Oral squamous cell carcinoma (OSCC) has a high incidence of metastasis. Tumour immunotherapy targeting PD-L1 or PD-1 has been revolutionary; however, only a few patients with OSCC respond to this treatment. Therefore, it is essential to gain insights into the molecular mechanisms underlying the growth and metastasis of OSCC. In this study, we analysed the expression levels of protein kinase D3 (PKD3) and PD-L1 and their correlation with the expression of mesenchymal and epithelial markers. We found that the expression of PKD3 and PD-L1 in OSCC cells and tissues was significantly increased, which correlated positively with that of mesenchymal markers but negatively with that of epithelial markers. Silencing PKD3 significantly inhibited the growth, metastasis and invasion of OSCC cells, while its overexpression promoted these processes. Our further analyses revealed that there was positive feedback regulation between PKD3 and PD-L1, which could drive EMT of OSCC cells via the ERK/STAT1/3 pathway, thereby promoting tumour growth and metastasis. Furthermore, silencing PKD3 significantly inhibited the expression of PD-L1, and lymph node metastasis of OSCC was investigated with a mouse footpad xenograft model. Thus, our findings provide a theoretical basis for targeting PKD3 as an alternative method to block EMT for regulating PD-L1 expression and inhibiting OSCC growth and metastasis.

Salivary analysis can be used to assess the severity of caries. Of the known salivary proteins, a paucity of information exists concerning the role of proteinase 3 (PR3), a serine protease of the chymotrypsin family, in dental caries. Whole, unstimulated saliva was collected from children with varying degrees of active caries and tested using a Human Protease Array Kit and an enzyme-linked immunosorbent assay. A significantly decreased concentration of salivary PR3 was noted with increasing severity of dental caries (P<0.01); a positive correlation (r=0.87; P<0.01; Pearson’s correlation analysis) was also observed between salivary pH and PR3 concentration. In an antibacterial test, a PR3 concentration of 250 ng·mL⁻¹ or higher significantly inhibited Streptococcus mutans UA159 growth after 12 h of incubation (P<0.05). These studies indicate that PR3 is a salivary factor associated with the severity of dental caries, as suggested by the negative relationship between salivary PR3 concentration and the severity of caries as well as the susceptibility of S. mutans to PR3.

Cytolytic T lymphocytes (CTL) and natural killer (NK) cells have been implicated as important cells in antitumor responses. Our previous research has shown that high mobility group nucleosomal-binding domain 2 (HMGN2) could be released by IL-2 and PHA stimulated peripheral blood mononuclear cells (PBMCs) and also induced tumor cells apoptosis at low doses. In this study, we isolated and cultured PBMCs and CD8⁺ T cells to analyze the expression and antitumor effects of HMGN2. PBMCs from healthy donors were isolated using Human Lymphocyte Separation tube. CD8⁺ T cells were separated from the PBMCs using MoFlo XDP high-speed flow cytometry sorter. Activation of PBMCs and CD8⁺ T cells were achieved by stimulating with Phytohemagglutinin (PHA) or tumor antigen. In addition, the methods of ELISA, intracellular staining, and fluorescence-labeling assays were used. PHA induced PBMCs to release high levels of HMGN2, and CD8⁺ T cells was the major cell population in PBMCs that release HMGN2 after PHA activation. Tumor antigen-activated CD8⁺ T cells also released high levels of HMGN2. Supernatants of tumor antigen-activated CD8⁺ T cells were able to kill tumor cells in a dose-dependent manner. This antitumor effect could be significantly blocked by using an anti-HMGN2 antibody. Fluorescence-labeling assays showed that the supernatant proteins of activated CD8⁺ T cells could be transported into tumor cells, and the transport visibly decreased after HMGN2 was depleted by anti-HMGN2 antibody. These results suggest that HMGN2 is an anti-tumor effector molecule of CD8⁺ T cells.

Oral squamous cell carcinoma (OSCC) is capable of constructing a favorable immune escape environment through interactions of cells with cells and of cells with the environment. Programmed death ligand‑1 (PD‑L1) is a well‑recognized inhibitor of anti‑tumor immunity that plays an important role in tumor immune escape. However, the molecular mechanisms regulating PD‑L1 expression are not yet fully understood. In this study, to investigate the role of protein kinase D3 (PKD3) in the regulation of PD‑L1 expression, the expression and correlation of PKD3 and PD‑L1 were first analyzed by the immunostaining of human OSCC tissue sections, cell experiments and TCGA gene expression databases. The expression levels of PKD3 and PD‑L1 were found to be significantly higher in OSCC cells than in normal tissues or cells. In addition, the expression levels of PKD3 and PD‑L1 were found to be significantly positively correlated. Subsequently, it was found that the levsel of PD‑L1 expression decreased following the silencing of PKD3 and that the ability of interferon (IFN)‑γ to induce PD‑L1 expression was also decreased in OSCC. The opposite phenomenon occurred following the overexpression of PKD3. It was also found that the phosphorylation of signal transducer and activator of transcription (STAT)1/STAT3 was reduced by the knockdown of PKD3 in OSCC. Moreover, the expression level of PD‑L1 was decreased after the use of siRNA to knockdown STAT1 or STAT3. On the whole, the findings of this study confirm that PKD3 regulates the expression of PD‑L1 induced by IFN‑γ by regulating the phosphorylation of STAT1/STAT3. These findings broaden the understanding of the biological function of PKD3, suggesting that PKD is a potential therapeutic target for OSCC.

The ubiquitin-editing enzyme A20 is known to regulate inflammation and maintain homeostasis, but its role in self-DNA-mediated inflammation in acute kidney injury (AKI) is not well understood. Here, our study demonstrated that oxidized self-DNA accumulates in the serum of AKI mice and patients. This oxidized self-DNA exacerbates the progression of AKI by activating the cGAS-STING pathway and NLRP3 inflammasome. While inhibition of the STING pathway only slightly attenuates AKI progression, suppression of NLRP3 inflammasome-mediated pyroptosis significantly alleviates AKI progression and improves the survival of AKI mice. Subsequently, we found that Tnfaip3 (encoding A20) is significantly upregulated following oxidized self-DNA treatment. A20 significantly alleviates AKI development by dampening STING signaling pathway and NLRP3-mediated pyroptosis. Moreover, A20-derived peptide (P-II) also significantly alleviates ox-dsDNA-induced pyroptosis and improves the survival and renal injury of AKI mice. Mechanistically, A20 competitively binds with NEK7 and thus inhibiting NLRP3 inflammasome. A20 and P-II interfere with the interaction between NEK7 and NLRP3 through Lys140 of NEK7. Mutation of Lys140 effects on the interaction of NEK7 with A20 and/or NLRP3 complex. Conditional knockout of NEK7 in macrophages or pharmacological inhibition of NEK7 both significantly rescue AKI mouse models. This study reveals a new mechanism by which A20 attenuates oxidized self-DNA-mediated inflammation and provides a new therapeutic strategy for AKI.

Targeted drug delivery is urgently needed for cancer therapy, and green synthesis is important for the biomedical use of drug delivery systems in the human body. In this work, we report two targeted delivery systems for anticancer drugs based on tea polyphenol functionalized and reduced graphene oxide (TPGs). The obtained TPGs demonstrated an efficient doxorubicin loading capacity as high as 3.430 × 10⁶mg g⁻¹ and 3.932 × 10⁴mg g⁻¹, and exhibited pH-triggered release. Furthermore, the kinetic models, adsorption isotherms, and possible loading mechanisms were investigated in details. Compared to TPG1 and free doxorubicin, TPG2 is biocompatible to normal cells even at high concentrations and promotes tumor cells death by delivering the doxorubicin mainly to the nuclei. These results were confirmed using cell viability tests and confocal laser microscopy. Moreover, apoptosis tests showed that the mechanism of cancer cell death induced by TPG1 and TPG2 might follow the similar mechanisms. Taken together, these results demonstrate that TPGs provide a multifunctional drug delivery system with a greater loading capacity and pH-sensitive drug release for enhanced cancer therapy. The high drug payload capability and enhanced antitumor efficacy demonstrate that we developed systems are promising for various biomedical applications and cancer therapy.