Introduction
Oral cancer is one of the most prevalent cancers worldwide[
1]. Despite improvements in diagnosis and treatment in recent decades, the survival rate for oral cancer has not significantly changed due to the development of distant metastases and therapeutic resistance[
2‐
4]. It is essential to thoroughly investigate the pathogenesis of this disease to provide fundamental knowledge for future clinical applications.
MicroRNAs (miRNAs) constitute an abundant class of small, non-coding RNA molecules that regulate gene expression by targeting mRNAs to induce translational repression or mRNA degradation[
5]. Increasing evidence indicates that miRNAs contribute to the development of cancer by negatively regulating target gene expression, and therefore they can function as tumor suppressors or oncogenes[
6,
7]. Recently, miRNA screening in several types of cancer has identified unique expression profiles associated with specific tissues or clinical features, including head and neck cancer[
8,
9]. To improve the understanding of the role of miRNAs in oral cancer, we previously performed global miRNA profiling of normal keratinocyte and cancer cell lines. We discovered 23 miRNAs with significantly altered expression in cancer cells, including miR-196[
10]. miR-196 has been reported to be aberrantly expressed in various malignancies, including melanoma, leukemia, and glioblastoma[
11‐
21]. However, the underlying mechanism by which these molecules cause malignancy remains unclear.
In the present study, we characterized the function of miR-196 and elucidated it’s molecular mechanism in oral cancer. We found that the miR-196 family positively regulated cell invasion and migration, and had no effect on cell growth. Mechanistically, miR-196 exerted their effects by directly targeting and inhibiting non-metastatic cells 4 (NME4) protein expression to regulate the JNK-TIMP1-matrix metalloproteinase (MMP) signaling pathway. We revealed that both miR-196a and miR-196b were highly over-expressed in the cancer tissues of patients with oral cancer, demonstrating the clinical significance of these molecules during cancer progression.
Discussion
The dysregulation of miRNAs is associated with malignant transformation. Previously, miR-196 expression was shown to be altered in several cancers. Although some investigators have reported decreased miR-196 expression in cancers, others have observed increased miR-196 expression. For example, miR-196a and miR-196b are down-regulated (or function as tumor suppressors) in melanoma[
11] and acute lymphoblastic leukemia[
17,
19]. However, miR-196a and miR-196b over-expression has been observed in several malignant diseases, including cancers of the esophagus[
21], pancreas[
20], colorectum[
12], glioblastoma[
14], and several types of leukemia[
13,
18,
32]. High miR-196a levels have also been associated with a poor prognosis in pancreatic cancer, glioblastoma, and oral squamous cell carcinoma. Furthermore, the polymorphism of pre-miR-196a2 gene was observed in several malignant diseases, including head and neck cancer, and has been associated with cancer susceptibility or prognosis[
33‐
37]. These studies indicate that miR-196 dysregulation plays an important role in carcinogenesis. Consistent with other reports, we previously observed miR-196 overexpression in oral cancer cell lines[
10]. In that study, we further identified miR-196 overexpression in the cancer tissues of approximately 90% of patients with oral cancer compared to their expression in normal tissue (Figure
5), and this overexpression was associated with an aggressive phenotype with lymph node invasion (Table
1). These results demonstrate the significance of miR-196 in the development of oral cancer.
There are limited reports on the role of miR-196 in cancer development. In a study of breast cancer, ectopic miR-196a expression suppressed cell invasion, but the expression level of miR-196 was not correlated with the clinical metastatic status[
15]. This result is different from the findings in gastrointestinal cancers, in which miR-196 overexpression promotes cell migration and invasion in colorectal and gastric cancer cells[
12,
38,
39]. In another study, transfection of miR-196a mimic oligonucleotides into esophagus cells revealed that miR-196a promoted cell proliferation and suppressed apoptosis[
40]. Thus far, the function of miR-196 in cancer remains obscure. In this study, we determined the role of miR-196 in oral cancer using both silencing and overexpression approaches. We found that both miR-196a and miR-196b actively promoted cell migration and invasion (Figures
1B-C), which were supported by the altered expression of fibronectin and N-cadherin (Figures
1D). However, neither miR-196a nor miR-196b affected cell growth and colony formation (Additional file
2: Figure S1). Hence, the main function of the miR-196 family in oral cancer is the regulation of cell mobility and invasiveness.
Identification of the target molecules of these miRNAs is of high interest. Previously, several molecules were reported as regulatory targets of miR-196. HOX family members have been reported primarily, including HOX-B7 (miR-196a target) in melanoma[
16], HOX-A (miR-196b target) in acute lymphoblastic leukemia[
18], and HOX-C8 (miR-196 family target) in melanoma[
11] and breast cancer[
15]. In this study, four HOX family genes (HOXA5, HOXB6, HOXB7, HOXC8) were identified as targets of miR-196. However, none of these genes responded to miR-196 perturbation (Additional file
2: Figure S4). This difference may be due to the distinct tissue specificity among the various regulatory targets of miR-196. In this study, after clarifying the correlation between NME4 and miR-196 expression in cells (Figures
2A-C), assessing the response of NME4 to miR-196 modulation (Figures
2D), we identified NME4 as a direct target of miR-196a and miR-196b in oral cancer using a luciferase reporter assay (Figure
2E),
NME4, also named nm23-H4, is a member of the nm23 family[
41]. The proteins in this family possess nucleoside diphosphate kinase activity, which is believed to be involved in DNA repair mechanisms[
42]. Nm23 family proteins also contain the RGD domain, as they can bind to integrin, and this family has been postulated to be involved in cell adhesion and migration[
43]. Thus far, only a few studies assessed the association of NME4 with cancer, but genomic aberration or altered gene expression has been observed for NME4 in several types of cancers[
44‐
47]. Although the function of NME4 is unclear, it was reported that an nm23 family member, NEM1, is regulated by TP53[
48] and that it acts as a metastatic suppressor[
49]. In this study, we also found that ectopic expression of NME4 has no significant effect on cell invasion and migration (Figures
3B-C), indicating that a certain level of NME4 protein is sufficient for maintaining cellular mobility. However, restoration of silenced NME4 suppressed these effects induced by miR-196 (Figures
3B-C), suggesting that NME4 participates in the miR-196 regulatory pathway by inhibiting these functions. Collectively, miR-196 plays an oncogenic role by degrading NME4, thus accelerating cell migration and invasion.
The downstream regulatory mechanism of the miR-196–NME4 interaction was further investigated. In examining three MAPK family molecules, we found that p-JNK, but not p-Erk or p-p38, responded to miR-196 expression and NME4 inhibition, whereas miR-196 and NME4 had minimal effects on the expression of MAPK proteins (Figure
4A). These results indicate that miR-196–NME4 signaling could result in JNK phosphorylation and activation. In addition, TIMP1 and MMP1/9 displayed opposite responses to miR-196 suppression and NME4 augmentation (Figure
4A). These results suggest that TIMP1 and MMP1/9 are the downstream regulatory molecules of the miR-196–NME4 signaling axis. Additionally, we found that p-JNK inhibition increased TIMP1 expression and decreased MMP1/9 expression (Additional file
2: Figure S5). Hence, TIMP1 and MMP1/9 could be regulated by JNK phosphorylation. Moreover, the role of the NME4-pJNK-TIMP1-MMP1/9 signaling pathway in miR-196 function was further demonstrated by immunofluorescence staining and confocal microscopy (Figure
4B). Furthermore, this molecular pathway was also confirmed in another oral cell line (SAS) (Additional file
2: Figure S6) and paired normal and cancerous oral cancer tissues (Figure
5D). Thus, miR-196 appear to fine-tune the invasion mechanism in oral cancer by inhibiting NME4, leading to the activation of p-JNK and MMP1/9 and suppression of TIMP1 (Figure
4C).
In conclusion, we clarified that miR-196 promotes invasive and migratory phenotypes in oral cancer. Mechanistically, miR-196 exerted its functions by targeting to NME4, leading to the regulation of downstream molecules, including activating p-JNK, suppressing TIMP1, and augmenting MMP1/9. Consistently, clinical studies have revealed that both miR-196a and miR-196b are remarkably up-regulated in cancer tissue and correlated with lymph node metastasis. Thus, our findings provide new knowledge of the underlying mechanism of cancer metastasis. miR-196 may serve as a promising marker for better oral cancer management.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
YCL performed all experiments; she involved in the design of study, developing assay methodology, acquisition and analysis of laboratory data, and writing the manuscript. JTC participated in the design of study, acquisition of clinical data, performing clinicopathologic analysis, discussion of the laboratory data and revision of manuscript. CTL, CJK, SFH, IHC, CCH, and YCH participated in acquisition of clinical data and clinicopathologic analysis. CYT, HMW and TCY involved in data analysis, discussion and interpretation. GRY and CHC participated in the design of the study, developing assay methodology, and performed statistical analysis. AJC is the corresponding author as well as the study supervisor; she involved in conception and design of this work, analysis and interpretation of data, writing and revision of the manuscript. All authors have read and approved the final manuscript.