LACC patients commonly exhibit clinical manifestations such as ocular pain, globe dystopia, proptosis, and ‘S’ shaped ptosis [17], and have a grave prognosis characterized by frequent local recurrences, distant metastases, and tumor-related mortality [9]. Moreover, LACC destroys the lacrimal gland which is the pivotal contributor to the tear film to protect and lubricate the ocular surface [18, 19]. Although tears are known to contain miRNAs, proteins and other molecules [14], there have been extremely limited studies on tears as a source of biomarkers for LACC. As reported, while LACC and SACC are morphologically and genetically similar, they exhibit distinct microRNA expression profiles [9] and LACC has a poor prognosis. In our present study, we screened LACC-specific miRNAs using microarray data. We then verified the data by qPCR experiments in tissues, SACC cell lines, tears and serum. Ultimately, we identified hsa-miR-200b-3p, hsa-miR-200c-3p and hsa-miR-141-3p as the most significantly upregulated miRNAs.

Changes in tissue miRNAs can be reflected in the serum, offering a non-invasive alternative to direct tissue measurement which is intrusive and inconvenient. Emerging investigations have focused on the development of non-invasive and more convenient biomarkers that enable earlier diagnosis of tumors that with local recurrences and distant metastases. These three identified miRNAs belong to the miRNA-200 family which consists of five members (miRNA-200a, -200b, -200c, -141 and -429) that have been reported in several types of advanced carcinoma [20]. These miRNAs are thought to be implicated in the early stages of tumor metastasis [21, 22]. For instance, hsa-miR-141-3p and hsa-miR-200c-3p have been identified as potential biomarkers for predicting colon cancer metastasis [23]. Additionally, the expression level of hsa-miR-200a-3p, hsa-miR-200b-3p, hsa-miR-200c-3p and hsa-miR-141-3p correlate with lymph node metastasis in breast cancer [24]. Moreover, hsa-miR-200c-3p in serum has been suggested as a novel non-invasive early biomarker for bladder cancer [25]. These 3 investigated miRNAs were selected based on the results of experiments in which we analyzed the expression in clinical tissue samples and SACC cell lines. Whereas the highly metastatic potential of the ACC-M cell line, we observed these three miRNAs were highly expressed in ACC-M compared to HSG cell line, suggesting that these 3 differentially expressed miRNAs were metastasis-related miRNAs directly or indirectly involved in the progression of metastasis. Furthermore, our microarray data revealed that the three miRNAs significantly upregulated in the recurrence group compared with the LACC primary group, thus highlighting their potential relevance to tumor metastasis and prognosis. Consistent with their expression in tissues, these three miRNAs were also upregulated in the serum and tears of LACC patients compared to controls. Therefore, it is reasonable to propose that these three miRNAs in tear fluid or serum might be used for the early detection and diagnosis of LACC.

Following the resection or radiotherapy of LACC lesions, it is essential to conduct these three miRNAs detection in tears and serum at different stages of disease progression to monitor prognosis in patients who have been cured or experienced relapse assessed by the imaging and clinical manifestations, with the goal of developing a correlation analysis along with a diagnostic evaluation system and facilitating their translation into clinical practice for early diagnosis and intervention. Meanwhile, detecting molecular changes in tear fluid can help us to be vigilant against lacrimal gland diseases whenever necessary. However, our study has some limitations. Only 10 LACC tissues that lacked systems-level identification were used to investigate the differentially expressed miRNAs, preventing us from correlating expression levels with TNM staging and overall survival time. Future research should involve a large sample size to better determine the correlation between miRNAs expression and clinical stages. In addition, we did not conducted an in-depth investigation on the functions and mechanisms of these miRNAs. As miRNAs act as oncogenes or tumor suppressors depending on their target gene functions, we plan to utilize databases of miRNA:mRNA target interactions to establish a network profile of these three miRNAs and further evaluate their impact on LACC.