[Study on the invasion of

Objective: To explore the differences in bacterial communities within tissues during the process of oral mucosal carcinogenesis, and analyze the relationship between the high-abundance species Prevotella intermedia (Pi) and the occurrence and development of oral mucosal carcinogenesis. Methods: Fresh tissue samples were collected from patients diagnosed with oral leukoplakia (OLK), oral squamous cell carcinoma (OSCC), and healthy controls (HC) at Nanjing Stomatological Hospital from January 2022 to November 2024, following strict inclusion criteria. Bacterial DNA was extracted from these specimens, and the 2bRAD sequencing for microbiome (2bRAD-M) was employed to analyze and compare the α and β diversity, as well as the community composition of bacteria within tissues, aiming to identify specifically expressed bacteria. Subsequently, paraffin-embedded clinical specimens were collected: 15 cases in the OLK group (including 4 cases of simple hyperplasia, 6 cases of mild dysplasia, and 5 cases of moderate to severe dysplasia), 12 cases in the OSCC group, and 5 cases in the HC group. A 4-nitroquinoline n-oxide (4NQO)-induced OLK progression mouse model was also constructed. Mice were randomly divided into three groups using a random number table, with six in each group. The negative control group was given distilled water to drink; Group 1 was given distilled water containing 4NQO to drink until week 12, while Group 2 was given distilled water containing 4NQO to drink until week 22. After the mice were sacrificed, their tongue tissue were collected and fixed. Fluorescence in situ hybridization (FISH) with specific probes was used to validate the presence of Pi in human and mouse tissue sections, analyzing the correlation between histopathological grading and the invasion depth of Pi. Results: The 2bRAD-M microbial analysis revealed that the relative abundance of Pi in OSCC tissues (10.80%) was significantly higher than in the HC group (0.50%) (P=0.001) and OLK group (0.70%) (P=0.002). FISH probe detection showed that the fluorescence intensity of Pi in human OSCC tissues (125.00±25.13) was higher than in the HC group (11.40±25.49), simple hyperplasia OLK (11.75±23.50), and mild dysplasia OLK (26.83±29.51) groups, with statistically significant differences respectively (P=0.002, P=0.003, P=0.005). However, there was no significant difference compared to moderate to severe dysplasia OLK (47.40±26.88) (P=0.210). The fluorescence area of Pi in human OSCC tissues (9 255.00±2 048.00) was significantly larger than in the HC group (18.00±40.25), simple hyperplasia OLK group (27.00±54.00), mild dysplasia (76.00±95.19), and moderate to severe dysplasia (1 628.00±1 265.00) groups, with highly significant differences (P<0.001). There was a significant correlation between the invasive depth of Pi and the degree of histopathological grading (P<0.001). In mice, the fluorescence intensity of Pi in OSCC tissues (119.00±8.54) was significantly higher than in the HC group (8.17±20.00) (P<0.01), but showed no significant difference compared with the OLK group (34.33±28.28) (P>0.05). The fluorescence area of Pi in mice OSCC tissues (9 971.0±2 807.0) was significantly larger than in the HC group (37.83±92.67) and the OLK group (451.6±780.1) (P=0.006, P=0.043). There is a significant correlation between the depth of invasion of Pi and the degree of histopathological grading (P<0.001). Conclusions: This study suggests that Pi in oral mucosal tissue may be a potential biomarker for early detection of OSCC and play an important role in the carcinogenesis process of oral mucosa.