the fourth most common childhood cancer overall. It is treated with a combination of surgery, chemotherapy, and radiation, and, while most children are cured, survival remains poor in those with advanced-stage disease. Known driver mutations (in WT1, WTX, and CTNNB1) account for only one-third of Wilms tumor cases. To better define the genomic landscape of Wilms tumor, we performed whole-exome sequencing of 44 Wilms tumors. We also sequenced matched germline DNA by whole-exome sequencing (15 cases) or targeted Sanger sequencing of candidate mutations (29 cases) to identify somatic mutations. Through this approach, we identified recurrent somatic mutations in MYCN, DROSHA, and DICER1 which were mutually exclusive with known Wilms tumor mutations in WT1 and CTNNB1. The ribonucleases DROSHA and DICER1 perform two key steps in the biogenesis of microRNAs (miRNAs), which are ∼22-nt non-coding RNAs that regulate the stability and translation of target mRNAs. DROSHA and DICER1 mutations occurred at or near conserved metal-binding residues in their ribonuclease (RNase) IIIB domains. In vitro processing assays modeling these mutations showed that they partially or completely disrupt the ribonuclease activity of DROSHA and DICER1. In addition, next-generation sequencing of small RNAs in these tumors revealed that the DICER1 mutations preferentially impaired processing of miRNAs derived from the 5′ arm of pre-miRNA hairpins, including the let-7 tumor suppressor miRNA family. Although DROSHA-mutant tumors did not exhibit skewed expression of 5′ vs. 3′-derived miRNAs, expression of let-7 and other putative tumor suppressor miRNAs was reduced in these tumors as well. While germline and somatic mutations in DICER1 have been reported in some cancers, this is the first report of recurrent DROSHA mutations in any human tumor type. Moreover, these results suggest that impairment of miRNA processing by somatic mutation of DROSHA and DICER1 defines a novel subclass of Wilms tumor. Citation Format: Dinesh Rakheja, Kenneth S. Chen, Yangjian Liu, Tsung-Cheng Chang, Abhay A. Shukla, James Malter, Joshua T. Mendell, James F. Amatruda. Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis in Wilms tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-204. doi:10.1158/1538-7445.AM2014-LB-204

Abstract 4387: Up-regulation of VHL by miR-1273C inhibits renal cell carcinoma

Abstract VHL (Von Hippel-Lindau) is a known tumor suppressor that binds to hypoxia inducible factor (HIF) and induces ubiquitin-mediated degradation. VHL is also involved in the regulation of tumor angigoenesis, cell cycle regulation, and apoptosis. Recent studies have reported that double strand RNA complementary to target promoter sequence can induce gene expression in several cancer cells and this is called small activating RNA (saRNA). By scanning gene promoters in silico for sequences complementary to known miRNAs, we identified 32 putative miRNAs target site in the promoter of VHL. Experimental validation identified two miRNAs (miR-1273C and miR-4512) that induce VHL expression in 769-P renal cancer cells. Another dsRNA molecule (dsVHL-101) complementary to the putative miR-1273C target site also induced VHL expression. Functional studies revealed that transfection of miR-1273C and dsVHL-101 inhibited cell proliferation and induced apoptosis in dose dependent manner. Furthermore, knockdown experiments confirmed that induction of VHL and apoptosis by miR-1273C and dsVHL-101 required the miRNA maturation protein Ago2 and nuclear transport protein TNRC6A. This study suggests that miRNAs play an inhibiting role in the progression of cancer cells by VHL gene expression, which may be a potential therapeutic approach in the future. Citation Format: Chang Woo Lee, Moo Rim Kang, Jieun Yun, Soo Jin Oh, Jong Soon Kang. Up-regulation of VHL by miR-1273C inhibits renal cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4387. doi:10.1158/1538-7445.AM2014-4387

MicroRNA-193b-3p acts as a tumor suppressor by targeting the MYB oncogene in T-cell acute lymphoblastic leukemia.

The MYB oncogene is a leucine zipper transcription factor essential for normal and malignant hematopoiesis. In T-cell acute lymphoblastic leukemia (T-ALL), elevated MYB levels can arise directly through T-cell receptor-mediated MYB translocations, genomic MYB duplications or enhanced TAL1 complex binding at the MYB locus or indirectly through the TAL1/miR-223/FBXW7 regulatory axis. In this study, we used an unbiased MYB 3'untranslated region-microRNA (miRNA) library screen and identified 33 putative MYB-targeting miRNAs. Subsequently, transcriptome data from two independent T-ALL cohorts and different subsets of normal T-cells were used to select miRNAs with relevance in the context of normal and malignant T-cell transformation. Hereby, miR-193b-3p was identified as a novel bona fide tumor-suppressor miRNA that targets MYB during malignant T-cell transformation thereby offering an entry point for efficient MYB targeting-oriented therapies for human T-ALL.

Identification and validation of miRNA target sites within nontraditional miRNA targets.

miRNAs are endogenous small RNAs that regulate gene expression through recognition of complementary RNA sequences. While miRNAs have generally been understood to repress gene expression posttranscriptionally through recognition of 3'-untranslated regions (3'-UTRs) of mRNA transcripts, they have the potential to target additional classes of RNAs. Understanding the expanding pool of potential miRNA targets has been hindered by the lack of tools for predicting target sites within these RNAs. Here, the principles for developing computational algorithms for identifying putative miRNA target sites outside of mRNA are discussed. Laboratory techniques for validating computational miRNA target predictions are described.

Identification and characterization of novel serum microRNA candidates from deep sequencing in cervical cancer patients.

Small non-coding microRNAs (miRNAs) are involved in cancer development and progression, and serum profiles of cervical cancer patients may be useful for identifying novel miRNAs. We performed deep sequencing on serum pools of cervical cancer patients and healthy controls with 3 replicates and constructed a small RNA library. We used MIREAP to predict novel miRNAs and identified 2 putative novel miRNAs between serum pools of cervical cancer patients and healthy controls after filtering out pseudo-pre-miRNAs using Triplet-SVM analysis. The 2 putative novel miRNAs were validated by real time PCR and were significantly decreased in cervical cancer patients compared with healthy controls. One novel miRNA had an area under curve (AUC) of 0.921 (95% CI: 0.883, 0.959) with a sensitivity of 85.7% and a specificity of 88.2% when discriminating between cervical cancer patients and healthy controls. Our results suggest that characterizing serum profiles of cervical cancers by Solexa sequencing may be a good method for identifying novel miRNAs and that the validated novel miRNAs described here may be cervical cancer-associated biomarkers.

Identification and characterization of microRNAs related to salt stress in broccoli, using high-throughput sequencing and bioinformatics analysis.

BackgroundMicroRNAs (miRNAs) are a new class of endogenous regulators of a broad range of physiological processes, which act by regulating gene expression post-transcriptionally. The brassica vegetable, broccoli (Brassica oleracea var. italica), is very popular with a wide range of consumers, but environmental stresses such as salinity are a problem worldwide in restricting its growth and yield. Little is known about the role of miRNAs in the response of broccoli to salt stress. In this study, broccoli subjected to salt stress and broccoli grown under control conditions were analyzed by high-throughput sequencing. Differential miRNA expression was confirmed by real-time reverse transcription polymerase chain reaction (RT-PCR). The prediction of miRNA targets was undertaken using the Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology (KO) database and Gene Ontology (GO)-enrichment analyses.ResultsTwo libraries of small (or short) RNAs (sRNAs) were constructed and sequenced by high-throughput Solexa sequencing. A total of 24,511,963 and 21,034,728 clean reads, representing 9,861,236 (40.23%) and 8,574,665 (40.76%) unique reads, were obtained for control and salt-stressed broccoli, respectively. Furthermore, 42 putative known and 39 putative candidate miRNAs that were differentially expressed between control and salt-stressed broccoli were revealed by their read counts and confirmed by the use of stem-loop real-time RT-PCR. Amongst these, the putative conserved miRNAs, miR393 and miR855, and two putative candidate miRNAs, miR3 and miR34, were the most strongly down-regulated when broccoli was salt-stressed, whereas the putative conserved miRNA, miR396a, and the putative candidate miRNA, miR37, were the most up-regulated. Finally, analysis of the predicted gene targets of miRNAs using the GO and KO databases indicated that a range of metabolic and other cellular functions known to be associated with salt stress were up-regulated in broccoli treated with salt.ConclusionA comprehensive study of broccoli miRNA in relation to salt stress has been performed. We report significant data on the miRNA profile of broccoli that will underpin further studies on stress responses in broccoli and related species. The differential regulation of miRNAs between control and salt-stressed broccoli indicates that miRNAs play an integral role in the regulation of responses to salt stress.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0226-2) contains supplementary material, which is available to authorized users.

Global knockdown of microRNAs affects the expression of growth factors and cytokines in human adipose-derived mesenchymal stem cells.

Cell therapies utilizing mesenchymal stem cells (MSCs) have a great potential in many research and clinical settings. The mechanisms underlying the therapeutic effects of MSCs have been studied previously and the paracrine effects elicited by their production of various growth factors and cytokines were recognized as being crucial. However, the molecular controls that govern these paracrine effects remain poorly understood. To elucidate the molecular regulators of this process, we performed a global knockdown of microRNAs (miRNAs) in human adipose-derived mesenchymal stem cells (hADSCs) by inhibiting DGCR8, a key protein in miRNA biogenesis. Global disruption of miRNA biogenesis in hADSCs caused dramatic changes in the expression of subsets of growth factors and cytokines. By performing an extensive bioinformatic analysis, we were able to associate numerous putative miRNAs with these genes. Taken together, our results strongly suggest that miRNAs are essential for the production of growth factors and cytokines in hADSCs. [BMB Reports 2014; 47(8): 469-474]

Developmental regulation of Ran 3untranslated region during somatic embryogenesis in Dimocarpus longan Lour

Plant somatic embryogenesis (SE) is similar to zygotic embryogenesis and considered as a potential model for studying the regulation of gene expression during plant embryogenesis. Ran protein was shown to be crucial in longan SE. Ran is a highly conserved Small GTPase that plays crucial roles in multiple biological processes, such as cell proliferation. The function of Ran has been extensively studied in past years, but regulation of Ran expression remains elusive. In this study, 11 Ran 3′-untranslated regions (3′UTR) differing in their sequences and length were demonstrated and long 3′UTR contain more putative miRNA target sites. Quantitative reverse transcription PCR (qPCR) was employed to study expression of Ran 3′UTRs during longan SE. We showed that Ran 3′UTRs developmentally expressed during longan SE without lengthening as SE progress. Our results demonstrated that Ran 3′UTRs were highly expressed at the developmental stages of somatic embryogenesis that the cells divided rapidly and supported the role of Ran in cell proliferation. Developmental regulation of Ran 3′UTRs lengthen during longan SE suggested that the polymorphism of be Ran 3′UTRs could be functional.

Regulation of human growth hormone receptor expression by microRNAs.

Human GH binds to its receptor (GHR) on target cells and activates multiple intracellular pathways, leading to changes in gene expression, differentiation, and metabolism. GHR deficiency is associated with growth and metabolic disorders whereas increased GHR expression has been reported in certain cancers, suggesting that the GHR gene requires tight controls. Several regulatory mechanisms have been found within its 5'-untranslated region (UTR) promoter and coding regions. However, the 3'-UTR has not been previously examined. MicroRNAs (miRNAs) are small (19-22 nucleotides) noncoding RNAs that downregulate gene expression mainly through targeting the 3'-UTR of mRNAs and enhancing their degradation or inhibiting translation. In the present study, we investigated whether miRNAs regulate GHR expression. To define putative miRNA binding sites in the GHR 3'-UTR, we used multiple in silico prediction tools, analyzed conservation across species and the presence of parallel sites in GH/IGF axis-related genes, and searched for reports linking miRNAs to GHR-related physiological or pathophysiological activities. To test prioritized sites, we cotransfected a wild-type GHR 3'-UTR luciferase reporter vector as well as miRNA binding site mutants into HEK293 cells with miRNA mimics. Furthermore, we tested whether the miRNAs altered endogenous GHR mRNA and protein levels in HEK293 cells and in 2 cancer cell lines (MCF7 and LNCaP). Our experiments have identified miRNA (miR)-129-5p, miR-142-3p, miR-202, and miR-16 as potent inhibitors of human GHR expression in normal (HEK293) and cancer (MCF7 and LNCaP) cells. This study paves the way for the development of miRNA inhibitors as therapeutic agents in GH/GHR-related pathophysiologies, including cancer.

Hormone replacement therapy enhances IGF-1 signaling in skeletal muscle by diminishing miR-182 and miR-223 expressions: a study on postmenopausal monozygotic twin pairs.

MiRNAs are fine-tuning modifiers of skeletal muscle regulation, but knowledge of their hormonal control is lacking. We used a co-twin case–control study design, that is, monozygotic postmenopausal twin pairs discordant for estrogen-based hormone replacement therapy (HRT) to explore estrogen-dependent skeletal muscle regulation via miRNAs. MiRNA profiles were determined from vastus lateralis muscle of nine healthy 54–62-years-old monozygotic female twin pairs discordant for HRT (median 7 years). MCF-7 cells, human myoblast cultures and mouse muscle experiments were used to confirm estrogen’s causal role on the expression of specific miRNAs, their target mRNAs and proteins and finally the activation of related signaling pathway. Of the 230 miRNAs expressed at detectable levels in muscle samples, qPCR confirmed significantly lower miR-182, miR-223 and miR-142-3p expressions in HRT using than in their nonusing co-twins. Insulin/IGF-1 signaling emerged one common pathway targeted by these miRNAs. IGF-1R and FOXO3A mRNA and protein were more abundantly expressed in muscle samples of HRT users than nonusers. In vitro assays confirmed effective targeting of miR-182 and miR-223 on IGF-1R and FOXO3A mRNA as well as a dose-dependent miR-182 and miR-223 down-regulations concomitantly with up-regulation of FOXO3A and IGF-1R expression. Novel finding is the postmenopausal HRT-reduced miRs-182, miR-223 and miR-142-3p expression in female skeletal muscle. The observed miRNA-mediated enhancement of the target genes’ IGF-1R and FOXO3A expression as well as the activation of insulin/IGF-1 pathway signaling via phosphorylation of AKT and mTOR is an important mechanism for positive estrogen impact on skeletal muscle of postmenopausal women.

MicroRNAs targeting Nicastrin regulate Aβ production and are affected by target site polymorphisms.

Despite the growing number of genome-wide association studies, the involvement of polymorphisms in microRNA target sites (polymiRTS) in Alzheimer’s disease (AD) remains poorly investigated. Recently, we have shown that AD-associated single-nucleotide polymorphisms (SNPs) present in the 3′ untranslated region (3′UTR) of amyloid precursor protein (APP) could directly affect miRNA function. In theory, loss of microRNA (miRNA) function could lead to risk for AD by increasing APP expression and Aβ peptide production. In this study, we tested the hypothesis that Nicastrin, a γ-secretase subunit involved in Aβ generation, could be regulated by miRNAs, and consequently affected by 3′UTR polymorphisms. Bioinformatic analysis identified 22 putative miRNA binding sites located in or near Nicastrin 3′UTR polymorphisms. From these miRNA candidates, six were previously shown to be expressed in human brain. We identified miR-24, miR-186, and miR-455 as regulators of Nicastrin expression, both in vitro and under physiological conditions in human cells, which resulted in altered Aβ secretion. Using luciferase-based assays, we further demonstrated that rs113810300 and rs141849450 SNPs affected miRNA-mediated repression of Nicastrin. Notably, rs141849450 completely abolished the miR-455-mediated repression of Nicastrin. Finally, the rs141849450 variant was identified in 1 out of 511 AD cases but not in 631 controls. These observations set the stage for future studies exploring the role of miRNAs and 3′UTR polymorphisms in AD.

Identification and validation of a novel microRNA-like molecule derived from a cytoplasmic RNA virus antigenome by bioinformatics and experimental approaches.

BackgroundIt is generally believed that RNA virus replicating in the cell cytoplasm would not encode microRNAs (miRNAs) due to nucleus inaccessibility. Recent studies have described cytoplasmic RNA virus genome-derived miRNAs in West Nile virus (WNV) and Dengue virus (DENV). However, naturally occurring miRNAs derived from the antigenome of a cytoplasmic RNA virus have not been described.MethodsHepatitis A virus (HAV) was served as a model virus to investigate whether the antigenome of a cytoplasmic RNA virus would be processed into miRNAs or miRNA-like small RNAs upon infection. HAV antigenome was queried for putative miRNA precursors (pre-miRNA) with the VMir analyzer program. Mature miRNA prediction was performed using MatureBayes and Bayes-SVM-MiRNA web server v1.0. Finally, multiple experimental approaches, including cloning and sequencing-, RNAi-, plasmid-based miRNA expression- and luciferase reporter assays, were performed to identify and validate naturally occurring viral antigenome-derived miRNAs.ResultsUsing human HAV genotype IA (isolate H2) (HAVH2), a virally encoded miRNA-like small RNA was detected on the antigenome and named hav-miR-N1-3p. Transcription of viral pre-miRNA in KMB17 and HEK293T cells led to mature hav-miR-N1-3p production. In addition, silencing of the miRNA-processing enzyme Dicer or Drosha caused a dramatic reduction in miRNA levels. Furthermore, artificial target of hav-miR-N1-3p was silenced by synthesized viral miRNA mimics and the HAVH2 naturally-derived hav-miR-N1-3p.ConclusionThese results suggested that the antigenome of a cytoplasmic RNA virus could be processed into functional miRNAs. Our findings provide new evidence supporting the hypothesis that cytoplasmic RNA viruses naturally encode miRNAs through cellular miRNA processing machinery.

Plasma exosome microRNA profiling unravels a new potential modulator of adiponectin pathway in diabetes: effect of glycemic control.

Type 2 diabetes is a chronic disease characterized by inadequate β-cell response to the progressive insulin resistance. MicroRNAs (miRNAs) are short, endogenous, noncoding RNAs representing a class of powerful gene expression modulators. Previous population studies observed a modulation of circulating miRNAs in diabetic patients; however, few data are presently available on miRNA modulation in diabetic patients naïve to pharmacological treatment as well as the effect of glycemic control on this. We aimed at studying circulating miRNA expression in diabetic patients naïve to treatment and at investigating the influence on this of glycemic control. This was a case-control study. Eighteen treatment-naïve diabetic patients with poor metabolic control and 12 control patients participated in the study. Wide miRNA expression profiling was performed, and the expression of miRNAs found to be dysregulated was then validated by quantitative RT-PCR. Finally, algorithm-identified putative miRNA targets were evaluated by quantitative RT-PCR and ELISA. In diabetic patients, microarray analysis showed that four miRNAs are increased, whereas 21 miRNAs are decreased. Quantitative RT-PCR validation confirmed the significant up-regulation of miR-326 (P = .004) and down-regulation of let-7a (P < .001) and let-7f (P = .003). Notably, an inverse negative correlation was found between circulating miR-326 and its putative target adiponectin (p = -0.479, P = .009). After 12 months of antidiabetic treatment, quantitative RT-PCR data analysis showed that miR-326 levels were unaffected, whereas the levels of let-7a and let-7f were significantly increased. Treatment-naïve, poorly controlled diabetic patients show a significant dysregulation of miRNAs involved in the regulation of the adiponectin pathway, a phenomenon that may be reversed, at least in part, by improved glycemic control.

An eight-miRNA signature as a potential biomarker for predicting survival in lung adenocarcinoma.

BackgroundLung adenocarcinoma is a heterogernous disease that creates challenges for classification and management. The purpose of this study is to identify specific miRNA markers closely associated with the survival of LUAD patients from a large dataset of significantly altered miRNAs, and to assess the prognostic value of this miRNA expression profile for OS in patients with LUAD.MethodsWe obtained miRNA expression profiles and corresponding clinical information for 372 LUAD patients from The Cancer Genome Atlas (TCGA), and identified the most significantly altered miRNAs between tumor and normal samples. Using survival analysis and supervised principal components method, we identified an eight-miRNA signature for the prediction of overall survival (OS) of LUAD patients. The relationship between OS and the identified miRNA signature was self-validated in the TCGA cohort (randomly classified into two subgroups: n = 186 for the training set and n = 186 for the testing set). Survival receiver operating characteristic (ROC) analysis was used to assess the performance of survival prediction. The biological relevance of putative miRNA targets was also analyzed using bioinformatics.ResultsSixteen of the 111 most significantly altered miRNAs were associated with OS across different clinical subclasses of the TCGA-derived LUAD cohort. A linear prognostic model of eight miRNAs (miR-31, miR-196b, miR-766, miR-519a-1, miR-375, miR-187, miR-331 and miR-101-1) was constructed and weighted by the importance scores from the supervised principal component method to divide patients into high- and low-risk groups. Patients assigned to the high-risk group exhibited poor OS compared with patients in the low-risk group (hazard ratio [HR] = 1.99, P <0.001). The eight-miRNA signature is an independent prognostic marker of OS of LUAD patients and demonstrates good performance for predicting 5-year OS (Area Under the respective ROC Curves [AUC] = 0.626, P = 0.003), especially for non-smokers (AUC = 0.686, P = 0.023).ConclusionsWe identified an eight-miRNA signature that is prognostic of LUAD. The miRNA signature, if validated in other prospective studies, may have important implications in clinical practice, in particular identifying a subgroup of patients with LUAD who are at high risk of mortality.

THU0460 Charaterization of Micrornas Involved in the Regulation of Atherotrhombosis in Antiphospholipid Syndrome and Systemic Lupus Erythematosus

BackgroundmiRNAs are key players in pathophysiological processes, but no previous studies have investigated their asscociation with the cardiovascular and atherothrombotic risks observed in primary antiphospholipid syndrome (APS) and systemic lupus...

The isolation and identification of salt-responsive novel microRNAs from Salicornia brachiata, an extreme halophyte

The small, non-coding RNA species (20–24 nucleotides), including microRNAs (miRNAs) and small interfering RNAs, are key regulators of gene expression in eukaryotic cells. Mostly, miRNAs have been identified from glycophytes under different abiotic stresses. The isolation and identification of miRNAs from halophytes are challenging due to the presence of high salt concentrations. In this study, small RNA cDNA libraries were constructed using an improved method to identify salt-responsive novel miRNAs from Salicornia brachiata. A total of 159 sequences were cloned and analysed. There was no adapter contamination and low-abundance small RNAs were amplified efficiently. Twelve putative miRNAs/pre-miRNAs and a small RNA were identified and confirmed by northern blot. Among the putative miRNAs, nine are novel and three belong to the conserved miRNA families (MIR169g, MIR1433, MIR138b). Northern hybridisation and real-time PCR showed that nine miRNAs (seven novel and two conserved) and one small RNA were highly expressed at 2.0 M NaCl, two novel miRNAs at 1.5 M NaCl, and one conserved miRNA at 1.0 M NaCl. Furthermore, 67 putative target genes of the isolated miRNA were predicted on the basis of sequence homology, of which more than 50 % are associated with abiotic stresses. The target genes of miRNA 169g (Sb-miRNA10), and Sb-miRNA7 (NF-YA transcription factor and cytochrome P450-like TATA box binding protein respectively) were analysed by real-time PCR. The study of miRNAs from an extreme halophyte broadens our understanding of the important role played by miRNAs in plant abiotic stress.

The analysis of the inflorescence miRNome of the orchid Orchis italica reveals a DEF-like MADS-box gene as a new miRNA target.

Plant microRNAs (miRNAs) are small, regulatory non-coding RNAs involved in a wide range of biological processes, from organ development to response to stimuli. In recent years, an increasing number of studies on model plant species have highlighted the evolutionary conservation of a high number of miRNA families and the existence of taxon-specific ones. However, few studies have examined miRNAs in non-model species such as orchids, which are characterized by highly diversified floral structures and pollination strategies. Therefore, we analysed a small RNA library of inflorescence tissue of the Mediterranean orchid Orchis italica to increase the knowledge on miRNAs in a non-model plant species. The high-throughput sequencing and analysis of a small RNA library of inflorescence of O. italica revealed 23 conserved and 161 putative novel miRNA families. Among the putative miRNA targets, experimental validation demonstrated that a DEF-like MADS-box transcript is cleaved by the homolog of miR5179 of O. italica. The presence of conserved miRNA families in the inflorescence of O. italica indicates that the basic developmental flower regulatory mechanisms mediated by miRNAs are maintained through evolution. Because, according to the “orchid code” theory, DEF-like genes exert a key function in the diversification of tepals and lip, the cleavage-mediated inhibitory activity of miR5179 on a OitaDEF-like transcript suggests that, in orchids, miRNAs play an important role in the diversification of the perianth organs.