Naa10p has tumor-suppressive function in vitro and in vivo
Our previous study revealed that Naa10 is overexpressed in OSCC tissues, and Naa10p expression correlates to TNM stage and lymph node status. Moreover, our data confirmed that Naa10p expression is associated with better prognosis, suggesting that Naa10p functions as an independent prognostic factor for OSCC patients[35]. To further explore the role of Naa10p in OSCC cells, CAL 27 and SCC-15 cells with stable interference of Naa10p expression were generated by lentiviral infection of shRNA targeting Naa10p. As illustrated in Fig. 1A, silencing of Naa10p was confirmed by western blot analysis in the OSCC cell lines (Left panel, Fig. 1A). Next, cell proliferation capacity was assessed by colony forming assay. Notably, Naa10p knockdown resulted in an increased number of colonies compared with cells transfected with control shRNA. Subsequently, we found that knock-down of Naa10p significantly elevated the migration (Fig. 1B) and invasion (Fig. 1C) of CAL 27 and SCC-15 cells by using transwell assay.
Next, we evaluated the potential effects of Naa10p on tumorigenicity in BALB/c nude mice. Specifically, the growth rates of tumor xenografts were elevated by stable knockdown of Naa10p (Fig. 1D, 1E and 1F). Consequently, mice inoculated with Naa10p-silenced cells had shorter survival time (Fig. 1G). Taken together, these data indicated that Naa10p suppresses tumorigenesis and progression of OSCC in vitro and in vivo.
Naa10p knockdown inhibits P53 signaling pathway
After elucidating Naa10p’s inhibitory roles on OSCC, we sought to further gain insight into the mechanism by which Naa10p regulates invasion and metastasis phenotype in OSCC. We performed cellular Gene Expression Profile with Naa10p stably silenced CAL27 cells and control cells, and the differentially regulated genes were selected for KEGG pathway analysis. The analysis result showed that the P53 signaling pathway was the most relevant downstream signaling pathway of Naa10p (Fig. 2A). Next, we performed Gene Set Enrichment Analysis (GSEA) and found that P53 signaling pathway was enriched in this dataset (Fig. 2B). Subsequently, we performed the genetic variations of P53 signaling pathways of clustering Analysis (Fig. 2C). Consequently, some genes in P53 signaling pathway were determined by qRT–PCR, and Pirh2 was upregulated and p53 downregulated after knocking down Naa10p (Fig.2D). p53 is a major substrate of Pirh2, and Pirh2 promotes p53 degradation[32]. Furthermore, we previously demonstrated that the expression of Naa10p was negatively correlated with that of Pirh2 in OSCC[35]. Therefore, the Pirh2-p53 signaling pathway is selected for verification and study in the next step.
Naa10p attenuates MMPs expression via the Pirh2-p53 signaling pathway
To uncover whether Naa10p was involved in regulating P53 signaling pathway, we verified the expression correlation of Naa10p, Pirh2 and p53 by immunohistochemical staining, which suggested that Naa10p abundance was negatively associated with that of Pirh2, but positively associated with that of p53 (Fig. 3A). Moreover, tumor invasion is often associated with the enhanced synthesis of matrix metalloproteinases (MMPs), among which MMP-2 and MMP-9 are of central importance[37]. Thus, we sought to determine the expression level of Pirh2, p53, MMP-2 and MMP-9 protein in CAL 27 and SCC-15 cells after silencing Naa10p. Western blot showed that Naa10p stable knockdown significantly increased Pirh2, MMP-2 and MMP-9 expression, and decreased p53 expression. Consistently, Naa10p stable overexpression inhibited the level of Pirh2, MMP-2 and MMP-9, and elevated p53 expression (Fig. 3B).
To confirm whether Pirh2 inhibited the expression of p53 and modulated p53-dependent expression of MMPs in OSCC cells, the expression of Pirh2 in OSCC cells was silenced by RNA interference (RNAi). The result showed that Pirh2 knockdown could dramatically increase p53 and decrease MMP-2 in CAL 27 and SCC-15 cells (Fig. 3C). These results emphasized the important role of Naa10p in the p53 pathway and suggested that Naa10p induces Pirh2 reduction and rescues p53 expression.
Naa10p interacts with RelA/p65 and attenuates phosphorylation of p65 in OSCC
In the previous study, there was a significantly inverse correlation of the expression of Naa10p and Pirh2 in OSCC patient tissues[35]. Furthermore, the effect of Naa10p on the expression of Pirh2 was determined by qRT-PCR in OSCC cells. The results indicated that Naa10 down-regulates the mRNA expression of Pirh2. (Fig. S1). These data raised a possibility that Naa10p regulates Pirh2 expression at the transcriptional level. Accumulating evidence demonstrated that Naa10p interacts with various transcription factors to regulate the expression of tumor-related target genes[10,11,13]. Interestingly, we scanned the proximal 1907bp of the promoter region of Pirh2 using Promo software (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/ promoi nit.cgi?dirDB=TF_8.3) and identified potential RelA/p65 binding sites. Thus, we concluded whether RelA/p65 has a transcriptional activation effect on Pirh2 and Naa10p suppresses RelA/p65-mediated transcription by interacting with the RelA/p65. Immunoprecipitation assays were performed to uncover the interaction between Naa10p with RelA/p65 in OSCC cells, and the results revealed endogenous interaction between Naa10p and RelA/p65 in OSCC cells (Fig. 4A). To further investigate the subcellular interaction of Naa10p and RelA/p65, CAL 27 and SCC15 cells were fractionated to acquire cytoplasmic and nuclear proteins. Next, we performed immunoprecipitation assay with cytoplasmic and nuclear proteins and demonstrated the presence of physical interaction was mainly in the cytoplasm (Fig. 4B). In vitro GST-Pull Down assay confirmed the association between GST-tagged Naa10p and His-RelA/p65, indicating a direct binding between these two molecules (Fig. 4C). By immunofluorescence staining and confocal microscopic observation, colocalization of Naa10p and p65 in CAL 27 and SCC-15 cells was revealed mainly in the cytoplasm (Fig. 4D).
Next, we sought to analyze whether Naa10p could affect the level of p65 and p65 phosphorylation. Naa10p was knockdown in CAL 27 cells through shRNA transfection. The results showed that knockdown of Naa10p significantly elevated p65 phosphorylation (serine-536), while p65 did not change (Fig. 4E). Similar results could be observed in SCC-15 cells (Fig. 4E).
Naa10p suppresses RelA/p65 activates Pirh2 transcription
Next, we explored whether RelA/p65 can affect the transcription of Pirh2, the human Pirh2 promoter-luciferase plasmid pGL3-Pirh2 and the RelA/p65 overexpression plasmid were cotransfected to CAL 27 cells, and the transcriptional activation was detected. The luciferase activity was significantly higher than in the control group after RelA/p65 overexpression (Lanes 1 and 3 in Left and Right panels; Fig. 5A). The result elucidated that RelA/p65 has a transcriptional activation effect on Pirh2. Futhermore, we showed that overexpression of RelA/p65 augmented Pirh2’s promoter activity, which was enhanced by silencing of Naa10p, but was compromised by co-expression of Naa10p (Lane 3 and 4 in Left and Right panels; Fig. 5A), indicating Naa10p could alleviate RelA/p65-regulated Pirh2 transcription.
To elucidate the RelA/p65 and Pirh2 promoter-specific binding sites, the truncated luciferase reporter plasmid: S1: pGL3-Pirh2-S1 (−1383 ~ +144); S2: pGL3-Pirh2-S2 (−916 ~ +144) of the core segment of the human Pirh2 promoter (length 1907bp) was further constructed based on identified three potential RelA/p65 binding sites on the promoter region of Pirh2 by using Promo software (Fig. 5B). Then, the truncated luciferase reporter plasmids were cotransfected to CAL 27 cells with p65 overexpression plasmid and Naa10p knockdown or overexpressin plasmid, respectively. The results showed that the luciferase activity of the full-length Pirh2 promoter region was increased following Naa10p knokdown and decreased after the Naa10p overexpression. Moreover, the luciferase activity of the truncated Pirh2 promoter regin-S1 was also increased after Naa10p knockdown and is comparable with the full-length Pirh2 promoter. However, the luciferase activity of the truncated S2 had no significant difference with that of the control group (Fig. 5C). Similar data could be observed following stable expression of Naa10p (Right panel, Fig. 5C).
We performed chromatin immunoprecipitation (ChIP)-qPCR assays in CAL 27 cells, and the results revealed that p65 bound to S1 site, but not to S2 site in qCHIP assays (Fig. 5D, Left panel). Once Naa10p was knockdown, binding of RelA/p65 to S1 site was dramatically inreased (Fig. 5D, Right panel), suggesting that binding of RelA/p65 to S1 site was negatively regulated by the cellular levels of Naa10p. Collectively, these data indicated that RelA/p65 binds to the promoter region S1 to regulate Pirh2 transcription and Naa10p suppresses RelA/p65 induced Pirh2 transcription.
The tumor suppressor function of Naa10p is dependent on Pirh2-p53 signaling pathway
To further confirm the migration and invasion effect of Naa10p on OSCC is dependent on the Pirh2-p53 signaling pathway, CAL 27 cells were transfected with Naa10p siRNA or Pirh2 siRNA, respectively or in combination. Notably, silencing of Naa10p promoted CAL 27 cells migration, invasion, and while silencing of Pirh2 inhibited CAL 27 cells migration, invasion. Pirh2 silencing indeed relieved the cell migration and invasion promoted by Naa10p knockdown (Fig. 6A). Similar results were also observed in cell invasion (Fig. 6B). These data suggested that Naa10p functions in the migration and invasion of OSCC cells by targeting Pirh2-p53 signaling pathway.