YBX3 Mediates the Metastasis of Nasopharyngeal Carcinoma via PI3K/AKT Signaling

Abstract

The metastasis of nasopharyngeal carcinoma (NPC) is a complex process associated with oncogenic dysfunction, the deciphering of which remains a challenge and requires more in-depth studies. Y-box protein 3 (YBX3) is a DNA/RNA binding protein associated with gene transcription, DNA repair, and the progression of various diseases. However, whether and how YBX3 affects the metastasis of NPC remains unknown. Thus, in this study, we aimed to investigate the role of YBX3 in the metastasis of NPC and determine its underlying mechanism. Interestingly, it was found that the expression of YBX3, which was associated with NPC metastasis, was upregulated in the clinical NPC tissues and cell lines. Moreover, we found that knockdown of YBX3 expression by lentivirus shRNA significantly suppressed NPC cells migration in vitro and metastasis in vivo. Mechanistically, RNA sequencing results suggested that the genes regulated by YBX3 were significantly enriched in cell adhesion molecules, cAMP signaling pathway, calcium signaling pathway, focal adhesion, PI3K/AKT signaling pathway, Ras signaling pathway, Rap1 signaling pathway, NF-κB signaling pathway, and Chemokine signaling pathway. Of these, PI3K/AKT signaling pathway contained the most genes. Accordingly, YBX3 knockdown decreased the activation of PI3K/AKT signaling pathway, thereby inhibit epithelial-to-mesenchymal transition (EMT) and MMP1. These results have demonstrated that YBX3 are involved in the metastasis of NPC through regulating PI3K/AKT signaling pathway, and serve as a potential therapeutic target for patients with NPC.

Keywords: MMP1; PI3K/AKT signaling pathway; Y-box protein 3; epithelial-to-mesenchymal transition (EMT); nasopharyngeal carcinoma; tumor metastasis.

Figure 1

High expression of YBX3 in NPC tissues and cell lines. (A) Representative histologic sections of nasopharyngeal tissue and nasopharyngeal carcinoma tissue in patients were strained with H&E and IHC-YBX3. H&E demonstrated a typical feature of Nasopharyngeal Carcinoma, and the expression of YBX3 protein was detected in the tumor cells. Scale bar, 50 μm. (B) WB analysis was used to detect the protein level expression of YBX3 and in normal nasopharyngeal tissues and patient derived nasopharyngeal carcinoma tissue. (C) Q-PCR analysis was used to detect the RNA level expression of YBX3 in normal nasopharyngeal tissues and patient derived nasopharyngeal carcinoma tissue. **p < 0.01. (D) YBX3 mRNA expression level in NPC tissues and normal tissues from Rhodes’s dataset in ONCOMINE database (n = 31). *p < 0.05. (E) WB analysis was used to detect the protein level expression of YBX3 in NP460 and NPC cells with 5-8F and S18. (F) Q-PCR analysis was used to detect the RNA level expression of YBX3 in NP460 and NPC cells with 5-8F and S18. **p < 0.01. (G) YBX3 expression levels in NPC tumor tissues with and without distant metastasis from GSE103611 in GEO database (n = 24). Three probes: 16761430, 16761436, and 16761437 targeted different positions of YBX3. *p < 0.05. NP tissue, normal nasopharyngeal tissues; NPC tissue, nasopharyngeal carcinoma tissues.

Figure 2

The efficiency of YBX3 in NPC cells interfered by lentiviruses. (A) WB analysis was used to detect the interfering effects of YBX3 shRNA in 5-8F. (B) Q-PCR analysis was used to detect the interfering effects of YBX3 shRNA in 5-8F. (C) WB analysis was used to detect the interfering effects of YBX3 shRNA in S18. **p < 0.01. (D) Q-PCR analysis was used to detect the interfering effects of YBX3 shRNA in S18. **p < 0.01.

Figure 3

Deficiency of YBX3 inhibits NPC cell migration. (A) The wound healing experiments was used to detect the migration ability in 5-8F. Scale bar, 50 μm; *p < 0.05; **p < 0.01. (B) The wound healing experiments were used to detect the migration ability in S18. Scale bar, 50 μm; *p < 0.05; **p < 0.01. (C) The transwell experiments were used to detect the migration ability in 5-8F. Scale bar, 50 μm; **p < 0.01. (D) The transwell experiments were used to detect the migration ability in S18 cells. Scale bar, 50 μm; **p < 0.01.

Figure 4

YBX3 affects NPC tumor metastasis. (A) Schematic diagram depicting the lung metastasis model of NPC by injecting 5-8F and S18 cells with stably depleting YBX3 into the tail vein. (B) The visible surface lung metastatic nodules were quantified with six lungs per group (n = 6). **p < 0.01. (C) Representative images of lung and quantifications of YBX3 control and YBX3 shRNA metastatic cancer models. Arrowheads point to metastatic nodules. (D, E) H&E staining analysis of lung metastasis. Scale bar, 500 μm (up); 100 μm (down).

Figure 5

Downstream transcriptional genes of YBX3 in NPC cells. (A) Volcano diagram depicting the 558 differentially expressed genes (DEGs) from the RNA sequencing in 5-8F cells transfected with shRNA-YBX3 or shRNA-control. Red represents upregulated genes, blue represents downregulated genes, gray represents no-signification. (B) KEGG pathway analysis of the DEGs between shRNA-YBX3 and shRNA-control groups.

Figure 6

YBX3 promotes NPC metastasis by activating PI3K/AKT signaling. (A) WB analysis was used to detect the activation of PI3K/AKT signaling and cell migration related marker molecules in 5-8F. (B) WB analysis was used to detect the activation of PI3K/AKT signaling and cell migration related marker molecules in S18. (C) Q-PCR analysis was used to detect the activation of PI3K/AKT signaling and cell migration related marker molecules in 5-8F. (D) Q-PCR analysis was used to detect the activation of PI3K/AKT signaling and cell migration related marker molecules in S18. **p < 0.01. (E) Schematic diagram depicting the YBX3 mediated regulation of target genes via PI3K/AKT signaling, which is involved in the modulation of NPC metastasis.