microRNA—Does it Presage the Malignant Change of Oral Potentially Malignant Disorders: A Systematic Review

INTRODUCTION

Early detection of oral cancer (OC) and commencement of apposite treatment is considered to be the ideal route to advance the survival of patients. Recently, noninvasive biomarkers have been used for premature detection of OC and to predict the rate of malignant progression of cancer have been implemented. One such noninvasive method to examine saliva, is a body fluid rich in proteins, nucleic acids, electrolytes, exfoliated mucosal cells, and hormones which shows variation in various local and systemic conditions.¹ Oral squamous cell carcinoma (OSCC) is the most predominant, approximately 90% of oral malignancies are associated with increased mortality and morbidity in terminal stages. Of the various etiological factors of OSCC, OPMD serves to play a vital role. Oral potentially malignant disorders are associated with an increased menace of malignant progression, thus the intervention of OPMDs is essential to prevent further progression. The main challenge is identifying the OPMDs with an increased risk of malignant progression. In accordance, vast areas of research demanding the discovery of various biomarkers are being undertaken. Recently, more precise and sensitive molecular biomarkers for early discovery of OPMD and to predict the therapeutic planning and progression of OSCC have been studied.

One such diagnostic and prognostic biomarker is microRNA (miRNA). Also, miRNAs are 22 nucleotides long, single-stranded, endogenous, non-coding RNA molecules,² which have the ability to intercede with the translational repression of messenger RNAs to regulate target gene expression.^3,4 They have been said to act as the ideal set of molecular biomarkers owing to their stability in body fluids and their disease-specific behavior.⁵ Furthermore, miRNA has the ability to adjust the expression of several target genes, as a result of this miRNA–messenger RNA (mRNA) interaction, RNA-induced slicing complex incorporated with mature miRNAs are formed which regulates cellular growth, apoptosis, differentiation and tumor suppression.⁴ Approximately 1,500 such miRNAs have been studied and further discovery is still an unending story. Also, miRNA shows variation between healthy and malignant conditions, which might be due to the genetic and epigenetic instability present inOC.⁶ This makes them human gene regulators.⁷ They tend to show a positive correlation with the clinical staging, metastasis, and survival outcome of various cancer and at instances they themselves act as oncogenes or tumor suppressor genes. In literature, various in vitro studies have been performed to figure out the role of miRNA in tumorigenesis. Tran et al.⁷ came up with their in vitro study that miRNAs are significant in tumorigenesis.⁸ Histopathological and clinical evaluation of lesions is one of the standard methods of diagnosis but is highly subjective and less objective to predict the malignant transformation. Use of adjuvant methods such as molecular biomarkers, miRNA, can help to overcome this drawback. Moreover, miRNA can be differentially expressed among OPMD, the presence of which can be correlated with malignant progression. They bring stable makes their use and reliance more advantageous than other unstable biomarkers.

Thus, this systematic review is targeted to analyze the currently available studies on altered miRNAs and to delineate their role in tumorigenesis with an aim to bridge the existing gap of considering miRNAs as a specific independent biomarker for malignant progression and to map cancer progression.

MATERIALS AND METHODS

This systematic review was done adopting PRISMA guidelines and PICO was used to frame the clinical questioning and search criteria.

RESULTS

DISCUSSION

Over the long run, OPMD can progress from mild dysplasia to in situ carcinoma and finally OSCC. The hazard rate of malignant transformation in the OPMD group is 8.19 times higher when compared to patients with no previous OPMD incidence.⁸ The rate of malignant progression can be reduced if OPMDs are diagnosed and treated at early stages. One of the effective ways is to identify the OPMDs with a high risk of progression and to treat them as most of them never show malignant progression and remain stable. For this, histopathological and clinical examinations have served as diagnostic tools. Although effective the histopathological grading of lesions is highly subjective and invasive which cannot be performed in all patients primarily patients with impaired coagulation. Considering this, other reliable yet simple methods have been studied. Evaluating biomarkers present in saliva has now been implemented, this serves to be a noninvasive yet reliable and stable diagnostic aid. One such biomarker in the saliva is miRNA, their structure enables its stable prevalence in all body fluids such as saliva, and plasma.²⁴

When looking into studies based on miRNA and its role in OPMD progression into cancer, it was found that the studies conducted were very scarce and the first ever study to be done was published in 2009.⁹ Since then, few studies either genome-wide or specific genome studies using salivary miRNA have been done. This resulted in figuring out the role and the regulation of miRNAs during cancer genesis. Apart from saliva, miRNA found in tissue samples and plasma serve to be effective, while saliva is a noninvasive and feasible method. Tissue samples serve to be direct evidence of miRNA status in lesions and studies conducted using saliva have concluded that utilizing saliva is of equal effectiveness.^2,9–18 It is a known fact that cellular debris and degraded RNAs are one of the components of saliva. Thus, miRNA detection can be enhanced by using the supernatant saliva, that is, cell-free nucleic acids can be identified through a centrifuge, one of the procedures done universally in all included studies. Body fluids considered were saliva and serum. In the included articles, either stimulated or unstimulated saliva as a whole or only supernatants were used for analysis. Further variation existed as salivary samples were collected at different stages of the lesion.

A vast range of miRNAs were analyzed and quantified in the included studies, miRNA-181a and b;¹⁹ miRNA 21,^9,11,18,20 miRNA-146a;²¹ miRNA 31;^9,11,18,23 miRNA 21-5p;²³ miRNA-24-3p,^12,23 miRNA-21-5p, miRNA-99a-5p, miRNA-100-5;²³ miRNA-10b, miRNA-372;^10,18 and miRNA-375;^10,18 mRNA 145, miRNA 184;¹¹ miRNA-10b-5p, miR-486-5p;¹³ miRNA-136, miRNA-147, miRNA-1250, miRNA-148a, miRNA-632, miRNA-646, miRNA-668, miRNA-877, miRNA-503, miRNA-220a, miRNA-323-5p, miRNA-24, miRNA- 27b;¹⁴ miRNA-412-3p, miRNA-489-3p, miRNA-512-3p, miRNA-597-5p, and miRNA-603, miR-193b-3p, miRNA-30e-3p, miRNA-376c-3p, miRNA-484, miRNA-720, and miRNA-93-3p;¹⁷ miRNA-423-5p;¹⁷ miRNA 125a, miRNA200a.¹⁸ Added up, a total of 43 miRNA were studied for their role in tumorigenesis their regulation pattern, and their difference in patients with OPMD or OSCC than normal counterparts.

Until now, histopathological examination and dysplasia grading are one of the standard methods used to predict malignant progression and have been used as a standard comparator for new emerging methods. In such a way, the analyzed miRNAs were compared with dysplasia grading. Kai Feng et al.⁸ concluded that patients with epithelial dysplasia showed early disease progression which was found to be similar with miRNA 31 levels, found to be higher in OPMD patients. The Cox proportional hazard ratio reveals that miRNA 31 can be used as an independent marker for OPMD progression.⁹ Maheshwari et al.⁹ concluded that statistically significant (p = 0.001) result was obtained with miRNA 21 and 31 among patients with dysplasia and controls.¹⁰ Tu et al.¹⁰ came up with the results that miRNA 10b, miRNA 372, and miRNA 375 showed no significant relation with epithelial dysplasia. While the receiver operating characteristics (ROC) results demonstrated that miRNA 372 could distinguish dysplastic changes in OPMD lesions with a sensitivity of 57.1% and a high specificity of 92.3%.²¹ Tu et al.¹⁰ in their upgraded study showed that miRNA 375 levels showed significance similarly with a sensitivity of 71% and specificity of 83%¹⁰. Gai C et al. showed among different diagnostic tools, nodal metastasis, vascular invasion, and unfortunate survival showed significant relation with miRNA 181.¹⁶

The main objective of all included studies was to evaluate the role of miRNAs in disease progression in OPMD and OSCC patients. As a result, few potential and independent diagnostic markers were identified. Furthermore, miRNA 21, a biomarker, studied by most of the authors proved to be an effective one despite having controversial results among some.⁹ Also, miRNA 21 showed a significant difference among patients with OPMD and controls (p = 0.002), and upregulation of miRNA 21 was found more in oral leukoplakia than oral lichen planus^10,18 and in OSCC. The receiver operating characteristic curve was designed resulting in quadrupled miRNA 21 levels with 65% sensitivity and specificity.¹¹ In tongue OSCC cases, miRNA is much higher in progressed lesions than early ones (p < 0.05), and miRNA 21 is ∼39.51-fold higher in advanced tumors as compared to normal counterparts. Thus, miRNA upregulation is correlated with tumor staging and progression. Further TPM1 and PTEN tumor-suppressing genes were found to be correlated with miRNA 21 levels, where miRNA 21 target these genes and silence them leading to cancer progression.²⁰

When looking into miRNA 31, it was found from the ROC curve that it effectively differentiated disease progression among patients with disease progression and non-progressing patients with an area under the curve (AUC) of 0.81.¹ Similar upregulation was found in patients with OPMD and OSCC.¹⁰ with a 2.5-fold increase.^10,11 Thus, stable levels of miRNA 31 indicate no progression of OPMD into malignancy.¹⁸ Furthermore, miRNA 10b and miRNA 372 show upregulation in OSCC and OPMD samples. While the increase in miRNA 372 levels is directly associated with poor prognosis,¹⁸ miRNA 184 levels proved to be one of its kind to discriminate between OSCC and OPMD, the ROC curve showed a 3-fold increase in levels of miRNA among patients with OPMD and OSCC while having a sensitivity and specificity of 75% and 80%, respectively.¹¹ Salivary miRNA 24p-3p which has been shown to originate from the tumor cells shows a characteristic high level in OSCC patients with ROC AUC of 0.738. At the same time, overexpression of miRNA 24p-3p has been shown to promote the proliferation of OSCC cells and regulate cell cycle-related gene expression.¹² Stage II OSCC shows high expression levels of miRNA 486-5p, where an increase in exosome dimension was seen compared to control samples.¹³ Overexpression of miRNA-27b is a hopeful marker indicating initiation of OSCC.¹⁴

During the progression of OSCC, cell motility, cell proliferation, and maturation takes place. miRNA 181 upregulation can be accompanied by the above-mentioned genesis process thus targeting such miRNAs during treatment procedures can result in a significant result.¹⁵ The survival rate after cancer treatment plays an important role during treatment planning and for which numerous predictive factors have been discovered and implemented. Adding to that miRNA 146a levels can be used to predict the survival rate. The decline in levels of miRNA 146a after initiation of treatment indicates a better survival rate.²⁴

Furthermore, miRNA 375,⁴ and miRNA 10b-5p¹³ showed downregulation in OPMD patients more than their normal counterparts. A –ΔCt value of 8.97 was observed in OPMD patients, lower than the value observed in controls (10.17).¹⁰ Comparably, miRNA 145 levels exhibited a statistically significant (p < 0.001) decrease in levels of OPMD than normal counterparts predicting disease progression with a specificity of 70% and sensitivity of 60%.¹¹

Overall, when analyzing the included articles, it can be put forth despite their limitations that miRNAs serve as a suitable biomarker for disease progression and can be relied upon.

CONCLUSION

From this review, it can be understood that salivary miRNAs are regulated in OPMDs and OSCC which has correlations with the clinical and histopathological staging, predicting malignant progression, and also aid in treatment outcomes. They have the potential to be used as a noninvasive independent biomarker for OPMD and related oral lesions. Further, in future, the use of salivary biomarkers such as miRNA in early predicting OPMDs in smokers can be implemented which might serve to be a breakdown in smoking cessation and to plan therapy accordingly.

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