Up-regulation of miR-182-5p is associated with NPC development and poor prognosis
To evaluate the clinical significance of miR-182-5p in human NPC, the expression levels of miR-182-5p were analyzed in seventy-five NPC samples using qRT-PCR. The relationship between miR-182-5p expression status and clinicopathological features of NPC was evaluated. miR-182-5p overexpression was significantly associated with advanced T classification, poor N classification, high Clinical Stage (Fig. 1a and Supplementary Table 1). No significant differences were found in the expression of miR-182-5p in regards to age and gender (Supplementary Table 1). In addition, miR-182-5p up-regulation was closely associated with lymph node metastasis (p = 0.0070) (Fig. 1b). Moreover, Kaplan–Meier analysis indicated that high miR-182-5p expression was closely associated with NPC poor overall survival (Fig. 1c). These results indicated that miR-182-5p overexpression might play an important role in the regulation of NPC progression.
miR-182-5p promotes NPC cell proliferation and migration in vitro and tumorigenesis and tumor metastasis in vivo
In order to explore the biological function of miR-182-5p in NPC, miR-182-5p mimic was transfected into HK1 and CNE1 cells by using Lipofectamine 2000, and Control-mimic transfected cells were used as controls (Fig. 2a). Then, the effects of miR-182-5p on cell growth and migration were evaluated. CCK-8 assays showed that miR-182-5p overexpression increased the cell growth (Fig. 2b). Besides, cell migration assay revealed that miR-182-5p mimic could dramatically increase cell motility compared with the Control-mimic treated cells (Fig. 2c and Supplementary Fig. 1). In contrast, suppression of miR-182-5p by using the microRNA shRNA lentivirus impeded cell growth and migration in both NPC cell lines (Fig. 2d-f and Supplementary Fig. 1).
To further determine the function of miR-182-5p in the growth of NPC in vivo, we performed NPC xenograft growth in the nude mice. HK1 cells stably infected with lentivirus containing miR-182-5p shRNA were inoculated subcutaneously into double dorsal flanks of 4-week-old nude mice. As shown in Fig. 2g, h, the growth of tumors derived from HK1 cells stably infected with the miR-182-5p shRNA were significantly suppressed compared with that of xenografts derived from cells infected with control negative microRNA shRNA. Besides, knockdown of miR-182-5p induced a significant decrease of the numbers of lung metastatic lesions compared with the group treated with Control-shRNA (Fig. 2i, j).
miR-182-5p directly targets ZFP36L1 by interacting with its 3’ UTR
To identify the potential targets of miR-182-5p, we integrated bioinformatic algorithms using the publicly available databases miRanda (http://www.microrna.org), miRDB (http://www.mirdb.org), TargetScan (http://www.targetscan.org). According to the bioinformatics analysis, two putative binding sites for miR-182-5p was found at the 3’ UTR of ZFP36 ring finger protein like 1 (ZFP36L1; also known as TIS11B, BRF1, Berg36, ERF-1) (Fig. 3a). Then, we performed luciferase reporter assays using the 3’ UTR of ZFP36L1. As shown in Fig. 3b, c, decreased luciferase activity of the wild-type ZFP36L1 3’ UTR in miR-182-5p overexpressed cells was observed. Besides, mutation in potential interaction sites 1 or 2 abrogated the miR-182-5p-induced effect on the luciferase activity of ZFP36L1 3’ UTR (Fig. 3b, c). Moreover, the protein levels of ZFP36L1 were down-regulated in cells transfected with the miR-182-5p mimic (Fig. 3d), whereas knockdown of miR-182-5p resulted in up-regulation of the ZFP36L1 protein (Figure 3e). Taken together, miR-182-5p directly targets ZFP36L1 in NPC cells.
ZFP36L1 may function as a tumor-suppressor in NPC and its expression levels are inversely correlated with that of miR-182-5p
Previously, Hodson's group have established a role for ZFP36L1 in the prevention of malignant transformation . To determine the function of ZFP36L1 in NPC, we generated cell lines expressing ectopic ZFP36L1 in HK1 and CNE1 cells (Supplementary Fig. 2a). On the other hand, siRNA specifically targeting ZFP36L1 were also used in these cells (Supplementary Fig. 2a). Overexpression of ZFP36L1 suppressed the cell growth and migration compared with the controls (Supplementary Fig. 2b, d). Conversely, ZFP36L1 knockdown enhanced cell proliferation and migration in both HK1 and CNE1 cells (Supplementary Fig. 2c, e).
To further measure the relationship between ZFP36L1 and miR-182-5p in NPC, the ZFP36L1 protein expression in primary NPC samples was evaluated using IHC. As shown in Fig. 4a and Supplementary Table 2, ZFP36L1 down-regulation was closely associated with advanced tumor invasion (p = 0.013), advanced lymph node metastasis (p = 0.018), advanced clinical stage (p = 0.001). Notably, the expression of ZFP36L1 in NPC patient with lymph node metastasis was significantly down-regulated (Fig. 4b, c). Patients with low ZFP36L1 expression showed shortening overall survival when compared to that with high ZFP36L1 expression (p < 0.001, Fig. 4d). Furthermore, the expression levels of miR-182-5p and ZFP36L1 were inversely correlated in NPC tissues (Fig. 4e).
miR-182-5p enhances NPC cell proliferation and migration depends on the down-regulation of ZFP36L1
Next, we sought to determine whether miR-182-5p promoted NPC cell proliferation and migration by down-regulating ZFP36L1. ZFP36L1 was transiently transfected into HK1 and CNE1 cells expressing miR-182-5p mimic (Fig. 5a). Results of CCK-8 assays showed that re-introduction of ZFP36L1 reversed the increase in cell growth induced by miR-182-5p overexpression (Fig. 5b, c). Moreover, the migratory ability was almost rescued to the original levels in miR-182-5p-transfected cells with ectopic overexpressing ZFP36L1 (Fig. 5d). Collectively, these results indicated that miR-182-5p-mediated promotion of cell proliferation and migration depends on down-regulation of ZFP36L1 in NPC.
Up-regulation of miR-182-5p is induced by HIF-1α in NPC cells
Given that hypoxia is associated with increased malignancy, resistance to therapy and distant metastasis in solid tumors, including NPC [20, 21], we hypothesized that hypoxia may mediate the up-regulation of miR-182-5p in NPC. To this end, miR-182-5p levels were measured by qPCR in cells cultured in the hypoxic incubator at different time points. Interestingly, miR-182-5p was significantly induced in a time-dependent manner in response to hypoxia (Fig. 6a, b). As the cellular adaptive responses to hypoxic stress are mainly mediated by the hypoxia-inducible factors (HIFs), of which HIF-1α is the best-examined subunit binding to hypoxia-responsive element (HRE) sites in the promoters of target genes [22, 23], we tested whether miR-182-5p was transcriptionally induced by the HIF-1α. Indeed, by searching the ~2400bp promoter sequence of miR-182-5p, three potential HRE sites were identified (Fig. 6c). As illustrated in Fig. 6d, e, ectopic overexpression of HIF-1α significantly increased miR-182-5p promoter activity. Consistently, the expression levels of miR-182-5p were increased by HIF-1α overexpression (Fig. 6f). These data suggested that up-regulation of miR-182-5p in NPC was attributable to the transcriptional activation effect induced by HIF-1α.