MiRNAs bind to specific sites in the 3’untranslated region (UTR) of target mRNAs and are closely associated to all major cellular processes of life, ranging from embryonic development to tissue homeostasis and cancer[33–36]. NPC is an EBV cancer and it is different from other head and neck cancers due to distinctive geographical, etiological, and biological features[37, 38]. Many studies have shown that the pathogenesis of NPC is closely related to miRNAs[39, 40].
MiR-2110 was cloned from EBV-positive NPC samples, but its role and molecular basis have not been reported in NPC. We have previously reported that reduced levels of miR-2110 are negatively related to T, N, and clinical stages. Moreover, miR-2110 is considered as an unfavorable independent predictive factor promoting a shorter overall survival time of NPC patients. These data suggest that miR-2110 acts as a tumor suppressor in NPC.
Distant metastasis of tumors is the main cause of death in patients with NPC[41, 42]. Interestingly, we found that the reduced expression of miR-2110 was negatively related to lymph node metastasis of NPC, suggesting that miR-2110 is a metastasis suppressor in NPC. Previous studies have demonstrated that miR-2110 is absorbed by the long noncoding RNAs, AFAP1-AS1 and ARAP1-AS1, to reduce the targeting of SP1 and HDAC2, respectively, thereby promoting breast invasion[25, 26]. However, these studies lack animal models to verify the functions of miR-2110. In the present study, we established stable overexpression of miR-2110 in NPC cells. In vitro and in vivo studies showed that miR-2110 significantly inhibited the migration, invasion, and metastasis of NPC cells. After inhibiting the expression of miR-2110, the migration and invasion abilities of NPC cells were significantly restored. These data supported that miR-2110 functions as a metastasis suppressor in NPC.
PTEN is a classical tumor suppressor that regulates important cell functions, including cell proliferation, differentiation, and metastasis[43–45]. Most of the anticancer effects of PTEN depend on its lipid phosphatase activity. PI3K/AKT signaling is one of the best-characterized pathways negatively targeted by PTEN through its phosphatase activity[46–48]. In line with these reports, we observed that miR-2110 activated PTEN and further suppressed the PI3K/AKT pathway, which inactivated downstream EMT signaling. To further explore the detailed mechanism of miR-2110 as a tumor suppressor in modulating PTEN/PI3K/AKT to inactivate EMT signaling, bioinformatics analysis was applied to predict the target of miR-2110, which predicted FGFR1 as its potential target. FGFR1 belongs to the FGFR family and has been reported to act as a tumor gene in various cancers, including breast cancer, lung cancer, colorectal cancer, and NPC[49–53]. In the present study, we confirmed that miR-2110 directly downregulated FGFR1 protein expression by binding to its 3’UTR in NPC cells. Interestingly, a previous study has reported that FGFR1 downregulates PTEN protein expression in stem cell leukemia/lymphoma syndrome[54]. However, the specific molecular mechanism of FGFR1 inhibiting PTEN protein expression has not been reported.
Previous studies have reported that NEDD4, a ubiquitin ligase, ubiquitinates and degrades PTEN protein expression in tumors[55–57]. In addition, NEDD4 has also been reported as a tumor promoter in NPC[58]. Interestingly, both NEDD4 and PTEN were predicted as the interacting proteins of FGFR1 based on GeneMANIA. Subsequently, we identified the combination of FGFR1 and NEDD4 or PTEN. FGFR1 overexpression upregulated the protein expression of PTEN in NPC cells. Yet, PTEN mRNA level did not show the significant change. Further, overexpressed FGFR1 increased the interaction between PTEN and NEDD4, which promoted the NEDD4-mediated ubiquitination degradation of PTEN. Finally, we demonstrated that FGFR1 overexpression decreased the protein stability of PTEN in miR-2110-overexpressing NPC cells. These data demonstrated that miR-2110 directly targets FGFR1 to decrease the recruitment of NEDD4 to ubiquitinate and degrade PTEN protein, which upregulates PTEN expression to suppress the PI3K/AKT pathway and activate EMT signaling, ultimately suppressing NPC metastasis.
CB, a cardiotonic steroid or bufotalin, is isolated from toad venom and has an antitumor effect on many cancers[59, 60]. Our group recently identified a chemically synthesized CB that significantly inhibits the proliferation, metastasis, and tumor stemness of NPC, lung adenocarcinoma, and hepatocellular carcinoma[29, 30, 61]. In the present study, we demonstrated that CB also significantly upregulated miR-2110 expression in NPC. Further, we showed that chemically synthesized CB suppressed miR-2110 expression via inactivating PI3K/AKT-induced c-Jun expression, which is an oncogenic transcriptional factor that binds to the miR-2110 promoter and negatively modulates its expression in NPC.
We have previously demonstrated that CB inhibits the migration and invasion of NPC. To determine whether miR-2110 participates in CB-mediated inhibition of NPC migration and invasion, CB-treated NPC cells were exposed to a miR-2110 inhibitor in the present study, which significantly increased the invasive and migratory abilities of CB-treated NPC cells. In addition, the expression levels of PTEN and E-cadherin were downregulated, and the expression levels of FGFR1, N-cadherin, c-Jun, and Snail were upregulated. Finally, in vivo experiments indicated that inhibition of miR-2110 partially restores the metastatic ability of NPC cells in CB-treated NPC cells. These data demonstrated that miR-2110-modulated FGFR1/PTEN/PI3K/AKT –mediated EMT signal participates in CB-mediated inhibition of NPC migration, invasion, and metastasis.
In summary, the present study revealed that reduced miR-2110 is an unfavorable factor promoting NPC progression and poor prognosis. Further, miR-2110 is induced by CB via suppressing the PI3K/AKT/c-Jun pathway and directly targeting FGFR1 to attenuate its expression, thus decreasing the interaction between NEDD4 and PTEN, which suppresses the ubiquitination degradation level of PTEN. As a result, PI3K/AKT and downstream EMT signaling are further suppressed, which reduces NPC metastasis. The present findings emphasized the critical role of miR-2110/FGFR1/NEDD4/PTEN in NPC metastasis, providing a potential therapeutic target for NPC patients with distant metastasis.