Andrew Jakymiw

Disruption of GW bodies impairs mammalian RNA interference.

The GW182 RNA-binding protein was initially shown to associate with a specific subset of mRNAs and to reside within discrete cytoplasmic foci named GW bodies (GWBs). GWBs are enriched in proteins that are involved in mRNA degradation. Recent reports have shown that exogenously introduced human Argonaute-2 (Ago2) is also enriched in GWBs, indicating that RNA interference function may be somehow linked to these structures. In this report, we demonstrate that endogenous Ago2 and transfected small interfering RNAs (siRNAs) are also present within these same cytoplasmic bodies and that the GW182 protein interacts with Ago2. Disruption of these cytoplasmic foci in HeLa cells interferes with the silencing capability of a siRNA that is specific to lamin-A/C. Our data support a model in which GW182 and/or the microenvironment of the cytoplasmic GWBs contribute to the RNA-induced silencing complex and to RNA silencing.

GW182 is critical for the stability of GW bodies expressed during the cell cycle and cell proliferation

A novel cytoplasmic compartment referred to as GW bodies was initially identified using human autoantibodies to a 182 kDa protein named GW182. GW bodies are small, generally spherical, cytoplasmic domains that vary in number and size in several mammalian cell types examined to date. Based on our earlier studies, GW bodies were proposed to be cytoplasmic sites for mRNA storage and/or degradation. In the present study, immunogold electron microscopy identified electron dense structures of 100-300 nm diameter devoid of a lipid bilayer membrane. These structures appeared to comprise clusters of electron dense strands of 8-10 nm in diameter. By costaining with CENP-F and PCNA, and employing a double-thymidine block to synchronize HeLa cells, GW bodies were observed to be small in early S phase and larger during late S and G2 phases of the cell cycle. The majority of GW bodies disassembled prior to mitosis and small GW bodies reassembled in early G1. The analysis of GW bodies in two experimental models of cell proliferation using reversal of 3T3/serum-starvation and concanavalin A stimulation of mouse splenocytes and T cells, revealed that proliferating cells contained larger, brighter, and more numerous GW bodies as well as up to a fivefold more total GW182 protein than quiescent cells. In vitro gene knockdown of GW182 led to the disappearance of GW bodies demonstrating that GW182 is a critical component of GW bodies. The incremental expression of the GW182 protein in cells induced to proliferate and the cyclic formation and breakdown of GW bodies during mitosis are intriguing in view of the notion that GW bodies are specialized centers involved in maintaining stability and/or controlling degradation of mRNA.

Formation of GW bodies is a consequence of microRNA genesis

GW bodies (GWBs), or mammalian P bodies, proposed to be involved in messenger RNA storage and/or degradation, have recently been linked to RNA interference and microRNA (miRNA) processing. We report that endogenous let‐7 miRNA co‐precipitates with the GW182 protein complex. In addition, knockdown of two proteins, Drosha and its protein partner DGCR8, which are vital to the generation of mature miRNA, results in the loss of GWBs. Subsequent introduction of short interference RNA specific to lamin A/C is accompanied by reassembly of GWBs and concurrent knockdown of lamin A/C protein. Taken together, these studies show that miRNAs are crucial components in GWB formation.

The role of GW/P-bodies in RNA processing and silencing

GW bodies, also known as mammalian P-bodies, are cytoplasmic foci involved in the post-transcriptional regulation of eukaryotic gene expression. Recently, GW bodies have been linked to RNA interference and demonstrated to be important for short-interfering-RNA- and microRNA-mediated mRNA decay and translational repression. Evidence indicates that both passenger and guide strands of short-interfering RNA duplexes can localize to GW bodies, thereby indicating that RNA-induced silencing complexes may be activated within these cytoplasmic centers. Formation of GW bodies appears to depend on both specific protein factors and RNA, in particular, microRNA. Work over the past few years has significantly increased our understanding of the biology of GW bodies, revealing that they are specialized cell components that spatially regulate mRNA turnover in various biological processes. The formation of GW bodies appears to depend on both specific protein factors and RNA, in particular, microRNA. Here, we propose a working model for GW body assembly in terms of its relationship to RNA interference. In this process, one or more heteromeric protein complexes accumulate in successive steps into larger ribonucleoprotein structures.

Overexpression of Dicer as a Result of Reduced let-7 microRNA Levels Contributes to Increased Cell Proliferation of Oral Cancer Cells

Recent reports have demonstrated that Dicer, an RNase III endonuclease required for microRNA (miRNA) maturation, is aberrantly expressed in different types of cancer. Furthermore, Dicer has been reported to be regulated by the let‐7 family of miRNA genes. We hypothesize that Dicer is aberrantly expressed in oral cancer cells due to altered expressions of let‐7 and that Dicer contributes to the development and progression of the disease. Western blot examination of Dicer protein levels in four head and neck squamous cell carcinoma (HNSCC) cell lines, including two oral cancer cell lines, demonstrated that Dicer had between 4‐ and 24‐fold higher expression levels when compared to normal human primary gingival epithelial cells. Furthermore, five of six oral cancer tissues analyzed by indirect immunofluorescence had increased Dicer protein expression, compared to normal gingival epithelial tissue. The Dicer mRNA levels were not found to correlate well with protein expression in the HNSCC cell lines, suggesting that Dicer protein expression was post‐transcriptionally regulated. Analysis of let‐7a and let‐7b levels in HNSCC cell lines by real‐time PCR demonstrated that let‐7b, but not let‐7a, was significantly reduced in the HNSCC cell lines compared to control cells. Lastly, transfection of oral cancer cells with chemically synthesized let‐7b and small interfering RNAs targeting Dicer significantly inhibited cell proliferation up to 83% and >100%, respectively, as early as 3 days post‐transfection. Together, these data demonstrate that elevated expression levels of Dicer in oral cancer cells correlate with downregulation of let‐7b and increased cell proliferation.

Autoimmune targeting of key components of RNA interference

RNA interference (RNAi) is an evolutionarily conserved mechanism that is involved in the post-transcriptional silencing of genes. This process elicits the degradation or translational inhibition of mRNAs based on the complementarity with short interfering RNAs (siRNAs) or microRNAs (miRNAs). Recently, differential expression of specific miRNAs and disruption of the miRNA synthetic pathway have been implicated in cancer; however, their role in autoimmune disease remains largely unknown. Here, we report that anti-Su autoantibodies from human patients with rheumatic diseases and in a mouse model of autoimmunity recognize the human Argonaute (Ago) protein, hAgo2, the catalytic core enzyme in the RNAi pathway. More specifically, 91% (20/22) of the human anti-Su sera were shown to immunoprecipitate the full-length recombinant hAgo2 protein. Indirect immunofluorescence studies in HEp-2 cells demonstrated that anti-Su autoantibodies target cytoplasmic foci identified as GW bodies (GWBs) or mammalian P bodies, structures recently linked to RNAi function. Furthermore, anti-Su sera were also capable of immunoprecipitating additional key components of the RNAi pathway, including hAgo1, -3, -4, and Dicer. Together, these results demonstrate an autoimmune response to components of the RNAi pathway which could potentially implicate the involvement of an innate anti-viral response in the pathogenesis of autoantibody production.

High resolution of microRNA signatures in human whole saliva

OBJECTIVE Identifying discriminatory human salivary RNA biomarkers reflective of disease in a low-cost non-invasive screening assay is crucial to salivary diagnostics. Recent studies have reported both mRNA and microRNA (miRNA) in saliva, but little information has been documented on the quality and yield of RNA collected. Therefore, the aim of the present study was to develop an improved RNA isolation method from saliva and to identify major miRNA species in human whole saliva. DESIGN RNA samples were isolated from normal human saliva using a combined protocol based on the Oragene RNA collection kit and the mirVana miRNA isolation kit in tandem. RNA samples were analysed for quality and subjected to miRNA array analysis. RESULTS RNA samples isolated from twenty healthy donors ranged from 2.59 to 29.4 μg/ml saliva and with 1.92-2.16OD(260/280 nm) ratios. RNA yield and concentration of saliva samples were observed to be stable over 48 h at room temperature. Analysis of total salivary RNA isolated from these twenty donors showed no statistical significance between sexes; however, the presence of high-, medium-, and low-yield salivary RNA producers was detected. MiRNA array analysis of salivary RNA detected five abundantly expressed miRNAs, miR-223, miR-191, miR-16, miR-203, and miR-24, that were similarly described in other published reports. Additionally, many previously undetected miRNAs were also identified. CONCLUSION High quality miRNAs can be isolated from saliva using available commercial kits, and in future studies, the availability of this isolation protocol may allow specific changes in their levels to be measured accurately in various relevant diseases.

Keratinization-associated miR-7 and miR-21 Regulate Tumor Suppressor Reversion-inducing Cysteine-rich Protein with Kazal Motifs (RECK) in Oral Cancer

Background: The role of miRNA-mediated regulation of RECK in keratinized tumors is unclear. Results: miRNAs express differentially in subtypes of OSCCs, and keratinization-associated miRNAs inversely correlate with RECK in oral cancer cells. Conclusion: miR-7 and miR-21 negatively regulate the tumor suppressor gene RECK. Significance: Keratinization-associated miRNAs may serve as novel targets to reduce tumor aggressiveness. MicroRNAs (miRNAs) are small non-coding RNAs that posttranscriptionally regulate gene expression during many biological processes. Recently, the aberrant expressions of miRNAs have become a major focus in cancer research. The purpose of this study was to identify deregulated miRNAs in oral cancer and further focus on specific miRNAs that were related to patient survival. Here, we report that miRNA expression profiling provided more precise information when oral squamous cell carcinomas were subcategorized on the basis of clinicopathological parameters (tumor primary site, histological subtype, tumor stage, and HPV16 status). An innovative radar chart analysis method was developed to depict subcategories of cancers taking into consideration the expression patterns of multiple miRNAs combined with the clinicopathological parameters. Keratinization of tumors and the high expression of miR-21 were the major factors related to the poor prognosis of patients. Interestingly, a majority of the keratinized tumors expressed high levels of miR-21. Further investigations demonstrated the regulation of the tumor suppressor gene reversion-inducing cysteine-rich protein with kazal motifs (RECK) by two keratinization-associated miRNAs, miR-7 and miR-21. Transfection of miR-7 and miR-21-mimics reduced the expression of RECK through direct miRNA-mediated regulation, and these miRNAs were inversely correlated with RECK in CAL 27 orthotopic xenograft tumors. Furthermore, a similar inverse correlation was demonstrated in CAL 27 cells treated in vitro by different external stimuli such as trypsinization, cell density, and serum concentration. Taken together, our data show that keratinization is associated with poor prognosis of oral cancer patients and keratinization-associated miRNAs mediate deregulation of RECK which may contribute to the aggressiveness of tumors.

GW bodies, microRNAs and the cell cycle.

GW bodies (GWBs) are cytoplasmic foci initially identified through the use of an autoimmuneserum targeting the marker protein, GW182. GWBs were first considered as both storagecenters for a specific subset of mRNAs and degradation sites for mRNAs. Interestingly, theyare known to vary in size and number throughout the cell cycle and are largest in size and mostabundant in number during the late S and G2 phases. Recent studies have linked RNAinterference to GWBs, in that disruption or disassembly of GWBs was demonstrated to impairsiRNA and miRNA silencing activity. As miRNAs are implicated in the regulation of cellcycle progression and cell proliferation, it is very likely that GWBs, the critical intracellularstructures for miRNA function, may very well be also linked to this cellular process.

CIP2A expression and localization in oral carcinoma and dysplasia

Aims: Oral squamous cell carcinoma (OSCC) is the most prevalent malignancy of the oral cavity resulting in severe morbidity and mortality. To date only few proteins have been suggested as potential biomarkers or targets for this type of cancer. Cancerous inhibitor of PP2A (CIP2A) is a protein expressed in epithelial tissues that stabilizes the oncogene c-Myc and causes cell transformation. This study was designed to investigate the expression of CIP2A in OSCC cell lines and tissues representing human normal, dysplasia and OSCC. Methods: Using quantitative real time PCR, mRNA quantification for CIP2A was performed in a primary gingival cell line and OSCCs CAL 27 and SCC-25. Paraffin embedded human specimen classified as normal, dysplastic or OSCC were immunohistochemically stained for CIP2A expression. EGFR and CIP2A were also stained by immunofluorescence for co-localization. Samples of human normal oral tissue and OSCC were studied by PCR for mRNA expression of CIP2A. Results: CIP2A was significantly increased in the human carcinoma cell lines compared to the primary gingival cell line. CIP2A was overexpressed in the human oral dysplasia and OSCC tissues compared to normal oral tissues. CIP2A was also preferentially localized in the dysplastic and OSCC epithelial areas compared to EGFR that was expressed mainly in areas of relatively normal epithelium and in dysplastic tissues above the basal layers. Conclusions: CIP2A may play a significant role in oral malignant transformation and therefore, it may be a potential target for chemotherapy of OSCC.