Effects of gingipain extract on the biological characteristics of oral squamous cell carcinoma cell HN6

doi:10.3969/j.issn.1674-8115.2024.02.002

Abstract

Abstract:

Objective ·To observe the effects of gingipain extract on the biological characteristics of oral squamous cell carcinoma cell HN6. Methods ·The HN6 cell line was selected, cultivated, and divided into different groups based on the protein concentration of gingipain extract from Porphyromonas gingivalis: control group, 3.125 μg/mL group, 6.25 μg/mL group, 12.5 μg/mL group, 25 μg/mL group, 50 μg/mL group, and 100 μg/mL group. After 24 and 48 h of cultivation, CCK-8 assay was used to detect the effects of gingipain extract on HN6 cell proliferation activity. Subsequent experiments were divided into control group, 25 μg/mL group and 50 μg/mL group. Flow cytometry was used to examine the effects of gingipain extract on cell cycle. Scratch assay and Transwell assay were performed to evaluate cell migration and invasion ability. Real-time PCR (RT-PCR) and Western blotting were used to measure the expression of E-cadherin and N-cadherin proteins and genes in cells. Results ·Stimulated with gingipain extract for 24 h, the HN6 cells showed significantly increased proliferation activity in the 25 μg/mL (P=0.025), 50 μg/mL (P=0.000), and 100 μg/mL (P=0.049) groups compared to the control group. After 48 h, proliferation activity was significantly higher in the 6.25 μg/mL(P=0.024), 12.5 μg/mL (P=0.006), 25 μg/mL (P=0.000), 50 μg/mL (P=0.000), and 100 μg/mL (P=0.000) groups compared to the control group. Cell cycle analysis revealed that, after 24 h of gingipain stimulation, the proportion of HN6 cells in the G1 phase decreased, while the proportion in the S+G2 phase significantly increased compared to the control group (25 μg/mL group: P=0.024; 50 μg/mL group: P=0.001). Compared to the control group, the scratch assay demonstrated a significant increase in the percentage of scratch closure as the concentration of gingipain extract increased (P=0.001). Compared to the control group, the Transwell invasion assay showed a significant increase in the number of cells passing through the bottom of the chamber as the concentration of gingipain extract increased. RT-PCR and Western blotting results indicated that as the concentration of gingipain extract increased, the expression levels of N-cadherin mRNA and protein in HN6 cells significantly increased, while the expression levels of E-cadherin mRNA and protein significantly decreased compared to the control group. Conclusion ·Gingipain extract could promote proliferation, migration, and invasion of oral squamous cell carcinoma HN6 cells.

Key words: gingipain, periodontitis, oral squamous cell carcinoma (OSCC), cell proliferation, cell migration, cell invasion

CLC Number:

R781.4

LI Huxiao, LI Xiaotian, ZHAO Xuri, ZHANG Huanyu, ZHOU Wei, SONG Zhongchen. Effects of gingipain extract on the biological characteristics of oral squamous cell carcinoma cell HN6[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(2): 161-168.

Figures/Tables 7

Tab 1 Primer sequences for real-time PCR

Gene	Forward primer (5´→3´)	Reverse primer (5´→3´)
GAPDH	GAAGGTGAAGGTCGGTC	GAAGATGGTGATGGGATTTC
N-cadherin	CAGGGACCAGTTGAAGCACT	TGCCGTGGCCTTAAAGTTAT
E-cadherin	CGAAGATGTAAACGAAGCC	GCCATTTCCAGTGACAATC

Fig 1 Effects of gingipain extract on HN6 cell proliferation

Fig 2 Effects of gingipain extract on HN6 cell cycle

Fig 3 Effects of gingipain extract on HN6 cell migration

Fig 4 Effects of gingipain extract on HN6 cell invasion (×100)

Fig 5 Effects of gingipain extract on invasion-associated genes of HN6 cells

Fig 6 Effects of gingipain extract on invasion-associated proteins of HN6 cells

TrendMD

References 30

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