[Expression Level of Protein Kinase D in Oral Squamous Cell Carcinoma with Diverse Differentiation].

OBJECTIVE: This study aimed to investigate the expression level of protein kinase D (PKD) in oral squamous cell carcinoma (OSCC) and its relationship with differentiation of OSCC. METHODS: Sample was collected from 10 healthy control subjects and 40 OSCC confirmed by histopathological diagnosis, and the immunohistochemical staining was adopted to detect the expression of PKDs in OSCC tissues. The proportion of stained cell and staining intensity were evaluated to get a score, which used to analyze the difference among PKD1, PKD2 and PKD3 in various differentiation OSCC tissues. The correlations between the staining score of PKDs and differentiation of OSCC were further analyzed. RESULTS: PKD1 and PKD3 were high expression in OSCC tissues. There were statistical significance among the staining score of PKD1, PKD2 and PKD3 in various differentiation OSCC tissues ( P<0.001). In addition, there was a significantly negative correlation between the staining score of PKD1 and PKD2 with the differentiation of OSCC ( r=-0.574, -0.341, P<0.001). CONCLUSION: In OSCC tissues with different degree of differentiation, there might be some differences among PKDs which play a major role. The expression of PKD1 and PKD2 was correlated with the differentiation of OSCC, the poor differentiation of OSCC, the higher expression of PKD1 and PKD2.

[Protein kinase D1 regulates the growth and metabolism of oral squamous carcinoma cells in tumor microenvironment].

OBJECTIVE: To observe the effect of protein kinase D1 (PKD1) on the growth and metabolism of oral squamous cell carcinoma HSC-4 cells and related molecular mechanisms in the tumor microenvironment. METHODS: HSC-4 cell lines were transfected with shRNA plasmids. Three groups (Wild, control-shRNA, and PKD1-shRNA) were cultured under acidic or hypoxic environment for a certain time. Western blot was used to detect the expression of autophagy-related and glycolytic-related proteins. The proliferation changes were detected by CCK-8 kits. RESULTS: The PKD1-knockdown HSC-4 cell line was established. PKD1 silencing increased autophagy activity. Under hypoxic and acidic conditions, the PKD1-knockdown HSC-4 cells showed lower proliferation than the parental cells. PKD1-knockdown also decreased the expression of hypoxia induciblefactor 1α (HIF-1α) and pyruvate kinase M2 (PKM2). CONCLUSIONS: Under hypoxic and acidic conditions, PKD1 gene silencing can increase apoptotic autophagy activity. Downregulated PKD1 gene expression can reduce the glycolysis of oral squamous cell carcinoma cells and inhibit tumor cell proliferation. This study revealed the important role of PKD1 in the metabolism and growth of oral squamous cell carcinoma, making it a possible target for the treatment of oral squamous cell carcinoma.

[Role of protein kinase D1 in regulating the growth, apoptosis and drug sensitivity of oral squamous carcinoma cells].

OBJECTIVE: This study aimed to investigate the role of protein kinase D (PKD)1 in regulating the growth, apop-tosis, and drug sensitivity of the squamous carcinoma cell line SCC-25. METHODS: The SCC-25 cell line was transfected with either the control-shRNA or PKD1-shRNA plasmids. The stable transfected cells were selected, and the efficiency of PKD1 knockdown was detected by Western blot. The growth and apoptosis of SCC-25 were analyzed with a cell counting kit-8 (CCK8) and flow cytometry. The 50% inhibitory concentrations (IC50) of paclitaxel in the control and PKD1 knockdown cell lines were detected by CCK-8. The expression levels of Bax, Bcl-2, and P-gp were detected by Western blot. RESULTS: PKD1 was constitutively expressed and phosphorylated in various cancer cell lines. Inhibiting the expression of PKD1 in SCC-25 cells by RNA interference could inhibit the growth and promote the apoptosis of SCC-25 cells via downregulating Bcl-2 expression. Additionally, inhibiting PKD1 expression could downregulate the expression of P-gp, thereby decreasing both the IC50 and resistance index of paclitaxel. CONCLUSIONS: PKD1 plays an important role in regulating the biobehavior of SCC-25. It is a potential therapeutic target for oral squamous carcinoma.

[Saliva of periodontitis patients promotes macrophage differentiation and activation].

OBJECTIVE: The aim of this study was to investigate the effect of saliva of patients with chronic periodontitis (CPD) on the differentiation, activation, and secretion of osteoclast-maturing mediators of macrophages. METHODS: A total of 40 saliva samples were collected from healthy donors (n=20) and severe periodontitis patients (n=20). Peripheral blood mononuclear cells (PBMCs) and THP-1 monocyte line cells were challenged with 15% saliva for 5 days. The phenotype, surface marker, and phagocytosis of macrophages were analyzed by flow cytometry and microscopy. Osteoclast-maturing mediators were assayed by using enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: When PBMCs were treated with CPD saliva for 5 days, 61.25%±11.33% of cells were transformed into large granular cells; 86.78%±13.69% of large granular cells were identified as CD14⁺⁺CD16⁺ macrophages. When THP-1 cells were treated with CPD saliva, most cells attached to the bottom of cell culture plates, thereby exhibiting macrophage morphology and releasing additional osteoclast-maturing mediators. Furthermore, the phagocytosis of THP-1 cells considerably increased in the presence of CPD saliva (66.35%±9.67%) compared with medium control (33.33%±7.52%), or healthy saliva (40.71%±3.52%). CONCLUSIONS: Saliva from patients with CPD can induce macrophage differentiation, activate phagocytose microorganisms, and secrete osteoclast-maturing mediators.

[Analysis of causes and whole microbial structure in a case of rampant caries].

OBJECTIVE: To analyze the whole microbial structure in a case of rampant caries to provide evidence for its prevention and treatment. METHODS: Clinical samples including blood, supragingival plaque, plaque in the caries cavity, saliva, and mucosal swabs were collected with the patient's consent. The blood sample was sent for routine immune test, and the others samples were stained using Gram method and cultured for identifying colonies and 16S rRNA sequencing. DNA was extracted from the samples and tested for the main cariogenic bacterium (Streptococcus mutans) with qPCR, and the whole microbial structure was analyzed using DGGE. RESULTS: The patient had a high levels of IgE and segmented neutrophils in his blood. Streptococci with extremely long chains were found in the saliva samples under microscope. Culture of the samples revealed the highest bacterial concentration in the saliva. The relative content of hemolytic bacterium was detected in the samples, the highest in the caries cavity; C. albicans was the highest in the dental plaque. In addition, 33 bacterial colonies were identified by VITEK system and 16S rDNA sequence phylogenetic analysis, and among them streptococci and Leptotrichia wade were enriched in the dental plaque sample, Streptococcus mutans, Fusobacterium nucleatum, and Streptococcus tigurinus in the caries cavity, and Lactobacillus in the saliva. S. mutans was significantly abundant in the mucosal swabs, saliva and plaque samples of the caries cavity as shown by qPCR. Compared to samples collected from a healthy individual and another two patients with rampant caries, the samples from this case showed a decreased bacterial diversity and increased bacterial abundance shown by PCR-DGGE profiling, and multiple Leptotrichia sp. were detected by gel sequencing. CONCLUSION: The outgrowth of such pathogenic microorganisms as S. mutans and Leptotrichia sp., and dysbiosis of oral microbial community might contribute to the pathogenesis of rampant caries in this case.