This preprint addresses the enormous challenges confronting researchers invested in serum miRNAs and their potential use as clinical biomarkers. We discuss some of the inherent technical obstacles and suggest several possible solutions. We hope to encourage all researchers to be united and collaborative in the pursuit of delivering clinically relevant biomarkers in the near future.
Mason, D, Zhang, X, Marques, TM, Rose, B, Khoury, S, Hill, M, Deutsch, F, Lyons, JG, Gama-Carvalho, M & Tran, N 2018, 'Human papillomavirus 16 E6 modulates the expression of miR-496 in oropharyngeal cancer.', Virology, vol. 521, pp. 149-157.View/Download from: UTS OPUS or Publisher's site
Human papillomavirus (HPV), notably type 16, is a risk factor for up to 75% of oropharyngeal squamous cell carcinomas (SCC). It has been demonstrated that small non-coding RNAs known as microRNAs play a vital role in the cellular transformation process. In this study, we used an LNA array to further investigate the impact of HPV16 on the expression of microRNAs in oropharyngeal (tonsillar) cancer. A number of miRNAs were found to be deregulated, with miR-496 showing a four-fold decrease. Over-expression of the high risk E6 oncoprotein down-regulated miR-496, impacting upon the post-transcriptional control of the transcription factor E2F2. These HPV specific miRNAs were integrated with the HPV16 interactome to identify possible mechanistic pathways. These analyses provide insights into novel molecular interactions between HPV16 and miRNAs in oropharyngeal cancers.
Zhang, X, Gee, HG, Rose, BR, Lee, CS, Clark, JC, Elliott, ME, Gamble, JG, Cairns, MJC, Harris, AH, Khoury, SK & Tran, NT 2016, 'Regulation of the tumour suppressor PDCD4 by miR-499 and miR-21 in oropharyngeal cancers', BMC Cancer, vol. 16.View/Download from: UTS OPUS or Publisher's site
The rates of oropharyngeal cancers such as tonsil cancers are increasing. The tumour suppressor protein Programmed Cell Death Protein 4 (PDCD4) has been implicated in the development of various human cancers and small RNAs such as microRNAs (miRNAs) can regulate its expression. However the exact regulation of PDCD4 by multiple miRNAs in oropharyngeal squamous cell carcinoma (SCC) is not well understood.
Using two independent oropharyngeal SCC cohorts with a focus on the tonsillar region, we identified a miRNA profile differentiating SCC tissue from normal. Both miR-21 and miR-499 were highly expressed in tonsil SCC tissues displaying a loss of PDCD4. Interestingly, expression of the miRNA machinery, Dicer1, Drosha, DDX5 (Dead Box Helicase 5) and DGCR8 (DiGeorge Syndrome Critical Region Gene 8) were all elevated by greater than 2 fold in the tonsil SCC tissue. The 3'UTR of PDCD4 contains three binding-sites for miR-499 and one for miR-21. Using a wild-type and truncated 3'UTR of PDCD4, we demonstrated that the initial suppression of PDCD4 was mediated by miR-21 whilst sustained suppression was mediated by miR-499. Moreover the single miR-21 site was able to elicit the same magnitude of suppression as the three miR-499 sites.
This study describes the regulation of PDCD4 specifically in tonsil SCC by miR-499 and miR-21 and has documented the loss of PDCD4 in tonsil SCCs. These findings highlight the complex interplay between miRNAs and tumour suppressor gene regulation and suggest that PDCD4 loss may be an important step in tonsillar carcinogenesis.
Ahadi, A, Khoury, S, Tran, N & Zhang, X 2016, 'Expression of microRNAs in HPV negative tonsil cancers and their regulation of PDCD4', Genomics Data, vol. 8, pp. 93-96.View/Download from: UTS OPUS or Publisher's site
Global rates of tonsil cancer have been increasing since the turn of the millennia, however we still have a limited understanding of the genes and pathways which control this disease. This array dataset which is linked to our publication (Zhang et al., 2015) describes the profiling of human miRNAs in tonsil and normal adjacent tissues. With this dataset, we identified a list of microRNA (miRNA) which were highly over represented in tonsil cancers and showed that several miRNAs were able to regulate the tumour suppressor PDCD4 in a temporal manner. The dataset has been deposited into Gene Expression Omnibus (GSE75630).
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 found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 µl of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/µl from serum but can also be adapted for other biological tissues.
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Background: Head and Neck Squamous Cell Carcinoma (HNSCC) is the 6th common malignancy in men. We currently have little understanding of the molecular events of this disease and no biomarkers currently exist for early detection. Recently, small non-coding RNAs such as microRNAs (miRNAs) were shown to be highly stable and could be found in body fluids such as serum. Given this, circulating miRNAs found in the blood of HNSCC patients could act as potential clinical biomarkers for early detection.
Methods: Using Agilent miRNA arrays we screened for the expression of circulating miRNAs in patient sera (n=52) showing the four representative subtypes of HNSCC and in sera isolated from normal individuals (n=11). A number of candidate miRNAs biomarkers were identified and validated using TaqMan qPCR. These biomarkers were then assessed for clinical relevance.
Results: Ninety-three dysregulated serum miRNAs were identified across all tumours in comparison to healthy sera. Specifically 166 serum miRNAs were deregulated in oral SCC serum, 22 in hypopharyngeal cancers and 34 in the oropharyngeal cohort. Unsupervised hierarchical clustering and principal component analysis indicated that sera profiles could clearly distinguish between HNSCC and control samples. A selection of these miRNAs was then validated using singleplex TaqMan qPCR.
Conclusions: Our study is the first to show that the expression levels of serum miRNAs can distinguish four different subtypes of HNSCC. QPCR analysis supported these findings with further studies now being validated in a larger cohort of clinical samples. Our findings provide a promising foundation for the application of small RNAs as biomarkers for the early detection of HNSCC.
Background: Head and Neck Squamous Cell Carcinoma (HNSCC) is the 5th common malignancy worldwide with a high morbidity rate. We propose that HNSCC cells release small RNAs into the circulation which can be potential biomarkers. Our comprehensive data set of miRNA expression from solid tumours and sera of the same patient provides an opportunity to identify biomarkers for early detection of HNSCC. This approach will assist in improving the prognostic outlook of HNSCC patients. Methods: MiRNA expression across tonsil tumours and serum was compared using Partek Genomics Suite. Results: We identified approximately 225 dysregulated miRNAs common to tumour and sera. A selection of these miRNAs was then validated using TaqMan qPCR. Conclusions: Our study is the first to compare expression levels of miRNAs across tumour and sera of the same patient with QPCR analysis supporting the findings. This is a promising foundation for further analysis of miRNA expression across tumour and sera in 3 remaining HNSCC subtypes.