Salivary Micro-RNA and Oral Squamous Cell Carcinoma: A Systematic Review

Oral squamous cell carcinoma (OSCC) is a widespread malignancy with high mortality. In particular, a delay in its diagnosis dramatically decreases the survival rate. The aim of this systematic review was to investigate and summarize clinical results in the literature, regarding the potential use of salivary microRNAs (miRNAs) as diagnostic and prognostic biomarkers for OSCC patients. Twelve papers were selected, including both case–control and cohort studies, and all of them detected significantly dysregulated miRNAs in OSCC patients compared to healthy controls. Based on our results, salivary miRNAs might provide a non-invasive and cost-effective method in the diagnosis of OSCC, and also to monitor more easily its evolution and therapeutic response and therefore aid in the establishment of specific therapeutic strategies.


Introduction
Oral cavity cancer is the most frequent malignancy of the head and neck. It represents the 16th most common malignancy and the 15th leading cause of death worldwide, with an incidence of oral cancer (age adjusted) in the world of four cases per 100,000 people, with a wide variation across the globe which depends on gender, age groups, countries, races and ethnic groups and socio-economic conditions [1].
Roughly 90% of oral cancers histologically originate from squamous cells and are classified as oral squamous cell carcinoma (OSCC). OSCC develops in the oral cavity and oropharynx and can occur due to many etiological factors; smoking and alcohol remain the most common risk factors especially in the western world. Other risk factors include diet, immunodeficiency and high-risk HPV 16/18 [2]. There are also several genetic alterations involved in oral carcinogenesis. Among these, alterations in oncosuppressors (APC, p53), proto-oncogenes (Myc), oncogenes (Ras), and genes that control normal cellular processes (EIF3E, GSTM1) play a fundamental role in cancer development [3].
Despite advances in therapies, the overall 5-year survival rate has remained unchanged during the past decades. While at early stages the survival rate is approximately 89%, at late stages it decreases to 39%. Unfortunately, oral cancer patients are still frequently diagnosed in advanced stages despite educational interventions for prevention and early diagnosis, that is the most important prognostic factor for predicting survival [4].
To date, the gold standard for OSCC diagnosis is represented by a clinical oral examination integrated by a histological investigation on tissue biopsies of suspicious lesions [5]. However, cancer research is currently focusing on finding less invasive and cost-effective methods to provide a more comprehensive view of the cancer profile, also to more easily monitor its evolution and therapeutic response and therefore aid in the establishment of

Bibliographic Search and Study Selection
The initial database search yielded a total of 1880 entries; of which 977 were found i PubMed ® /MEDLINE, 898 in Scopus, and five in Cochrane library. In addition, a partia research of the gray literature was carried out through Google Scholar. A flow chart tha depicts the screening process is displayed in Figure 1. After excluding all duplicates, th total number of entries was reduced to 1021. A total of 974 articles were excluded afte review of title and abstract. Hence, full-text examination was conducted for 47 articles. A total of 35 additional articles were excluded after full-text review and application of th eligibility criteria. The final selection consisted of 12 articles. Detailed data for the 12 in cluded studies are listed in Table 1.   Salivary miR-31 was significantly increased in patients with OSCC at all clinical stages, including very early stages. In addition, it was shown to be more abundant in saliva than in plasma, and after tumor surgical removal its expression was reduced. Salivary miR-139-5p was significantly reduced in TSCC patients compared to controls, and its level turned back to normal after surgery.
Mehdipour et al. 2018 [33]. 30  miR-21 levels were significantly increased in saliva samples derived from patients with OLP, dysplastic OLP and OSCC, compared to those from healthy controls. Conversely, significant decreases in miR-125a levels were found in the OLP, dysplastic OLP and OSCC samples, compared to those from healthy controls. Significant increases in miR-31 levels were found in samples derived from dysplastic OLP and OSCC patients, but not in nondysplastic OLP, compared to healthy controls. miR-200a levels were significantly decreased only in samples derived from OSCC patients The authors identified a significant increase of miR-24-3p in the salivary exosomes from 45 preoperative OSCC patients compared to healthy individuals OSCC-Oral squamous cell carcinoma; ND-Undeclared; OLP-Oral lichen planus; qRT-PCR-Quantitative reverse transcription polymerase chain reaction; RT-qPCR-Real-time quantitative polymerase chain reaction; qPCR-Quantitative polymerase chain reaction; RT-preamp-qPCR-reverse transcriptase preamplification-quantitative; FFPE-formalin-fixed paraffin-embedded; EV-extracellular vesicle.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et al. 2016;Seung-Ki Min et al. 2017;He L et al. 2020) also utilized microarray-based miRNA analysis.
All the papers included identified a set of significantly dysregulated miRNAs in OSCC patients compared to healthy controls. Different types of miRNAs were found to be upregulated and other downregulated in OSCC patients. In particular, mir21 was identified as upregulated in OSCC patients compared to healthy controls in three studies (Zahran et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  All the papers included identified a set of significantly dysregulated miRNAs in OSCC patients compared to healthy controls. Different types of miRNAs were found to be upregulated and other downregulated in OSCC patients. In particular, mir21 was identified as upregulated in OSCC patients compared to healthy controls in three studies The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  All the papers included identified a set of significantly dysregulated miRNAs in OSCC patients compared to healthy controls. Different types of miRNAs were found to be upregulated and other downregulated in OSCC patients. In particular, mir21 was identified as upregulated in OSCC patients compared to healthy controls in three studies The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  All the papers included identified a set of significantly dysregulated miRNAs in OSCC patients compared to healthy controls. Different types of miRNAs were found to be upregulated and other downregulated in OSCC patients. In particular, mir21 was iden-tified as upregulated in OSCC patients compared to healthy controls in three studies The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were ex-cluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regard-ing not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  All the papers included identified a set of significantly dysregulated miRNAs in OSCC patients compared to healthy controls. Different types of miRNAs were found to be upregulated and other downregulated in OSCC patients. In particular, mir21 was iden-tified as upregulated in OSCC patients compared to healthy controls in three studies The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were ex-cluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regard-ing not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were ex-cluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regard-ing not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Description of Included Studies
Detailed data for the 12 included studies are listed in Table 1. All studies included in the present review are studies performed on humans.
All the studies utilized real time quantitative polymerase chain reaction (RTq-PCR) to quantify salivary miRNAs. Three studies (Duz et  The other studies did not report superimposable outcomes.

Excluded Studies
Out of 47 papers for which the full-text was analyzed, 35 articles . were excluded from the systematic review. (Table A1). The main reasons for exclusion were the following: study type; miRNAs detected in samples different from saliva and data regarding not OSCC but oral cancer in general or premalignant conditions.

Quality Assessment of Included Studies
The risk of bias of included studies was assessed using the Newcastle Ottawa scale (NOS). Outcomes are reported in Table 2.

Discussion
The 5-year survival rates in OSCC depends on the stage at diagnosis. Patients have better survival and favorable outcomes if detected early, as compared to those diagnosed in advanced stages. There are several screening and awareness programs implemented, but they have not been able to lower the incidence of OSCC.
Traditional diagnostics for malignant tumors such as tissue biopsy and mucosal scraping examination can often be time-consuming and physically intrusive, adding excessive stress and pain to patients, thereby leading to poor patient compliance. In this context, the addition of a non-invasive diagnostic test based on a liquid biopsy would be beneficial in the early diagnosis and prognosis of such diseases as an alternative to solid biopsies. Liquid biopsy is a novel approach that relies on the study of circulating cells, circulating DNA, micro-RNA, microvesicles and exosomes in body fluids supporting the concept of personalized medicine [72,73]. In recent decades, saliva has been widely investigated as a promising source of OSCC biomarkers for liquid biopsy [74].
There are many advantages to employing saliva as a substrate for diagnostic analysis. Its sampling is inexpensive and non-invasive. Aberrant expression profiles of salivary miRNAs have been detected in different types of cancer, showing their power as a discriminatory clinical method [75]. A recent systematic review and meta-analysis demonstrated a high diagnostic accuracy of salivary and blood miRNAs to differentiate OSCC patients from healthy individuals, with sensitivity, specificity, and AUC (area under the SROC curve) values of 0.78, 0.82, and 0.91, respectively [43]. However, considerable heterogeneity was detected among the included studies and the authors suggested the need for further research on the topic.
A total of 12 papers were included in the present systematic review. Four of them were considered of good quality (NOS score equal to 7), and the remaining eight studies were considered at high risk of bias (NOS score 6 or 5), demonstrating the need for further studies with a more rigorous design on the topic.
The studies included applied heterogeneous methodologies to investigate the role of miRNAs in OSCC patients. In particular, the time of sampling (i.e., before and/or after surgery/radiotherapy) was not reported in all the studies. The research protocols included stimulated or unstimulated saliva for analysis of either whole saliva or salivary supernatant. In addition, salivary samples were taken at different clinical stages of OSCC and the studies differed regarding patients' demographic characteristics (i.e., smoke, alcohol consumption, positivity to HPV, patients' age and sex, etc.) and possible comorbidities possibly affecting the outcomes. Some of the studies included did not account for such patients' characteristics nor for the site of OSCC (buccal mucosa, gingiva, tongue, etc.). For this reason, the realization of a metanalysis was contraindicated and it is difficult to draw comprehensive conclusions on the topic. These heterogeneities might also explain why the type of miRNAs identified and the dysregulations detected in the various studies were mostly not superimposable.
Differently from the other investigations included, the study by He et al. analyzed miRNAs in salivary exosomes. The lipid bilayer of exosomes can protect miRNAs from degradation by RNase in body fluids. However, the use of salivary exosomal miRNAs as biomarkers for human disease remains controversial. A critical limitation of using salivary exosomes for cancer screening is that differences in isolation techniques may alter the composition of purified subpopulations and the purity of the exosome pellets. Gai et al. analyzed miRNAs in salivary extracellular vesicles (EVs, including exosomes, microvesicles or ectosomes, and apoptotic bodies). A previous study [76]. showed that most of the salivary RNA was associated with EVs. However, miRNAs can be differentially represented in whole saliva and salivary EVs, as it has been previously described for total plasma or plasma-derived EVs [77,78].
Despite the limitations listed above, our data overall demonstrate that there is strong evidence for a role of miRNAs in OSCC behavior. In fact, all the 12 papers included detected significantly dysregulated miRNAs in OSCC patients compared to healthy controls. In particular, some miRNAs were identified as differently expressed in OSCC patients in different papers: mir21, mir31, mir200a and mir125a. This panel of four miRNAs appears to have a significant predictive value in OSCC. Further longitudinal studies are needed in order to confirm which specific salivary miRNAs are the most effective biomarkers for the diagnosis and management of OSCC patients.
In conclusion, the present systematic review suggests that salivary miRNAs might provide a non-invasive and cost-effective method in the diagnosis of OSCC, and also to monitor more easily its evolution and therapeutic response and therefore aid in the establishment of specific therapeutic strategies.