METTL13 expression was upregulated in HNSCC and negatively correlated with clinical survival.
To investigate the expression pattern of METTL13 in HNSCC, we firstly analyzed its mRNA expression level by qPCR in 67 HNSCC and paired para-carcinoma tissues obtained from Hospital of Stomatology of Sun Yat-sen University. Notably, significant upregulation of METTL13 was observed (p < 0.001, Fig. 1A). Consistently, TCGA analysis data also showed that METTL13 was upregulated in the HNSCC samples (Fig. 1B). Next, we examined the METTL13 expression at protein level by immunohistochemistry (IHC). Higher expression of METTL13 was observed in human HNSCC samples than paired normal tissues (Fig. 1C). To further explore the potential prognostic value in HNSCC, a total of 44 patients were segmented into METTL13 low and high expression groups based on the qPCR results. The data showed METTL13 significantly negatively correlated with overall survival of HNSCC patients (p < 0.05, Fig. 1D). Collectively, we showed that the expression of METTL13 was upregulated in HNSCC, and it might play a key role in HNSCC development and progression.
Knockdown of METTL13 inhibited HNSCC cell malignant phenotypes in vitro.
Aberrant cellular activities often occur in cancer cells, such as continuous proliferative, evading growth inhibition, resisting cell apoptosis and active invasion and metastasis . To explore the functional role of METTL13 in HNSCC cells, we first selected eight HNSCC cell lines and compared their METTL13 expression by the Western blotting assay (Fig. 2A). The results revealed high METTL13 protein level in two HNSCC cancer cell lines (Cal27 and Cal33) and low METTL13 protein level in SCC1 cell line (Fig. 2A). Hence, we chose Cal27, Cal33 and SCC1 cell lines for further functional study. We then generated stable METTL13 knockdown Cal27 and Cal33 cell lines using shRNA targeting METTL13 and confirmed the successful knockdown of METTL13 gene at mRNA and protein levels (Fig. 2B, C). Next, we performed clone formation assay and CCK-8 assay to explore the effect of METTL13 gene knockdown on the proliferation in HNSCC cell lines. The results showed that the depletion of METTL13 significantly not only inhibited the clone formation ability, but also inhibited cell growth rate in these two cell lines (Fig. 2D, E). Next, we utilized a flow cytometer to study the role of METTL13 in cell apoptosis. Our results demonstrated a higher apoptosis rate after ablation of METTL13 in HNSCC cells (Fig. 2F). Subsequently, wound healing assay results revealed that silencing of METTL13 significantly suppressed cell migration and prolonged the repair time (Fig. 2G). Transwell cell migration assay further proved knockdown of METTL13 attenuated the invasion ability of HNSCC cells (Fig. 2H). Taken together, these results demonstrated that METTL13 is critical for proliferation, apoptosis and invasion of HNSCC cell lines.
Mettl13 Downregulation Negatively Correlates With Hnscc Csc-like Properties
We also investigated whether METTL13 played a role in HNSCC cancer stem cells (CSCs)-like properties. First, we determined HNSCC cellular self-renewal capacity by conducting the sphere-forming assay. Downregulation of METTL13 greatly reduced the size and number of tumor spheres when compared with those formed by control cells in both Cal27 and Cal33 cell lines (Fig. 3A, B), indicating that their ability to self-renew was impaired. We then further assessed the CSC-like properties of METTL13 using assay to detect aldehyde dehydrogenase (ALDH) activity, which was regarded as a reliable marker for CSCs . As showed in Fig. 3C-D, ALDH activity was significantly reduced in cells treated with sh-METTL13. Therefore, METTL13 played an essential part in maintaining HNSCC cells self-renewal ability.
Overexpression of METTL13 could promote HNSCC tumorigenicity.
In further explore the function of METTL13, a stable METTL13 overexpression SCC1 cell line was generated. And the up-regulation efficiency was verified by western blotting and qPCR assays (Fig. 4A, B). The CCK-8 assay revealed overexpression of METTL13 remarkably increased the capacity of HNSCC cells to proliferate (Fig. 4C). Consistently, lower rate of apoptotic cells was captured in METTL13over group by the flow cytometry (Fig. 4D). Next, we found that extra acquisition of METTL13 could accelerated cell migration of SCC1 in the transwell assay (Fig. 4E). Furthermore, METTL13over group tended to form larger size of tumor spheres after having cultured for seven days (Fig. 4F). On the other hand, overexpression of METTL13 endowed SCC1 cells with higher ALDH activities (Fig. 4G). To sum up, these data show that METTL13 plays a potential oncogenic role by increasing or inducing the malignant proliferation, migration and CSC-like properties in HNSCC cells.
METTL13 regulated EMT signaling pathway by enhancing translation efficiency of Snail to facilitate HNSCC progress
We have demonstrated that METTL13 expressly promoted HNSCC proliferation and metastasis, but the specific mechanism was unclear. Previous studies have reported that METTL13-mediated methylation of eukaryotic elongation factor 1A (eEF1A) promote oncogenesis via increased translation elongation and protein synthesis in cancer cells . And indeed, our polysome profile assay results revealed that decrease of METTL13 suppressed total protein translation in both Cal27 and Cal33 cell lines (Fig. 5A). In addition, the surface sensing of translation (SUnSET) monitoring method confirmed protein synthesis was inhibited after METTL13 depletion (Fig. 5B).
Previous study has indicated that METTL13 is involved in RAS-driven cancer development, we wonder whether depletion of METTL13 in HNSCC is also related to RAS signaling pathway . Therefore, RNA sequencing was performed using the METTL13 control and knockdown groups in both Cal27 and Cal33 cell lines. Interestingly, results from Gene Set Enrichment Analysis (GSEA) indicated that EMT signaling pathway was significantly negatively correlated with the downregulation of METTL13 in HNSCC cell lines (Fig. 5C), suggesting that downregulation of protein synthesis might prone to EMT-related factors. To examine whether this is the case, a series of key proteins associated with EMT pathway were examined by western blotting (Fig. 5D, data not shown). Notably, as a key transcription factor in EMT, Snail was significantly downregulated in METTL13-deficient cells (Fig. 5D). Therefore, we hypothesized that METTL13 could regulate EMT signaling pathway by regulating the translation activity of Snail mRNA. To test that, we performed polysome profile assay using sucrose gradient centrifugation to separate the ribosome and polysome segments. Based on these results, we used qPCR to detect the distribution of METTL13 affected mRNAs in the polysome segment. And the results showed Snail mRNA was significantly down-regulated in the METTL13 deficient groups in the polysome levels (Fig. 5E), indicating that the METTL13 is required for translation of Snail, which consistent with our previous conjecture. Altogether our results support that Snail is involved in METTL13-regulated EMT in HNSCC, and METTL13 regulated EMT signaling pathway by enhancing translation efficiency of Snail.
Decreased METTL13 expression weakened malignant phenotypes in vivo.
Xenograft studies were then conducted to investigate whether METTL13 could recapitulate the in vitro malignant phenotypes in vivo. Control and two METTL13 shRNA Cal33 cell lines were subcutaneous injected into nude mice. Mice were sacrificed after four weeks. In accordance with our in vitro results, deficiency of METTL13 in Cal33 cell dramatically suppressed tumor growth when compared with the control in vivo (Fig. 6A). And the tumor volume and tumor weight results further confirmed this effect (Fig. 6B, C). In order to get a more profound understanding of the function of METTL13 in vivo, we obtain the tumor sections. Next, hematoxylin and eosin (H&E) staining and METTL13 IHC results verified that METTL13 expression was decreased in sh-METTL13 tumor cells compared to the control (Fig. 6D, E). The staining of Ki67, a proliferation marker, showed alleviated cell growth after METTL13 gene knockdown, which was consistent with in vitro results (Fig. 6E). Consistently, IHC staining of nude mice showed the expression of Snail protein was reduced by the silencing of METTL13 (Fig. 6E).
Collectively, these data demonstrated that METTL13 is required for tumorigenic ability of HNSCC cells in vivo.