Salivary micro RNAs as Potential Biomarkers for Oral and Head and Neck Cancers

Abstract

Head and neck cancer (HNC) could include several squamous cell carcinoma (SCC) cancers that are considered a serious health problem worldwide today. The incidence rate of HNC has seen a sustained increase in Australia. For example, in Queensland, there is a 10.6 % increase of oral squamous cell carcinoma (OSCC) in male annually. Head and neck squamous cell carcinomas (HNSCC) patients may be asymptomatic, therefore, poor prognosis and metastasis are usually common in the patients. Subsequently, late diagnosis has a low rate of survival which most likely results in extreme severity of tumours at the time of diagnosis. The survival rate of patients with HNC is very poor, currently assessed at not more than five years with a high probability of local recurrence (40%). Therefore, a delay in diagnosis is still valid while early diagnostic methods are still needed. Traditional diagnostic methods such as radiology and endoscopic biopsy are major techniques performed for this type of cancer. However, these clinical methods are stressful, relatively invasive in nature and extremely expensive. Thus, there is an urgent need for more specific, sensitive and straight forward early diagnostic procedures for investigating HNC. Furthermore, there is a necessity to discover novel biomarkers for early HNC diagnosis. MicroRNAs which are small noncoding molecules can be tested in different bodily fluids for e.g. blood, urine, saliva and serum. These have been verified as significant regulators of gene expression in cancer biology and may play a significant role in the early diagnosis of HNC. Several studies have revealed the importance of miRNAs in the diagnosis of different types of cancer. However, few studies have investigated the expression of salivary profile miRNAs in HNC patients before treatment, and the possible utility of this easily accessible biological fluid as a diagnostic biomarker for HNC. Salivary biomarkers for HNC are still relatively limited compared with other types of cancers such as breast, lung and colon. Regarding existing knowledge, the literature review is inadequate for Australian HNC researchers. Furthermore, conflicting results have been reported in miRNAs expression levels in different types of cancer, including HNC and these might be as a result of contrasting experiment design, validation methods or sample variations. To address this literature gap, this study was designed to profile the expression of different salivary miRNA in HNSSC patients in Australian as compared to healthy individuals; as well as comparing grades and stages of cancer, cancer sites and habit status. Significant variation in salivary miRNAs expression has been hypothesised between HNC patients and healthy individuals which is invaluable in the diagnosis of HNC. To test the hypothesis, several studies have been carried out. The aims of these studies are: i) To analyse the profile miRNAs expressions and their significance in HNC Australian patients and healthy adults using oral rinse samples; and ii) to test the possible utility of saliva sample as an easily accessible biological fluid and stress free for diagnostic biomarker isolation for HNC. Chapter one explores the scientific problem and aims of this study. Chapter two provides a comprehensive literature review of the background in this field of work and the methodology used to conduct our research. Chapter three detected the potential identification of the significant variation of salivary miRNAs in OSCC. Five different miRNAs (miR-21, miR-10b, miR-125a, miR-31 and miR-200a) were selected from the literature review to optimise the conditions of RNA extraction and thermocycles of real time PCR. Besides, these miRNAs have been highlighted in conflicting results both oncogenic and suppressor genic in different types of cancer. However, very little is known about the role and expression of these miRNAs in saliva samples of oral cancer. Thus, the study of this chapter documented the role of these miRNAs that is specifically associated with HNSCC. In the above study, the result revealed that the overall expression of salivary miR-125a was significantly lower in OSCC patients compared with healthy individuals, while salivary miR-21 in OSCC patients was much higher than healthy individuals. Interestingly, upregulated salivary miR-21 was associated with tumour’s stage of OSCC (p≤0.05). A Receiver Operator Curve (ROC) was also constructed to estimate the discriminatory power of miR- 21 and miR- 125a for their potential to distinguish between healthy adults and the OSCC groups. These two miRNAs were shown to have good discriminatory ability with AUC values of 0.95 and 1, respectively. In chapter four, next generation sequencing was used to investigate the variation of miRNAs profile expression levels between OSCC patients and healthy individuals, as a more efficient and precise technique. This study aimed to detect potentially significant miRNAs in OSCC. The salivary miRNA expression profiling identified 2565 miRNAs differentially co-expressed. However, 1927 miRNAs expressions are nonsignificantly differentiated between OSCC and the healthy individuals’ group. There are 638 miRNAs significantly co-expressed in the supernatant saliva of OSCC patients compared with healthy individuals group where p-value ≤0.05; 114 miRNAs with a pvalue ≤ 0.001; while 27 miRNAs were significantly different with a p-value ≤ 0.0001. This study contributes to knowledge of the association between 27 dysregulated salivary miRNAs in OSCC and the potential to use them for OSCC diagnosis (p = 0.0001). 15 of these miRNAs were up-regulated (miR-7703, miR-3928-5p, miR-889- 5p, miR-3147, miR-4474-3p, miR-3170, miR-6895-5p, miR-1238-5p, miR-4521, miR- 548ac, miR-3158-3p, miR-1343-3p, miR-7152-5p, miR-3148, miR-3124-3p, ) and 12 miRNAs were down-regulated (miR-let-7f-5p, miR-let-7a-5p, miR-1247-5p, miR-574- 3p, miR-194-5p, miR-200c-3p, miR-32-5p, miR-6126, miR-99a-5p, miR-345-5p, miR- 301a-3p, miR-101-3p). Also, this study pointed out for the first time that overexpression of miR-7703 is a great and significant biomarker that could be used for cancer diagnosis. Hence, to confirm these results, it is reasonable to validate these miRNAs as signature diagnostic biomarkers in a large majority of patients. Chapter five was aimed to validate the previous findings of chapter four in relation to the five dysregulated miRNAs in 150 patients with head and neck cancer (HNC) along with 80 healthy individuals. Thus, five dysregulated miRNAs (miR-7703, miR- let-7a- 5p, miR- 345-5p, miR- 3928 and miR- 1470) were selected for the validation study using a real time PCR technique. In this study, significant expression differences of miR-let-7a-5p and miR-3928 (p≤0.05) in HNC patients compared with healthy individuals were confirmed. These miRNAs were revealed to provide a good discriminative ability with AUC values of 0.85 and 0.74, respectively. Taken together, the data reveals that the availability of significant miRNA could play a great role in HNC early diagnosis. Those studies contribute to knowledge on the correlation of miRNAs expression with OSCC and HNC and improve the availability of current diagnostic methods of cancer. In addition, the literature findings of miRNA studies supported our results and reported consistent results in the role of miRNA in cancer. However, this significance of the dysregulated expressions of miRNAs requires further investigation and additional research.