Porphyromonas gingivalis outer membrane vesicles augments proliferation and metastasis of oral squamous cell carcinoma cells

Abstract

Background: Porphyromonas gingivalis (P. gingivalis) is closely related to Oral squamous cell carcinoma (OSCC), and P. gingivalis outer membrane vesicles (OMVs) is the main pathogenic factor, which is associated with periodontitis, atherosclerosis and other diseases. However, few studies have reported an association between P. gingivalis OMVs and OSCC. The purpose of this study was to establish the clinical relationship between P. gingivalis and OSCC based on clinical samples. Further, the effect of P. gingivalis OMVs on OSCC was observed with cell model in vitro, and the possible molecular mechanism was discussed.

Methods: Immunohistochemistry was used to detect the abundance of P. gingivalis in OSCC and its paired paracancer tissues, and to analyze the correlation between P. gingivalis and clinicopathological parameters of patients. P. gingivalis OMVs were isolated to observe its effects on the proliferation and migration of OSCC cell lines. RNA-seq was performed and the expression of differentially expressed genes (DEGs) was detected by real-time quantitative PCR (RT-qPCR) to explore the potential mechnism of P. gingivalis OMVs on OSCC progression.

Results: The abundance of P. gingivalis in OSCC was higher than that in para-cancerous tissues, and was positively correlated with the degree of tissue differentiation (P = 0.028), T stage (P < 0.001), and clinical stage (P = 0.011). P. gingivalis OMVs promoted the proliferation and migration of HN6 cells, and promoted the proliferation of CAL27 cells, but had no significant effect on its migration. P. gingivalis OMVs treatment attenuated the expressions of TNFSF15, ZNF292, ATRX, ASPM and KIF20B in CAL27 and HN6 cells.

Conclusion: This study suggests that P. gingivalis may be an indicator of poor prognosis for OSCC. P. gingivalis OMVs may down-regulate the expression of TNFSF15, ZNF292, ATRX, ASPM, KIF20B and participate in the occurrence and development of OSCC.

Keywords: Porphyromonas gingivalis; Extracelular vesicles; Squamous cell carcinoma of head and neck.

Fig. 1

The abundance of P. gingivalis in OSCC tissues was higher than that in paracancer tissues. (A, D and G are overall views of tissue sections, scale bar = 1000 μm; B, E, H scale bar = 100 μm; C, F, I scale bar = 60 μm). A-C: IHC image of P. gingivalis in OSCC cancer tissue; D-F: IHC images of P. gingivalis in paired paracancerous tissues; G-I: IHC image of P. gingivalis in cancerous nest; J: IHC scores of P. gingivalis in OSCC tissues and paracancerous tissues

Fig. 2

Identification of P. gingivalis OMVs. A: TEM image of P. gingivalis OMVs (red arrows: indicate typical OMV structures, which are spherical or elliptical in shape; Blue arrows: the OMV membrane structures); B: NTA particle size distribution map

Fig. 3

P. gingivalis OMVs promotes the proliferation of CAL27 (A, C) and HN6 (B, D) cells in vitro (*: P < 0.05, **: P < 0.01, ***: P < 0.001)

Fig. 4

EdU assay showed that P. gingivalis OMVs promoted the proliferation of CAL27 (A, C) and HN6 (B, D) cells. A, C: EdU expression in CAL27 cells at 72 h of treatment with P. gingivalis OMVs; B, D: EdU expression in HN6 cells at 72 h of treatment with P. gingivalis OMV. (200× magnification, scale bar = 50 μm, *:P < 0.05, **:P < 0.01, ***:P < 0.001)

Fig. 5

P. gingivalis OMVs had no significant effect on the horizontal migration of CAL27 cells (A), but promoted the horizontal migration of HN6 cells (B), 50× magnification, scale bar = 200 μm, *: P < 0.05, **: P < 0.01, ***: P < 0.001)

Fig. 6

P. gingivalis OMVs had no significant effect on the vertical migration of CAL27 cells (A), but promoted the vertical migration of HN6 cells (B) (100× magnification, scale bar = 200 μm, *: P < 0.05, **: P < 0.01, ***: P < 0.001)

Fig. 7

DEGs heatmap. A: Cal27 cells 50 µg/mL versus Control-50 µg/mL; B: Cal27 cells 50 µg/mL versus Blank; C: Cal27 cells Control-50 µg/mL group versus Blank group; D: HN6 cells 50 µg/mL versus Control-50 µg/mL; E: HN6 cells 50 µg/mL versus Blank group, F. HN6 cells Control-50 µg/mL Group versus Blank group. Cal27(HN6)-B indicates the Blank group, Cal27(HN6)-N50 indicates the Control-50 µg/mL group, and CAL27(HN6) -50 indicates the 50 µg/mL Group. Red indicates high expression and blue indicates low expression

Fig. 8

KEGG pathway analysis of DEGs in the 50 µg/mL group versus the Control-50 µg/mL group. (A) CAL27; (B) HN6

Fig. 9

Selection and validation of differentially expressed genes. A: The intersection of CAL27 cell DEGs (50 µg/mL group versus Control-50 µg/mL group) and HN6 cell DEGs (50 µg/mL group versus Control-50 µg/mL group); B: intersection of CAL27, HN6 cells in DEGs (50 µg/mL group versus Blank group) after subtracting genes that overlap with DEGs (Control-50 µg/mL group versus Blank group). Cal27(HN6)-B indicates the Blank group, Cal27(HN6)-N50 indicates the Control-50 µg/mL group, and CAL27(HN6) -50 indicates the 50 µg/mL Group; C-H: among the HNSCC patient information collected by TCGA, survival curve analysis by UALCAN revealed that low expression of TNFSF15 (C), ZNF292 (D), KIF20B (E), ANKRD12 (F), BRCA2 (G), and ASPM (H) was associated with survival of HNSCC patients. Red for high

Fig. 10

P. gingivalis OMVs decreased the expression of TNFSF15, ZNF292, ATRX, KIF20B and ASPM genes in CAL27 and HN6 cells. A: TNFSF15; B: NPPA; C: ZNF292; D: ASPM; E: ATRX; F: KIF20B; G: ANKRD12; H: BRCA2; I: GCC2(*: P < 0.05, **: P < 0.01, ***: P < 0.001)