Recent studies showed that lncRNA CASC9 was upregulated and acted as an oncogene in a variety of tumors. However, the expression and biological functions of CASC9 in oral squamous cell carcinoma (OSCC) remain unknown. In this study, we found for the first time that CASC9 was remarkably upregulated in OSCC tissues and cell lines compared with paired noncancerous tissues and normal oral epithelial cells. Highly expressed CASC9 is strongly associated with tumor size, clinical stage, regional lymph node metastasis and overall survival time in OSCC patients. In vitro, CASC9 knockdown in OSCC cells SCC15 and CAL27 significantly promotes autophagy and apoptosis, while inhibiting proliferation. Moreover, the expression levels of p-AKT, p-mTOR, P62 and BCL-2 were significantly decreased, while the expression levels of BAX and the LC3BII/LC3BI ratio were increased in CASC9-knockdown SCC15 and CAL27 cells. After the addition of the AKT activator SC79 in CASC9-knockdown SCC15 and CAL27 cells, we found that the increased autophagy and apoptosis were remarkably rescued. Furthermore, the increased apoptosis was remarkably rescued in CASC9-knockdown OSCC cells treated with the autophagy inhibitor Autophinib. In addition, CASC9 depletion suppressed tumor growth in vivo. In conclusion, our findings demonstrate that lncRNA CASC9 promotes OSCC progression through enhancing cell proliferation and suppressing autophagy-mediated cell apoptosis via the AKT/mTOR pathway. CASC9 could potentially be used as a valuable biomarker for OSCC diagnosis and prognosis.

There is increasing evidence that the core clock gene Period 1 (PER1) plays important roles in the formation of various tumors. However, the biological functions and mechanism of PER1 in promoting tumor progression remain largely unknown. Here, we discovered that PER1 was markedly downregulated in oral squamous cell carcinoma (OSCC). Then, OSCC cell lines with stable overexpression, knockdown, and mutation of PER1 were established. We found that PER1 overexpression significantly inhibited glycolysis, glucose uptake, proliferation, and the PI3K/AKT pathway in OSCC cells. The opposite effects were observed in PER1-knockdown OSCC cells. After treatment of PER1-overexpressing OSCC cells with an AKT activator or treatment of PER1-knockdown OSCC cells with an AKT inhibitor, glycolysis, glucose uptake, and proliferation were markedly rescued. In addition, after treatment of PER1-knockdown OSCC cells with a glycolysis inhibitor, the increase in cell proliferation was significantly reversed. Further, coimmunoprecipitation (Co-IP) and cycloheximide (CHX) chase experiment demonstrated that PER1 can bind with RACK1 and PI3K to form the PER1/RACK1/PI3K complex in OSCC cells. In PER1-overexpressing OSCC cells, the abundance of the PER1/RACK1/PI3K complex was significantly increased, the half-life of PI3K was markedly decreased, and glycolysis, proliferation, and the PI3K/AKT pathway were significantly inhibited. However, these effects were markedly reversed in PER1-mutant OSCC cells. In vivo tumorigenicity assays confirmed that PER1 overexpression inhibited tumor growth while suppressing glycolysis, proliferation, and the PI3K/AKT pathway. Collectively, this study generated the novel findings that PER1 suppresses OSCC progression by inhibiting glycolysis-mediated cell proliferation via the formation of the PER1/RACK1/PI3K complex to regulate the stability of PI3K and the PI3K/AKT pathway-dependent manner and that PER1 could potentially be a valuable therapeutic target in OSCC.

Background: Programmed death-ligand 1 (PD-L1) contributes to the immune escape of tumor cells and is a critical target for antitumor immunotherapy. However, the molecular mechanisms regulating PD-L1 expression remain unclear, hindering the development of effective therapies. Here we investigate the role and molecular mechanism of the core clock gene Period2 (PER2) in regulating PD-L1 expression and its role in the combination therapy of oral squamous cell carcinoma (OSCC). Methods: Quantitative real-time PCR, western blotting or immunohistochemistry to detect expression of PER2 and PD-L1 in OSCC tissues and cells. Overexpression and knockdown of PER2 detects the function of PER2. Bioinformatics, immunoprecipitation, GST pull-down, CHX chase assay and western blot and strip to detect the mechanism of PER2 regulation for PD-L1. A humanized immune reconstitution subcutaneous xenograft mouse model was established to investigate the combination therapy efficacy. Results: In OSCC tissues and cells, PER2 expression was reduced and PD-L1 expression was increased, the expression of PER2 was significantly negatively correlated with PD-L1. In vitro and in vivo experiments demonstrated that PER2 inhibited PD-L1 expression and enhanced T-cell-mediated OSCC cell killing by suppressing the IKK/NF-κB pathway. Mechanistically, PER2 binds to heat shock protein 90 (HSP90) through the PAS1 domain and reduces the interaction of HSP90 with inhibitors of kappa B kinase (IKKs), promoting the ubiquitination of IKKα/β and p65 nuclear translocation to inhibit IKK/NF-κB pathway, thereby suppressing PD-L1 expression. In humanized immune reconstitution subcutaneous xenograft mouse model, it was demonstrated that PER2 targeting combined with anti-PD-L1 treatment improved the inhibition of OSCC growth by promoting CD8⁺ T-cell infiltration into the tumor. Conclusions: Our findings reveal the role and mechanism of PD-L1 regulation by PER2 and support the potential clinical application of PER2 targeting in combination with anti-PD-L1 in OSCC immunotherapy.

Previous studies have shown that the aberrant expression of period circadian clock 2 (Per2) is closely related to the occurrence and development of cancers, but the specific mechanism remains unclear. In the present study, we used shRNA to downregulate Per2 in oral squamous cell carcinoma (OSCC) Tca8113 cells, and then detected the alterations in cell cycle, cell proliferation and apoptosis by flow cytometric analysis and mRNA expression alterations in all the important genes in the cyclin/cyclin-dependent protein kinase (CDK)/cyclin-dependent kinase inhibitor (CKI) cell cycle network by RT-qPCR. We found that in the Tca8113 cells, after Per2 downregulation, the mRNA expression levels of cyclin A2, B1 and D1, CDK4, CDK6 and E2F1 were significantly increased (P<0.05), the mRNA expression levels of p53, p16 and p21 were significantly decreased (P<0.05), cell proliferation was significantly higher (P<0.05), apoptosis was significantly lower (P<0.05) and the number of cells in the G1/G0 phase was significantly decreased (P<0.05). The present study proves that in OSCC, clock gene Per2 plays an important role in cell cycle progression and the balance of cell proliferation and apoptosis by regulation of the cyclin/CDK/CKI cell cycle network. Further research on Per2 may provide a new effective molecular target for cancer treatments.

Objectives: The purpose of this study was to investigate in-vivo visible imaging of oral squamous cell carcinoma (OSCC) by targeting epidermal growth factor receptor (EGFR) with near-infrared quantum dots. Materials and methods: Quantum dots with an emission wavelength of 800 nm (QD800) were conjugated to monoclonal antibodies against EGFR, resulting in the probe designated as QD800-EGFR Ab. OSCC cell line (BcaCD885) expressing high levels of EGFR was transplanted subcutaneously into nude mice cheeks to develop an OSCC animal model. QD800-EGFR Ab containing 100 pmol equivalent of QD800 was intravenously injected into the animal model, and in-situ and in-vivo imaging of cheek squamous cell carcinoma was analyzed at 10 different time points. Results and conclusion: In-vivo imaging and immunohistochemical examination of the tumors showed that intravenously injected QD800-EGFR Ab probe could bind EGFR expressed on BcaCD885 cells. Fluorescence signals of BcaCD885 cells labeled with QD800-EGFR Ab probe could be clarly detected, and these fluorescence signals lasted for 24 hours. The most complete tumor images with maximal signal-to-noise ratio were observed from 15 minutes to 6 hours after injection of the probe. To the best of the authors' knowledge, this is the first study that has obtained clear in-situ and in-vivo imaging of head and neck cancer by using QD800-EGFR Ab probe. The authors conclude that the combination of near-infrared quantum dots that are highly penetrating for tissues with EGFR monoclonal antibody has promising prospects in in-vivo imaging of OSCC and development of personalized surgical therapies.

Current studies have shown that the clock gene Period 1 (Per1) is downregulated in various tumors and plays an important role in promoting tumor progression. However, the biological functions and mechanism of Per1 in tumors remain largely unknown. In this study, 86 specimens of oral squamous cell carcinoma (OSCC) tissues and adjacent noncancerous tissues were collected to determine Per1 expression level and clinical significance of Per1 expression. Per1 was stably inhibited or overexpressed in OSCC cells to investigate its function and mechanism in vitro and in vivo. We found that Per1 was remarkably downregulated in OSCC and that low Per1 expression was significantly associated with TNM clinical stage and poor prognosis of OSCC patients. Per1 overexpression in SCC15 OSCC cells (Per1‐OE SCC15 cells) significantly promoted autophagy and apoptosis while inhibited proliferation and AKT/mTOR pathway. However, the results obtained in Per1‐silenced TSCCA OSCC cells were opposite those obtained in Per1‐OE SCC15 cells. After addition of the AKT activator SC79 to Per1‐OE SCC15 cells, the increased autophagy and apoptosis as well as decreased proliferation were remarkably rescued. Furthermore, increased apoptosis was significantly rescued in Per1‐OE SCC15 cells treated with the autophagy inhibitor autophinib. In vivo tumorigenicity assays also confirmed that Per1 overexpression suppressed tumor growth. Taken together, our findings demonstrate for the first time that Per1 promotes OSCC progression by inhibiting autophagy‐mediated cell apoptosis and enhancing cell proliferation in an AKT/mTOR pathway‐dependent manner, and Per1 could be used as a valuable therapeutic target for OSCC.