isolation-of-small-noncoding-rnas-from-human-serum

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

University of Technology, Sydney

Royal Prince Alfred Hospital

The use of small interfering RNAs (siRNAs) as genetic inhibitors of gene expression has been shown to be an effective way of studying gene function in mammalian cells. Recently, different DNA vectors for expression of small hairpin RNAs (shRNAs) or co-expression of sense and antisense RNAs have been developed that direct siRNA-mediated gene silencing. One expression cassette design that has been used to express long sense and antisense RNAs in non-mammalian cell types is symmetric transcription using convergent promoters. However, convergent transcription as a way to generate functional siRNAs in mammalian cells has not been reported. This vector design permits the generation of expression constructs containing no repeat sequences, but capable of inducing RNA interference (RNAi)-mediated gene silencing.

Expressing functional siRNAs in mammalian cells using convergent transcription.

The use of long double-stranded RNA (dsRNA) for gene silencing in mammalian cells has generally been restricted to embryonic cell types and proposed to induce non-specific effects on gene expression in differentiated cells. In this study, we report that foreign and endogenous gene expression can be regulated in immortalised human cell lines by co-expression of long complementary RNAs with the potential to form dsRNA. The observed gene silencing effect was transferable to recipient control cells, occurred independently of cytoplasmic Dicer and produced an epi-allelic series of clones suitable for gene function studies. This complementary RNA co-expression approach permits the use of long complementary RNAs for regulating specific gene expression in mammalian cells.

Control of specific gene expression in mammalian cells by co-expression of long complementary RNAs.

Head and neck cancer is the world's sixth most common cancer, but despite advances in treatment, there has been no significant decline in the mortality rate. In recent years, there has been mounting epidemiologic and experimental evidence of a role for human papillomavirus (HPV) as the etiologic agent of a subset of head and neck cancers. The association is strongest for oropharyngeal cancers, especially those of the tonsil. HPV 16 is invariably the predominant type. HPV-positive cancers have been shown to be biologically distinct, clustering among nonsmokers and light drinkers, and have been associated with a favorable prognosis. This review examines the current findings of HPV in head and neck cancers and discusses implications for developing new treatments.

Role of human papillomavirus in the etiology of head and neck cancer.

This study investigates human papillomavirus (HPV) infection in the oral cavities of 88 Australian renal transplant recipients and 88 immunocompetent controls. Oral cavities were examined for lesions and brushings collected for HPV analysis by consensus PCR. No warts were identified; HPV DNA was detected in 18% of transplant versus 1% control samples (P<0.001). Cutaneous HPVs predominated. One patient had HPV16 in samples taken four years apart without evidence of associated lesions or malignancy. Transplant recipients were more likely than controls to have current cutaneous warts (P<0.001), fewer sexual partners (P=0.001), and to have never consumed alcohol (P=0.01). Among the transplant group, the risk of an HPV-positive sample was higher among older patients (P=0.05), and those with past cutaneous warts (P=0.04). This study extends previous surveys by encompassing overt and asymptomatic infection, a broad spectrum of cutaneous and genital HPVs, and by providing new data on risk factors for oral HPV infection.

Human papillomavirus in the oral cavity of patients with and without renal transplantation.

Our previous studies of tonsillar cancers from New South Wales, Australia, and Jilin Province in the north-east of China, provided evidence that the proportion of these cancers attributable to human papillomavirus (HPV) varies geographically. This study provides the first data on HPV in tonsillar cancers from Hong Kong.

New evidence for geographic variation in the role of human papillomavirus in tonsillar carcinogenesis.

Non-coding RNA molecules such as microRNAs (miRNAs) may play an important role in human carcinogenesis. Their expression has been profiled in many human cancers but there are few published studies in head and neck cancer. In this study, the relative expression of 261 mature miRNA genes was determined in nine head and neck cancer cell lines using an oligonucleotide array platform. Thirty-three miRNAs in the array were found to be highly expressed and 22 showed low levels of expression in all cell lines. Notable was the high expression of miR-21 and miR-205. Expression of several miRNAs was validated using Northern blot analysis. Potential targets of validated miRNAs included tumor suppressor genes, kinesin family member 1B isoform alpha (KIF1B), and hypermethylated in cancer 2 (HIC2), and pleomorphic adenoma gene 1 (PLAG1). This study provides the largest genomewide survey of mature miRNA transcripts in head and neck cancer cell lines.

MicroRNA expression profiles in head and neck cancer cell lines.

Analysis of global microRNA (miRNA) expression in postmortem cortical grey matter from the superior temporal gyrus, revealed significant up-regulation of miR-181b expression in schizophrenia. This finding was supported by quantitative real-time RT-PCR analysis of miRNA expression in a cohort of 21 matched pairs of schizophrenia and non-psychiatric controls. The implications of this finding are substantial, as this miRNA is predicted to regulate many target genes with potential significance to the development of schizophrenia. They include the calcium sensor gene visinin-like 1 (VSNL1) and the ionotropic AMPA glutamate receptor subunit (GRIA2), which were found to be down-regulated in the same cortical tissue from the schizophrenia group. Both of these genes were also suppressed in miR-181b transfected cells and shown to contain functional miR-181b miRNA recognition elements by reporter gene assay. This study suggests altered miRNA levels could be a significant factor in the dysregulation of cortical gene expression in schizophrenia.

Dysregulation of miRNA 181b in the temporal cortex in schizophrenia.

RNA-mediated gene silencing, in the form of RNA interference (RNAi) or microRNAs (miRNAs) has provided novel tools for gene discovery and validation in mammalian cells. Here, we report on the construction and application of a random small RNA expression library for use in identifying small interfering RNA (siRNA) effectors that can modify complex cellular phenotypes in mammalian cells. The library is based in a retroviral vector and uses convergent promoters to produce unique small complementary RNAs. Using this library, we identify a range of small RNA-encoding gene inserts that overcome resistance to 5-fluorouracil (5-FU)- or tumour necrosis factor alpha (TNF-alpha)- induced cell death in colorectal cancer cells. We demonstrate the utility of this technology platform by identifying a key RNA effector, in the form of a siRNA, which overcomes cell death induced by the chemotherapeutic 5-FU. The technology described has the potential to identify both functional RNA modulators capable of altering physiological systems and the cellular target genes altered by these modulators.

RNA modulators of complex phenotypes in mammalian cells.

Genome-wide microRNA (miRNA) expression profiling of salivary gland pleomorphic adenomas revealed a distinct expression signature consisting largely of upregulated miRNAs compared with matched normal tissue. Microarray data were confirmed by quantitative real time RT-PCR (q-RTPCR). Five miRNA genes upregulated in the tumours were found in close proximity to fragile sites and/or cancer associated genomic regions. Interestingly, q-RTPCR revealed an increase in the expression of components of the miRNA processing machinery (Dicer, Drosha, DGCR8 and p68) in tumours suggesting that the deregulation of miRNA expression may result from increased miRNA biogenesis. Target gene prediction analysis of the altered miRNAs indicated that genes in a number of signalling pathways important in tumourigenesis including WNT, MAPK and JAK-STAT were overrepresented. Significantly, the oncogene PLAG1 was overexpressed in our cohort and may be potentially regulated by these miRNAs. This is the first study to examine changes in the miRNA milieu in pleomorphic adenoma, the most common salivary gland tumour. This study has demonstrated an upregulation of both miRNAs genes and an upregulation of the miRNA processing machinery. These changes may be potential underlying mechanisms for the development of these benign tumours.

Alterations in miRNA processing and expression in pleomorphic adenomas of the salivary gland.

Whole-genome microRNA and gene expression analyses were used to monitor changes during retinoic acid induced differentiation of neuroblasts in vitro. Interestingly, the entire miR-17 family was over-represented among the down-regulated miRNA. The implications of these changes are considerable, as target gene prediction suggests that the miR-17 family is involved in the regulation of the mitogen-activated protein kinase (MAPK) signaling pathway, synaptic plasticity and other markers of neuronal differentiation. Significantly, many of the target responses predicted by changes in miRNA expression were supported by the observed changes in gene expression. As expected, markers of neuronal differentiation such as anti-apoptotic protein B-cell lymphoma 2 (BCL2), myocyte enhancer factor-2D (MEF2D) and zipper protein kinase (MAP3K12; aka ZPK/MUK/DLK) were each up-regulated in response to differentiation. The expression of these genes was also reduced in response to miR-17 and miR-20a transfection, and more specifically they were also shown to contain functional miRNA recognition elements for members of the miR-17 family by reporter gene assay. This suggests that the miR-17 family have an integral role in fine-tuning the pathways involved in the regulation of neuronal differentiation.

Down-regulation of miR-17 family expression in response to retinoic acid induced neuronal differentiation.

MicroRNAs (MiRNAs) are short non-coding RNAs that control protein expression through various mechanisms. Their altered expression has been shown to be associated with various cancers. The aim of this study was to profile miRNA expression in colorectal cancer (CRC) and to analyze the function of specific miRNAs in CRC cells. MirVana miRNA Bioarrays were used to determine the miRNA expression profile in eight CRC cell line models, 45 human CRC samples of different stages, and four matched normal colon tissue samples. SW620 CRC cells were stably transduced with miR-143 or miR-145 expression vectors and analyzed in vitro for cell proliferation, cell differentiation and anchorage-independent growth. Signalling pathways associated with differentially expressed miRNAs were identified using a gene set enrichment analysis.

Characterization of global microRNA expression reveals oncogenic potential of miR-145 in metastatic colorectal cancer.

A new class of regulatory molecules known as microRNAs (miRNAs) is redefining our understanding of the molecular pathways associated with tumorigenesis. These miRNAs are small noncoding RNA (ncRNA) sequences with potent regulatory potential. The aberrant expression of miRNAs has been associated with the development of various tumors. It has been suggested that miRNAs can both regulate and act as tumor-suppressor genes and oncogenes. Our understanding of the role of miRNAs in head and neck tumorigenesis is in its infancy. However, several recent studies have revealed extensive dysregulation of miRNA in head and neck tumors and have highlighted the potential of certain miRNAs to act as diagnostic and prognostic markers and targets for new therapeutic agents. The intent of this review is to discuss and summarize current findings that point to a significant role for miRNAs in head and neck tumorigenesis.

Potential role of micro-RNAs in head and neck tumorigenesis.

There is increasing use of multiple molecular markers to predict prognosis in human cancer. Our aim was to examine the prognostic significance of cyclin D1 and retinoblastoma (pRb) expression in association with human papillomavirus (HPV) status in oropharyngeal squamous cell carcinoma. Clinical records and specimens of 226 patients with follow-up from 1 to 235 months postdiagnosis were retrieved. Tumor HPV status was determined by HPV E6-targeted multiplex real-time PCR/p16 semiquantitative immunohistochemistry and cyclin D1 and pRb expression by semiquantitative immunohistochemistry. Determinants of recurrence and mortality hazards were modeled using Cox regression with censoring at dates of last follow-up. The HPV-positivity rate was 37% (91% type 16). HPV was a predictor of recurrence, an event (recurrence or death) and death after adjustment for clinicopathological variables. There were inverse relationships between HPV status and cyclin D1 and pRb. On univariate analysis, cyclin D1 predicted locoregional recurrence, event and death and pRb predicted event and death. Within the HPV-positive group, after adjusting for clinicopathological factors, patients with cyclin D1-positive cancers had up to a eightfold increased risk of poor outcome relative to those with cyclin D1-negative tumors. However, within the HPV-negative group, there was only a very small adjusted increased risk. A combination of pRb and HPV did not provide additional prognostic information. Our data provide the first evidence that a combination of HPV and cyclin D1 provides more prognostic information in oropharyngeal cancer than HPV alone. If findings are confirmed, treatment based on HPV and cyclin D1 may improve outcomes.

Use of cyclin D1 in conjunction with human papillomavirus status to predict outcome in oropharyngeal cancer.

As part of a cell's inherent protection against carcinogenesis, p14ARF is upregulated in response to hyperproliferative signalling to induce cell cycle arrest. This property makes p14ARF a leading candidate for cancer therapy. This study explores the consequences of reactivating p14ARF in breast cancer and the potential of targeting p14ARF in breast cancer treatment. Our results show that activation of the p14ARF-p53-p21-Rb pathway in the estrogen sensitive MCF-7 breast cancer cells induces many hallmarks of senescence including a large flat cell morphology, multinucleation, senescence-associated-Î²-gal staining, and rapid G1 and G2/M phase cell cycle arrest. P14ARF also induces the expression of the proto-oncogene cyclin D1, which is most often associated with a transition from G1-S phase and is highly expressed in breast cancers with poor clinical prognosis. In this study, siRNA knockdown of cyclin D1, p21 and p53 show p21 plays a pivotal role in the maintenance of high cyclin D1 expression, cell cycle and growth arrest post-p14ARF induction. High p53 and p14ARF expression and low p21/cyclin D1 did not cause cell-cycle arrest. Knockdown of cyclin D1 stops proliferation but does not reverse senescence-associated cell growth. Furthermore, cyclin D1 accumulation in the nucleus post-p14ARF activation correlated with a rapid loss of nucleolar Ki-67 protein and inhibition of DNA synthesis. Latent effects of the p14ARF-induced cellular processes resulting from high nuclear cyclin D1 accumulation included a redistribution of Ki-67 into the nucleoli, aberrant nuclear growth (multinucleation), and cell proliferation. Lastly, downregulation of cyclin D1 through inhibition of ER abrogated latent recurrence. The mediation of these latent effects by continuous expression of p14ARF further suggests a novel mechanism whereby dysregulation of cyclin D1 could have a double-edged effect. Our results suggest that p14ARF induced-senescence is related to late-onset breast cancer in estrogen responsive breast cancers and/or the recurrence of more aggressive breast cancer post-therapy.

p14ARF post-transcriptional regulation of nuclear cyclin D1 in MCF-7 breast cancer cells: discrimination between a good and bad prognosis?

Dans le cadre de la protection inh&eacute;rente de la cellule contre la canc&eacute;rogen&egrave;se, p14ARF est augment&eacute;e en r&eacute;ponse &agrave; HYPERPROLIF&Eacute;RATIVES signalisation provoquer l&#39;arr&ecirc;t du cycle cellulaire. Cette propri&eacute;t&eacute; rend p14ARF, des principaux candidats pour le traitement du cancer. Cette &eacute;tude explore les cons&eacute;quences de r&eacute;activer p14ARF dans le cancer du sein et le potentiel de ciblage p14ARF dans le traitement du cancer. Nos r&eacute;sultats montrent que l&#39;activation de la voie p14ARF-p53-p21-Rb dans les cellules canc&eacute;reuses du sein MCF-7 sensibles de l&#39;oestrog&egrave;ne induit de nombreux poin&ccedil;ons de la s&eacute;nescence, y compris une morphologie plate &agrave; grandes cellules, multinucleation, taches de s&eacute;nescence-associ&eacute;s-&beta;-gal et arr&ecirc;t du cycle cellulaire phase rapide G1 et G2/M. P14ARF induit &eacute;galement l&#39;expression du proto-oncog&egrave;ne cycline D1, qui est le plus souvent associ&eacute;e &agrave; une transition de phase G1-S et est fortement exprim&eacute; dans les cancers du sein avec mauvais pronostic clinique. Dans cette &eacute;tude, siARN knockdown de cycline D1, p21 et p53 montrent p21 fois un r&ocirc;le essentiel dans le maintien de haute cyclin D1 expression, cycle cellulaire et la croissance arr&ecirc;tent post-p14ARF induction. Expression de p53 et p14ARF haute et basse p21/cycline D1 ne causent pas l&#39;arr&ecirc;t du cycle cellulaire. Pr&eacute;cipitation de la cycline D1 s&#39;arr&ecirc;te la prolif&eacute;ration mais n&#39;inverse pas la croissance associ&eacute;e &agrave; la s&eacute;nescence cellulaire. De plus, cycline D1 accumulation dans l&#39;activation de post-p14ARF noyau corr&eacute;l&eacute;e avec une perte rapide de la prot&eacute;ine Ki-67 et l&#39;inhibition de la synth&egrave;se de l&#39;ADN. Effets latents des processus cellulaires induites par p14ARF r&eacute;sultant de haute nucl&eacute;aire cycline D1 accumulation inclus une redistribution de Ki-67 dans les nucl&eacute;oles, aberrante croissance nucl&eacute;aire (multinucleation) et la prolif&eacute;ration cellulaire. Enfin, la r&eacute;gulation n&eacute;gative de la cycline D1 par inhibition de ER abrog&eacute; r&eacute;cidive latente. La m&eacute;diation de ces effets latents par expression continue de p14ARF outre sugg&egrave;re un nouveau m&eacute;canisme par lequel une dysr&eacute;gulation de la cycline D1 pourrait avoir un effet &agrave; double tranchant. Nos r&eacute;sultats sugg&egrave;rent que p14ARF induite-s&eacute;nescence est reli&eacute;e &agrave; d&eacute;but tardif le cancer du sein dans les cancers du sein sensible oestrog&egrave;ne et/ou de la r&eacute;apparition du post-therapy de cancer du sein plus agressive.

P14ARF post-transcriptionnelle du nucl&eacute;aire cycline D1 en cellules de cancer du sein MCF-7: discrimination entre un bon et de mauvais pronostic ?

Come parte della protezione intrinseca di una cellula contro la cancerogenesi, p14ARF &egrave; sovraregolati in risposta a iperproliferative segnalazione per indurre l&#39;arresto del ciclo cellulare. Questa propriet&agrave; rende p14ARF un candidato principale per la terapia del cancro. Questo studio Esplora le conseguenze della riattivazione p14ARF nel cancro al seno e il potenziale di targeting p14ARF nel trattamento del cancro al seno. I nostri risultati mostrano che l&#39;attivazione del percorso p14ARF-p53-p21-Rb in estrogeno sensibile MCF-7 del seno cancro cellule induce molti tratti distintivi della senescenza, tra cui una morfologia piatta a grandi cellule, multinucleation, senescenza-associati-&beta;-gal colorazione e rapido G1 e G2/M fase arresto del ciclo cellulare. P14ARF induce anche l&#39;espressione del proto-oncogene cyclin D1, che &egrave; pi&ugrave; spesso associato con una transizione dalla fase G1-S ed &egrave; altamente espresso nei cancri della mammella con prognosi clinica. In questo studio, siRNA atterramento di ciclina D1, p21 e p53 Visualizza p21 gioca un ruolo fondamentale nel mantenimento di alti cyclin D1 espressione, ciclo cellulare e la crescita di arresto post-p14ARF induzione. Espressione di p53 e p14ARF ad alta e bassa p21/ciclina D1 non ha causato l&#39;arresto del ciclo cellulare. Atterramento di cyclin D1 arresta la proliferazione, ma non invertire la crescita delle cellule senescenza-associati. Inoltre, cyclin D1 accumulo nell&#39;attivazione post-p14ARF nucleo correlata con una rapida perdita di proteina Ki-67 nucleolare e inibizione della sintesi del DNA. Gli effetti latenti dei processi cellulari indotta da p14ARF derivanti dall&#39;alta nucleare cyclin D1 accumulo includevano una ridistribuzione di Ki-67 in nucleoli, aberrante crescita nucleare (multinucleation) e la proliferazione cellulare. Infine, downregulation di cyclin D1 attraverso l&#39;inibizione di ER abrogata ricorrenza latente. La mediazione di questi effetti latenti dall&#39;espressione continua di p14ARF ulteriormente suggerisce un nuovo meccanismo per cui disregolazione del cyclin D1 potrebbe avere un effetto a doppio taglio. I nostri risultati suggeriscono che p14ARF indotta-senescenza &egrave; correlata al cancro al seno ad esordio tardivo nei cancri della mammella reattivo di estrogeni e/o reiterazione del post-therapy di cancro al seno pi&ugrave; aggressivi.

p14ARF regolamento espresione del nucleare cyclin D1 in cellule di cancro al seno MCF-7: discriminazione tra una buona e cattiva prognosi?

MicroRNA (miRNA) are small non-coding RNA molecules which function as nucleic acid-based specificity factors in the universal RNA binding complex known as the RNA induced silencing complex (RISC). In the canonical gene-silencing pathway, these activated RISC particles are associated with RNA decay and gene suppression, however, there is evidence to suggest that in some circumstances they may also stabilise their target RNA and even enhance translation. To further explore the role of miRNA in this context, we performed a genome-wide expression analysis to investigate the molecular consequences of bidirectional modulation of the disease-associated miRNAs miR-181b and miR-107 in multiple human cell lines.

Alternative mRNA fates identified in microRNA-associated transcriptome analysis.

Regulation of gene expression is a fundamental process in both prokaryotic and eukaryotic organisms. Multiple regulatory mechanisms are in place to control gene expression at the level of transcription, post-transcription and post-translation to maintain optimal RNA and protein expressions in cells. miRNAs (microRNAs) are abundant short 21-23 nt non-coding RNAs that are key regulators of virtually all eukaryotic biological processes. The levels of miRNAs in an organism are crucial for proper development and sustaining optimal cell functions. Therefore the processing and regulation of the processing of these miRNAs are critical. In the present chapter we highlight the most important steps of miRNA processing, describe the functions of key proteins involved in the maturation of miRNAs, and discuss how the generation and the stability of miRNAs are regulated.

Biogenesis and the regulation of the maturation of miRNAs.

Cellular junctions are essential to the normal functioning of the endothelium and control angiogenesis, tissue leak, and inflammation. From a screen of micro RNAs (miRNAs) altered in in vitro angiogenesis, we selected a subset predicted to target junctional molecules. MiR-27a was rapidly downregulated upon stimulation of in vitro angiogenesis, and its level of expression is reduced in neovessels in vivo. The downregulation of miR-27a was essential for angiogenesis because ectopic expression of miR-27a blocked capillary tube formation and angiogenesis. MiR-27a targets the junctional, endothelial-specific cadherin, VE-cadherin. Consistent with this, vascular permeability to vascular endothelial growth factor in mice is reduced by administration of a general miR-27 inhibitor. To determine that VE-cadherin was the dominant target of miR-27a function, we used a novel technology with "Blockmirs," inhibitors that bind to the miR-27 binding site in VE-cadherin. The Blockmir CD5-2 demonstrated specificity for VE-cadherin and inhibited vascular leak in vitro and in vivo. Furthermore, CD5-2 reduced edema, increased capillary density, and potently enhanced recovery from ischemic limb injury in mice. The Blockmir technology offers a refinement in the use of miRNAs, especially for therapy. Further, targeting of endothelial junctional molecules by miRNAs has clinical potential, especially in diseases associated with vascular leak.

Nham Tran

Centre for Health Technologies,
Faculty of Engineering and Information Technology,
Centre for Health Technologies, Faculty of Engineering and Information Technology

University of Technology, Sydney

Isolation of Small Noncoding RNAs from Human Serum

Nham Tran

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Centre for Health Technologies,Faculty of Engineering and Information Technology,Centre for Health Technologies, Faculty of Engineering and Information Technology

University of Technology, Sydney

Isolation of Small Noncoding RNAs from Human Serum

Centre for Health Technologies,
Faculty of Engineering and Information Technology,
Centre for Health Technologies, Faculty of Engineering and Information Technology