Differential expression of matrix metalloproteinases and miRNAs in the metastasis of oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is one of the most common malignancies of the head and neck region in the world [1, 2]. OSCC is particularly risky and is usually discovered when the cancer has metastasized to the lymph nodes of the neck since it progresses without producing pain or symptoms that might be readily recognized by the patients in its early stages [3]. Although surgical resection followed by postoperative radiotherapy and/or chemotherapy has made considerable treatment progress, the 5-year overall survival rate of OSCC patients still remains poor due to the common neighbouring tissue invasion and neck lymph node metastasis [4‐6]. Moreover, though many research groups have made great efforts to study OSCC pathogenesis, the underlying mechanisms of OSCC tumourigenesis and development have not been fully elucidated. Therefore, further studies focusing on the molecular mechanisms of OSCC are still urgently needed to improve early diagnosis, targeted therapy and prognosis.

Matrix metalloproteinases (MMPs) are a family of highly homologous extracellular zinc- and calcium-dependent endopeptidases with enzymatic activity and are capable of degrading many components from either the extracellular matrix (ECM) or basement membrane [7]. Studies have shown that MMPs are involved in numerous physiopathological processes, such as tissue remodelling, embryonic development, mammary involution, bone reabsorption, and wound healing [8, 9]. MMPs induced by both tumour cells and surrounding stromal cells are related to various processes associated with tumour cell proliferation, angiogenesis, neighbouring invasion and remote metastasis due to their ability to degrade ECM and alter cell migration [10, 11]. Increased levels of one or several MMPs have been found in most human cancers [12]. Overexpressed MMP-2 and MMP-9 are involved in the invasion process of OSCC, and MMP-9 is related to the poor prognosis of OSCC patients without neck node metastasis [13]. Significantly higher MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, MMP-11, and MMP-13 levels were found in tumours compared with normal mucosa, and MMP-9 might be useful for evaluating the malignant potential of head and neck squamous cell carcinoma [14]. It has also been reported that CXCR4 might promote OSCC cell migration and invasion by regulating MMP9 and MMP13 expression to activate the ERK signalling pathway [15]. Overall, increasing evidence has suggested that MMPs play critical roles in OSCC progression.

MicroRNAs (miRNAs) are a class of short noncoding RNAs that regulate the levels of posttranscriptional mRNAs by anchoring to the target sites on mRNA sequences in a complementary base-pairing manner [16, 17]. It has been reported that miR-222 inhibits OSCC cell invasion via the downregulation of MMP1 expression [18]. Moreover, miR-29a may play an inhibiting role in the progression of OSCC by negatively regulating MMP2 expression [19]. These findings provide important clues for potential therapeutic targets and approaches in the future. However, the miRNA regulatory mechanisms of MMP expression in OSCC metastasis remain unclear. Thus, the elucidation of aberrantly expressed MMPs and the related miRNA regulatory mechanisms in OSCC progression is critical.

In the present study, we aimed to reveal the possible regulatory mechanisms related to miRNAs and MMPs involved in the OSCC metastatic process. Samples collected from patients with nonmetastatic or metastatic tumours, as well as normal controls, were used for high-throughput mRNA and miRNA microarray analysis. The selected important markers involved in the metastatic process were also validated by experiments in another cohort of OSCC patients.

In our study, mRNA and miRNA microarrays were used to analyse the expression profiles of MMPs and miRNAs in nonmetastatic tumour samples, metastatic tumour samples, and normal tissues. The results showed that 10 MMP genes were upregulated and MMP27 was downregulated in nonmetastatic tumour samples compared with normal controls. MMP12, MMP1, MMP9, MMP7, MMP3, MMP14, MMP17, MMP13, and MMP10 were the 9 common upregulated genes in metastatic and nonmetastatic tumour samples compared with the normal controls. Moreover, MMP7, MMP13, and MMP10 were significantly upregulated in metastatic tumour samples compared with nonmetastatic tumour samples.

Deraz et al. [32] revealed that the overexpression of MMP-10 could promote the invasion and metastasis of head and neck squamous cell carcinoma, and invasion driven by MMP-10 is possibly associated with p38 MAPK inhibition. It has been reported that MMP-13 could promote the invasion, migration, and adhesion abilities of oral cancer cells [33]. The abnormal expression of MMP-7 has been found to be closely related to the biological behaviour of OSCC, and MMP-7 may be induced by COX-2 to contribute to the invasion and metastasis of OSCC [34]. The upregulation of glutamate decarboxylase 1 (GAD1) correlated with cellular invasiveness and migration in OSCC by regulating β-catenin translocation and MMP7 activation [35]. Functional enrichment analysis revealed that MMP7, MMP13, and MMP10 were related to the collagen catabolic process, extracellular matrix disassembly, extracellular matrix, and metalloendopeptidase activity. Therefore, the overexpression of MMP7, MMP13, and MMP10 might play important roles in controlling tumoural invasiveness and metastasis in OSCC.

The expression levels of MMP7, MMP13 and MMP10 were validated to be significantly upregulated in oral cancer compared with normal samples according to the data mining of published profiles in Oncomine. Among these differentially expressed MMPs, we focused on the roles of MMP7, MMP13 and MMP10. After the target genes of the DE-miRNAs were predicted, functional enrichment analysis showed that the target genes of the DE-miRNAs identified in metastatic tumour samples compared to nonmetastatic tumour samples were mainly enriched in organelle membrane contact sites, cell migration in the hindbrain, and the TGF-beta signalling pathway. TGF-β1-triggered epithelial-mesenchymal transition (EMT) may play important roles in OSCC progression by upregulating MMPs to promote EMT [36]. Thus, the identified DE-miRNAs might be important for the progression of OSCC by regulating the target gene levels to influence the TGF-beta signalling pathway.

In our study, the regulatory relationships related to MMPs were selected to construct a regulatory network. In the regulatory network, MMP7 was regulated by miR-4697-5p and miR-7109-5p. MMP13 was regulated by 10 DE-miRNAs (miR-4697-5p, miR-6826-5p, miR-6741-5p, miR-6793-5p, miR-34b, miR-6759-3p, miR-503-5p, miR-6753-3p, miR-7111-3p, and miR-129-1-3p). Moreover, qRT-PCR confirmed that the expression of miR-7109-5p and miR-34b was significantly downregulated in metastatic OSCC samples compared with nonmetastatic tumour samples using samples collected from another cohort of OSCC patients. The abnormal expression of miR-7109-5p has been reported to occur in breast cancer and is associated with cancer development in patients with chronic obstructive pulmonary disease [37, 38]. The reduced expression of miR-34b and miR-129-3p was observed in gastric cancers due to DNA hypermethylation and was associated with poor clinicopathological features [39]. It was also suggested that the reduced expression of miR-34b*/c may be particularly important for the progression to the most advanced stages of human epithelial ovarian cancer [40]. Low expression levels of miR-34b and miR-34c were associated with distant metastasis formation in lung cancer [41]. To our knowledge, few studies have reported the regulatory mechanisms of miR-7109-5p and miR-34b in OSCC. According to the results of our study, miR-7109-5p and miR-34b seem to have tumour suppressor functions in OSCC. The aggressive form of OSCC shows the downregulation of these two miRNAs and subsequent upregulation of MMP 7 and MMP13, which are normally inhibited by miR-7109-5p and miR-34b. The feasibility of miR-7109-5p and miR-34b as promising prognostic and diagnostic indicators or potential cancer therapeutic targets will be evaluated in further studies. In the future, we will focus on the interaction of miRNAs and MMPs and the mechanism of OSCC progression mediated by the miRNA axis to provide valuable strategies for the diagnosis, treatment and prognosis of OSCC.

Several limitations of this study should be addressed. The most important limitation in this regard is the limited number of study samples and inability to control for potential confounders. Another limitation is that the patients included in this study had varying T and N stages, and the two groups of cases were not selected in pairs. The homogeneity of the two groups of cases was different. The metastatic group appears to be more homogeneous than the nonmetastatic group. The results of this study are at the transcriptional level, and further experiments at the protein level are needed to verify the results of this experiment.

In conclusion, our study performed gene and miRNA microarray analysis to reveal the underlying regulatory mechanisms of miRNAs and MMPs involved in the OSCC metastatic process. The MMP-related DEGs and DE-miRNAs were identified among the nonmetastatic tumour samples, metastatic tumour samples, and normal tissues. MMP7, MMP13, and MMP10 were upregulated in metastatic tumours compared with nonmetastatic tumours. The reduced expression of miR-7109-5p and miR-34b might play important roles in the metastasis of OSCC by upregulating MMP7 and MMP13, respectively.