NPC is a distinctive type of head and neck cancer which is consistently associated with EBV infection and prominent lymphoplasmacytic infiltration. Based on the studies on premalignant lesions and invasive cancers, we have proposed a model of NPC tumorigenesis, in which EBV infection and inactivation of multiple tumor suppressors on chromosomes 3p and 9p play crucial roles in the initiation process. The inactivation of
RASSF1A on 3p21.3 was shown to be an early event in NPC development [
20]. On chromosome 9p, we have previously demonstrated the consistent inactivation of
p16 on 9p21.3 in the primary tumors and precancerous lesions. In addition to a loss of growth inhibitory effects,
p16 inactivation predisposed nasopharyngeal epithelial cells to persistent EBV latent infection [
15].
miR-31 is a cancer-associated microRNA at 0.5 Mb telomeric to the
p16 locus and commonly deleted in various human cancers including melanoma, mesothelioma and urothelial carcinoma [
7,
21,
22]. In this study, we clearly demonstrated that
miR-31 is consistently inactivated in NPC by either homozygous deletion or promoter methylation. As shown in the xenografts, both
miR-31 and
p16 loci are located in the common homozygous deletion region of NPC (Figure
2a). Notably, loss of
miR-31 expression was also detected in the pre-invasive lesions although the dysplastic lesions are rare and only limited cases were studied. The findings suggest the crucial role of
miR-31 in early development of NPC.
miR-31 acts as tumor suppressor in several human malignancies, such as ATL (adult T cell leukemia), gastric cancer, mesothelioma, and melanoma [
7,
12,
13,
23]. Ectopic expression of
miR-31 inhibited
in vitro cell proliferation and
in vivo tumor growth in prostate cancers [
8,
24]. In our study,
miR-31 was found capable of inhibiting NPC cell proliferation, anchorage-independent growth, cell migration, and
in vivo tumor development. The tumor suppressor properties of
miR-31 in EBV-associated NPC were confirmed. The phenotypes resulting from the tumor suppressor miRNA is dependent on its target genes. Although a number of experimental validated
miR-31 target genes have been reported, it is likely that the targets vary from one tumor type to another. Here, we confirmed
MCM2 and
FIH1 as
miR-31 target genes in NPC cells. The growth inhibitory effect of
miR-31 in NPC via modulating of MCM2 and FIH1 expression was demonstrated. MCM2 is one of the six components of MCM protein complex which is important in the initiation of DNA replication. Regulation of MCM2 protein expression by
miR-31 was recently reported in prostate cancer [
8]. Elevated expression of MCM proteins were detected in both dysplasia and malignancy of various tissues [
18]. It is believed that deregulation of MCM proteins contribute to the early stage in carcinogenesis. In our earlier study, we demonstrated that knockdown of MCM2 significantly inhibited the cell growth, migration, and invasion in medulloblastoma [
18]. Furthermore, the involvement of MCM2 in regulating filopodia and stress fiber formation through cdc42 and Rho activation respectively was shown. Through knocking down of MCM2 by siRNA, studies demonstrated that the MCM2 expression impaired the growth of the prostatic (LNCaP) and colon (HCT116) cancer cell lines [
18,
25,
26]. Similar growth inhibition was also observed in the C666-1 cells with MCM2 depletion. Occurrence of inhibitory phenotypes in the
miR-31-expressing NPC cells is suspected to be due to MCM2 repression. Aside from MCM2, the suppressive effect of
miR-31 on NPC tumor growth was also via repressing FIH1. Liu
et al. first reported
miR-31 target
FIH1 and thereby activates the HIF pathway in HNSCC [
27]. High FIH1 expression contributes to the development of colon carcinomas and melanoma through the suppression of the p53-p21 axis [
19]. Interestingly, FIH1 overexpression is also sufficient to inhibit differentiation of primary human corneal epithelial keratinocytes (HCEKs) [
28]. Knocking down of FIH1 suppressed the cell proliferation in the clear cell renal cell carcinoma (CCRCC) (RCC10 and RCC4) and colon adenocarcinoma (LS174) cell lines [
19,
29]. Silencing of
FIH1 results in the elevation of p53 activity and p21 expression under normoxia [
19]. Here, we also showed an increase in the expression of phospho-p53 (Ser15) and p21 in the NPC C666-1 cells with
FIH1 silencing. Since
p53 mutation is rare in EBV-associated NPC, impaired p53 function may be associated with high FIH1 expression in this
miR-31 deficient cells. Although
miR-31 modulated the expression of FIH1 in NPC cells, it did not alter HIF1α expression as shown in head and neck squamous cell carcinoma (HNSCC) [
27]. HIF1α expression in the C666-1 cells was also not affected by knockdown of
FIH1 (Figure
7e). The oncogenic function of
FIH1 in NPC cells is likely to be HIF-independent. EBV-encoded LMP1 is capable to upregulate HIF1α through inducing Siah1 E3 ubiquitin ligase which promotes the degradation of prolyl hydroxylases 1 and 3 in nasopharyngeal epithelial cells [
30]. In our study, high
miR-31 expression was detected in the xenograft C15 which shows homogeneous LMP1 expression (Additional file
5: Figure S5). The observation raises the possibility of interplay between these two proteins in NPC. Such potential crosstalk of viral protein and cellular microRNA needs to be further investigate in future study.