Viswanathan Palanisamy

Nanostructural and Transcriptomic Analyses of Human Saliva Derived Exosomes

Background Exosomes, derived from endocytic membrane vesicles are thought to participate in cell-cell communication and protein and RNA delivery. They are ubiquitous in most body fluids (breast milk, saliva, blood, urine, malignant ascites, amniotic, bronchoalveolar lavage, and synovial fluids). In particular, exosomes secreted in human saliva contain proteins and nucleic acids that could be exploited for diagnostic purposes. To investigate this potential use, we isolated exosomes from human saliva and characterized their structural and transcriptome contents. Methodology Exosomes were purified by differential ultracentrifugation and identified by immunoelectron microscopy (EM), flow cytometry, and Western blot with CD63 and Alix antibodies. We then described the morphology, shape, size distribution, and density using atomic force microscopy (AFM). Microarray analysis revealed that 509 mRNA core transcripts are relatively stable and present in the exosomes. Exosomal mRNA stability was determined by detergent lysis with RNase A treatment. In vitro, fluorescently labeled saliva exosomes could communicate with human keratinocytes, transferring their genetic information to human oral keratinocytes to alter gene expression at a new location. Conclusion Our findings are consistent with the hypothesis that exosomes shuttle RNA between cells and that the RNAs present in the exosomes may be a possible resource for disease diagnostics.

Structural-Mechanical Characterization of Nanoparticle Exosomes in Human Saliva, Using Correlative AFM, FESEM, and Force Spectroscopy

All living systems contain naturally occurring nanoparticles with unique structural, biochemical, and mechanical characteristics. Specifically, human saliva exosomes secreted by normal cells into saliva via exocytosis are novel biomarkers showing tumor-antigen enrichment during oral cancer. Here we show the substructure of single human saliva exosomes, using a new ultrasensitive low force atomic force microscopy (AFM) exhibiting substructural organization unresolvable in electron microscopy. We correlate the data with field emission scanning electron microscopy (FESEM) and AFM images to interpret the nanoscale structures of exosomes under varying forces. Single exosomes reveal reversible mechanical deformation displaying distinct elastic, 70-100 nm trilobed membrane with substructures carrying specific transmembrane receptors. Further, we imaged and investigated, using force spectroscopy with antiCD63 IgG functionalized AFM tips, highly specific and sensitive detection of antigenCD63, potentially useful cancer markers on individual exosomes. The quantitative nanoscale morphological, biomechanical, and surface biomolecular properties of single saliva exosomes are critical for the applications of exosomes for cancer diagnosis and as a model for developing new cell delivery systems.

Horizontal transfer of RNAs: exosomes as mediators of intercellular communication

Multicellular organisms are similar to biological communities, consisting of various cell types; thus, inter‐cell communication is critical for the functioning of the whole system that ultimately constitutes a living being. Conventional models of cellular exchange include signaling molecules and direct contact‐mediated cell communications. Exosomes, small vesicles originating from an inward budding of the plasma membrane, represent a new avenue for signaling between cells. This interchange is achieved by packaging RNA species into exosomes endowed with specific cell surface‐targeting motifs. The delivered RNA molecules are functional, and mRNA can be translated into new proteins, while microRNAs (miRNAs) target host mRNAs in the recipient cell. RNA involved in transmitting information or molecules between cells is called exosomal RNA (esRNA). This review summarizes the characteristics of exosomes, specifically focusing on their role in the horizontal transfer of cellular information. WIREs RNA 2012, 3:286–293. doi: 10.1002/wrna.115

Control of Cytokine mRNA Expression by RNA-binding Proteins and microRNAs

Cytokines are critical mediators of inflammation and host defenses. Regulation of cytokines can occur at various stages of gene expression, including transcription, mRNA export, and post- transcriptional and translational levels. Among these modes of regulation, post-transcriptional regulation has been shown to play a vital role in controlling the expression of cytokines by modulating mRNA stability. The stability of cytokine mRNAs, including TNFα, IL-6, and IL-8, has been reported to be altered by the presence of AU-rich elements (AREs) located in the 3′-untranslated regions (3′UTRs) of the mRNAs. Numerous RNA-binding proteins and microRNAs bind to these 3′UTRs to regulate the stability and/or translation of the mRNAs. Thus, this paper describes the cooperative function between RNA-binding proteins and miRNAs and how they regulate AU-rich elements containing cytokine mRNA stability/degradation and translation. These mRNA control mechanisms can potentially influence inflammation as it relates to oral biology, including periodontal diseases and oral pharyngeal cancer progression.

AUF1 and HuR Proteins Stabilize Interleukin-8 mRNA in Human Saliva

Human saliva contains thousands of mRNAs, some of which have translational value as diagnostic markers for human diseases. We have found that more than 30% of the mRNAs detected in human saliva contain AU-rich elements (ARE) in their 3′ untranslated regions (3′UTR). Since AREs are known to contribute to RNA turnover by forming complexes with ARE-binding proteins, we hypothesized that salivary mRNA stability is mediated by ARE-binding proteins in human saliva. To test this hypothesis, we monitored the in vitro degradation of a radiolabeled ARE-containing salivary mRNA (IL-8) in salivary protein extracts. The degradation of IL-8 mRNA was accelerated by competition for saliva ARE-binding proteins through the addition of excess unlabeled IL-8 mRNA fragments containing 4 tandem AREs. UV cross-linking and immunoprecipitation experiments revealed 2 ARE-binding proteins, AUF1 and HuR, associated with IL-8 mRNA in saliva. These results demonstrate that ARE-binding proteins contribute to the stability of ARE mRNAs in human saliva.

MKP-1 regulates cytokine mRNA stability through selectively modulation subcellular translocation of AUF1.

MAPK phosphatase-1 (MKP-1)/dual specificity protein phosphatase-1 (DUSP-1) is a negative regulator of the host inflammatory response to infection. However, the mechanisms underlying the regulation of cytokine expression by MKP-1, especially at the post-transcriptional level, have not been fully delineated. In the current study, MKP-1 specifically dephosphorylated activated MAPK responses and attenuated LPS-induced IL-6, IL-10, and TNF-α expression. In addition, MKP-1 was important in destabilizing cytokine mRNAs. In LPS-stimulated rat macrophages with overexpressed MKP-1, half-lives of IL-6, IL-10 and TNF-α mRNAs were significantly reduced compared to controls. Conversely, half-lives of IL-6, IL-10, and TNF-α mRNAs were significantly increased in bone marrow macrophages derived from MKP-1 knock out (KO) mice compared with macrophages derived from MKP-1 wild type (WT) mice. Furthermore, MKP-1 promoted translocation of RNA-binding protein (RNA-BP) ARE/poly-(U) binding degradation factor 1 (AUF1) from the nucleus to the cytoplasm in response to LPS stimulation as evidenced by Western blot and immunofluorescent staining. Knockdown AUF1 mRNA expression by AUF1 siRNA in MKP-1 WT bone marrow macrophages significantly delayed degradation of IL-6, IL-10 and TNF- α mRNAs compared with controls. Finally, AUF1 was immunoprecipitated with the RNA complex in cellular lysates derived from bone marrow macrophages of MKP-1 KO vs. WT mice, which had increased AUF1-bound target mRNAs, including IL-6, IL-10, and TNF-α in WT macrophages compared with MKP-1 KO macrophages. Thus, this work provides new mechanistic insight of MKP-1 signaling and regulation of cytokine mRNA stability through RNA binding proteins in response to inflammatory stimuli.

Caspase-mediated cleavage of RNA-binding protein HuR regulates c-Myc protein expression after hypoxic stress.

Altered expression of RNA-binding proteins modulates gene expression in association with mRNAs encoding many proto-oncogenes, cytokines, chemokines, and proinflammatory factors. Hu antigen R (HuR), a ubiquitously expressed protein, controls a range of cellular functions such as tumor progression, apoptosis, invasion, and metastasis by stabilizing the AU-rich element located at the 3′-untranslated region (UTR) of target mRNAs. Although significant progress has been made in understanding HuR regulation in gene expression, little is known about how HuR undergoes post-translational modifications and recruits target mRNAs during hypoxic stress. Here, we report that during CoCl2-induced hypoxic stress, HuR is significantly overexpressed and undergoes caspase-dependent cleavage in head and neck squamous cell carcinoma cells. Unexpectedly, the HuR-cleavage product 1 (HuR-CP1) was found to strongly associate with the 3′-UTR of c-myc mRNA and block mRNA translation. The binding efficiency of HuR to the 3′-UTR of c-myc mRNA was confirmed using ribonucleoprotein immunoprecipitation and site-directed mutagenesis at the AU-rich element sequences of the c-myc mRNA. Overexpression of a non-cleavable isoform, HuR-D226A, revealed a potent dominant-negative effect, repressing cleavage of endogenous HuR and promoting cell viability. Surprisingly, under hypoxia, siRNA knockdown of HuR elevated c-Myc protein expression. These findings suggest an important role for HuR in hypoxia, and we may have revealed a novel post-transcriptional mechanism that controls c-Myc expression in oral cancer progression.

Overexpression of RNA-binding protein CELF1 prevents apoptosis and destabilizes pro-apoptotic mRNAs in oral cancer cells.

CELF1 RNA-binding protein, otherwise called CUGBP1, associates and coordinates the degradation of GU-rich element (GRE) containing mRNA’s encoding factors important for cell growth, migration and apoptosis. Although many substrates of CELF1 have been identified, the biological significance of CELF1-mediated mRNA decay remains unclear. As the processes modulated by CELF1 are frequently disrupted in cancer, we investigated the expression and role of CELF1 in oral squamous cancer cells (OSCCs). We determined that CELF1 is reproducibly overexpressed in OSCC tissues and cell lines. Moreover, depletion of CELF1 reduced proliferation and increased apoptosis in OSCCs, but had negligible effect in non-transformed cells. We found that CELF1 associates directly with the 3′UTR of mRNAs encoding the pro-apoptotic factors BAD, BAX and JunD and mediates their rapid decay. Specifically, 3′UTR fragment analysis of JunD revealed that the GRE region is critical for binding with CELF1 and expression of JunD in oral cancer cells. In addition, silencing of CELF1 rendered BAD, BAX and JunD mRNAs stable and increased their protein expression in oral cancer cells. Taken together, these results support a critical role for CELF1 in modulating apoptosis and implicate this RNA-binding protein as a cancer marker and potential therapeutic target.

A regulated PNUTS mRNA to lncRNA splice switch mediates EMT and tumour progression

The contribution of lncRNAs to tumour progression and the regulatory mechanisms driving their expression are areas of intense investigation. Here, we characterize the binding of heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) to a nucleic acid structural element located in exon 12 of PNUTS (also known as PPP1R10) pre-RNA that regulates its alternative splicing. HnRNP E1 release from this structural element, following its silencing, nucleocytoplasmic translocation or in response to TGFβ, allows alternative splicing and generates a non-coding isoform of PNUTS. Functionally the lncRNA-PNUTS serves as a competitive sponge for miR-205 during epithelial–mesenchymal transition (EMT). In mesenchymal breast tumour cells and in breast tumour samples, the expression of lncRNA-PNUTS is elevated and correlates with levels of ZEB mRNAs. Thus, PNUTS is a bifunctional RNA encoding both PNUTS mRNA and lncRNA-PNUTS, each eliciting distinct biological functions. While PNUTS mRNA is ubiquitously expressed, lncRNA-PNUTS appears to be tightly regulated dependent on the status of hnRNP E1 and tumour context.

Transcriptomic Analyses of Saliva

Salivary biomarkers for diagnostic and prognostic assessments have become increasingly well established in recent years. Salivary mRNA transcriptomic analyses create a new paradigm in the emerging field for noninvasive molecular diagnosis. In this chapter, we will overview the development of sensitive and robust microarray and multiplex quantitative reverse transcriptase-PCR assays for the discovery and validation of mRNA biomarkers in human saliva. Total RNA isolated from human saliva is used for microarray profiling through Human Genome U133 Plus 2.0 and Exon 1.0 ST array platforms. A universal RNA linear amplification strategy was used to amplify RNA from nanogram scale followed by reverse transcription-PCR reaction, cleaned up enzymatically, and validated by quantitative PCR. Further, the integrity of RNA can be analyzed by the Agilent Bioanalyzer and quantified using a Nanodrop microvolume spectrophotometer. Using these invaluable technical tools, one can identify thousands of mRNA species in saliva. These methods indicate that salivary mRNA provides an efficient medium for biomarker discovery in oral and systemic diseases detection.