PER2 binding to HSP90 enhances immune response against oral squamous cell carcinoma by inhibiting IKK/NF-κB pathway and PD-L1 expression.

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/ß 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.

Development and Evaluation of a Nomogram for INCS Insensitivity in Chinese Adults with Allergic Rhinitis.

Objective: The objective of this study was to design and validate a nomogram of intranasal corticosteroid (INCS) insensitivity for adult patients with allergic rhinitis (AR). Methods: Training and validation datasets comprised randomly divided groups of AR patients diagnosed between 2019 and 2022, with a 7 : 3 ratio. These patients were categorized according to their INCS insensitivity status, and LASSO and multivariate logistic regression analyses were conducted to identify associated risk factors. These factors were incorporated into a nomogram for predicting INCS insensitivity. The performance of the nomogram was assessed using receiver operating characteristic (ROC) curves, calibration curves, and discrimination techniques. Results: In this study, 313 patients were included, of which 120 (38.3%) showed INCS insensitivity. The type of AR, comorbidities, family history of AR, and duration of AR were identified as predictors and incorporated into the nomogram using least absolute shrinkage and selection operator and multivariate logistic regression. The calibration curves showed excellent agreement between predicted and actual probabilities of INCS insensitivity in both the training and validation sets. The area under the curve values observed in the validation set were 0.918 (95% confidence interval, 0.859-0.943), and 0.932 (95% confidence interval, 0.849-0.953) in the training set, indicating strong performance on both sets. Decision curve analysis showed that the constructed nomogram yielded a net clinical benefit for AR patients. Conclusion: The nomogram constructed from risk predictors of INCS insensitivity in patients with AR demonstrated strong predictive power and enabled clinicians to identify high-risk patients, aiding them in developing an optimal treatment plan for AR.

A Novel Method Based on Hydrodynamic Cavitation for Improving Nitric Oxide Removal Performance of NaClO2.

In the removal of nitric oxide (NO) by sodium chlorite (NaClO2), the NaClO2 concentration is usually increased, and an alkaline absorbent is added to improve the NO removal efficiency. However, this increases the cost of denitrification. This study is the first to use hydrodynamic cavitation (HC) combined with NaClO2 for wet denitrification. Under optimal experimental conditions, when 3.0 L of NaClO2 with a concentration of 1.00 mmol/L was used to treat NO (concentration: 1000 ppmv and flow rate: 1.0 L/min), 100% of nitrogen oxides (NOx) could be removed in 8.22 min. Furthermore, the NO removal efficiency remained at 100% over the next 6.92 min. Furthermore, the formation of ClO2 by NaClO2 is affected by pH. The initial NOx removal efficiency was 84.8-54.8% for initial pH = 4.00-7.00. The initial NOx removal efficiency increases as the initial pH decreases. When the initial pH was 3.50, the initial NOx removal efficiency reached 100% under the synergistic effect of HC. Therefore, this method enhances the oxidation capacity of NaClO2 through HC, realizes high-efficiency denitrification with low NaClO2 concentration (1.00 mmol/L), and has better practicability for the treatment of NOx from ships.

Mediation of PKM2-dependent glycolytic and non-glycolytic pathways by ENO2 in head and neck cancer development.

BACKGROUND: Enolase 2 (ENO2) is a crucial glycolytic enzyme in cancer metabolic process and acts as a "moonlighting" protein to play various functions in diverse cellular processes unrelated to glycolysis. ENO2 is highly expressed in head and neck squamous cell carcinoma (HNSCC) tissues relative to normal tissues; however, its impact and underlying regulatory mechanisms in HNSCC malignancy remain unclear. METHODS: Molecular alterations were examined by bioinformatics, qRT-PCR, western blotting, immunofluorescence, immunohistochemistry, immunoprecipitation, and ChIP-PCR assays. Metabolic changes were assessed by intracellular levels of ATP and glucose. Animal study was used to evaluate the therapeutic efficacy of the ENO inhibitor. RESULTS: ENO2 is required for HNSCC cell proliferation and glycolysis, which, surprisingly, is partially achieved by controlling PKM2 protein stability and its nuclear translocation. Mechanistically, loss of ENO2 expression promotes PKM2 protein degradation via the ubiquitin-proteasome pathway and prevents the switch of cytoplasmic PKM2 to the nucleus by inactivating AKT signaling, leading to a blockade in PKM2-mediated glycolytic flux and CCND1-associated cell cycle progression. In addition, treatment with the ENO inhibitor AP-III-a4 significantly induces HNSCC remission in a preclinical mouse model. CONCLUSION: Our work elucidates the signaling basis underlying ENO2-dependent HNSCC development, providing evidence to establish a novel ENO2-targeted therapy for treating HNSCC.

GSK3B Overexpression Alleviates Posttraumatic Osteoarthritis in Mice by Promoting DNMT1-Mediated Hypermethylation of NR4A3 Promoter.

Background: Glycogen synthase kinase 3ß (GSK3B) is reported to be a protective factor for the degradation of chondrocytes by extracellular mechanisms. Nuclear receptor subfamily 4 group A member 3 (NR4A3) is a proinflammatory factor in osteoarthritis. Their regulation mechanism in posttraumatic osteoarthritis (PTOA) is not fully understood. Methods: GSK3B expression in the cartilage tissue of PTOA patients was analyzed by western blotting. IL-1ß-induced chondrocytes were transfected with pcDNA-GSK3B, and then, the cell viability, apoptosis, expression of the chondrocyte extracellular matrix degradation-related genes MMP13, aggrecan, and type II collagen, and secretion of inflammatory factors TNF-α and IL-6 were detected. Co-IP was used to analyze the interaction between GSK3B and DNMT1. Ch-IP and methylation-specific PCR assays were used for methylation. Also, cells were transfected with pcDNA-GSK3B or together with pcDNA-NR4A3, as well as transfected with si-NR4A3, and then, cell functions were tested. Then, the mice subjected to destabilization of medial meniscus (DMM) surgery were intra-articular injected with 100 µL of the following adeno-related virus vectors (empty vector, Ad-GSK3B, scrambled shRNA, and sh-NR4A3), respectively, and the virus titer was 2 × 108 TU/mL. Cartilage integrity was evaluated by safranin O/fast green staining, HE staining, and Osteoarthritis Research Society International (OARSI) score. Results: The expression of GSK3B protein was downregulated in PTOA patients. GSK3B overexpression alleviated IL-1ß-induced chondrocyte apoptosis and extracellular matrix degradation, as well as cartilage mineralization in PTOA model mice. NR4A3 overexpression reversed the effect of GSK3B on IL-1ß-induced chondrocyte functions. GSK3B could recruit DNMT1 to the NR4A3 promoter region to promote the methylation of NR4A3 and inhibit the expression of NR4A3 protein. Similarly, NR4A3 interference alleviated cartilage degradation under stimulating conditions by inhibiting the activation of the JAK2/STAT3 signaling pathway. Conclusion: GSK3B recruits DNMT1 to the NR4A3 promoter region and inhibits the activation of the NR4A3-mediated JAK2/STAT3 signaling pathway, thereby alleviating PTOA.

Circular RNA hsa_circ_0001649 suppresses the growth of osteosarcoma cells via sponging multiple miRNAs.

Osteosarcoma (OS) is a serious bone malignancy commonly occurred in childhood and adolescence. Circular RNA (circRNA) is a novel endogenous RNA that may be considered as a new biomarker for diseases' diagnosis or prognosis. This study explored the roles and mechanism of circ_0001649 in OS. The qRT-PCR was performed to test circ_0001649 expression in OS tissues and cells. Luciferase was used to confirm the binding of circ_0001649 with miR-338-5p, miR-647 and miR-942. OS cells were stably transfected with pEX-circ_0001649 or miRNAs mimic, CCK-8 kit, colony formation, apoptosis and western blot analysis were used to detect the roles of circ_0001649. Circ_0001649 was low-expressed in OS tissues and cell lines. Circ_0001649 overexpression suppressed U2OS and HOS cell viability and survival fraction, and induced apoptosis presented as the increasing levels of Apaf-1, cleaved-caspase-3 and cleaved-caspase-9. Further, circ_0001649 worked as a sponge to absorb miR-338-5p, miR-647 and miR-942 to suppress cell proliferation, induce apoptosis and inhibit STAT pathway. Circ_0001649 suppressed OS cell proliferation and STAT pathway and induced apoptosis through sponging miR-338-5p, miR-647 and miR-942.

Long non-coding RNA CCAT2 acts as an oncogene in osteosarcoma through regulation of miR-200b/VEGF.

Background/aim: Colon cancer-associated transcript 2 (CCAT2) is a new lncRNA, which is closely associated with risk of several cancers. The aim of this study was to explore the regulatory mechanism of CCAT2 in osteosarcoma (OSA). Methods: Cells were transfected with si-CCAT2, microRNA (miR)-200b inhibitor and the corresponding controls. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to measure the expression of CCAT2 and miR-200b in OSA tissues and cell lines. CCK8 and bromodeoxyuridine (BrdU) were conducted to examine cell proliferation. Apoptosis was detected by PI/FITC-Annexin V combining with flow cytometric analysis. Migration and invasion were respectively measured through transwell chambers assays. Western blot was used to examine expressions of relative proteins. Results: CCAT2 was highly expressed and miR-200b was lowly expressed in OSA tissues and cell lines. Knockdown of CCAT2 suppressed cell proliferation, migration and invasion but induced apoptosis and up-regulation of miR-200b. miR-200b inhibitor weakened the effect of si-CCAT2 on cell progression and cell mobility. Besides, knockdown of CCAT2 blocked the PI3K/Akt and AMPK pathways through up-regulating miR-200b. Conclusions: The CCAT2/miR-200b/vascular endothelial growth factor (VEGF) axis plays important regulating effect in OSA through the PI3K/Akt and AMPK pathways.

Regulatory role of microRNA-185 in the recovery process after ankle fracture.

The present study aimed to investigate the expression of microRNA (miR)-185 in the bone and blood tissues following ankle fracture, and its regulatory mechanism in the ankle fracture recovery process. In total, 28 patients with ankle fractures were included, including 15 cases receiving surgical treatment within 1-7 days after fracture, and 13 cases receiving surgery within 8-14 days after fracture. Reverse transcription-quantitative polymerase chain reaction was performed to detect the mRNA expression levels. Western blot analysis and ELISA were used to determine the protein expression levels. Bioinformatics analysis and dual-luciferase reporter assay were applied to predict and confirm the upstream regulator of tumor growth factor (TGF)-ß1. An MTT assay was performed to assess the cell proliferation. Compared with the 1-7-day surgery group, the mRNA and protein expression levels of TGF-ß1 were significantly elevated, while the expression levels of miR-185 were significantly declined in the bone and blood tissues in the 8-14-day surgery group. Bioinformatics analysis and dual-luciferase reporter assay predicted and confirmed that TGF-ß1 was the direct target gene of miR-185. Moreover, upregulated expression of miR-185 significantly decreased the protein expression levels of TGF-ß1 and reduced the proliferating activity of hFOB1.19 cells. Within two weeks after ankle fracture, the expression levels of TGF-ß1 are significantly upregulated in the bone and blood tissues, which may have been associated with the downregulated expression of miR-185. miR-185 may modulate TGF-ß1 to regulate the recovery of ankle fracture. These findings may contribute to the understanding of the biological functions and effects of miRNA-185 and TGF-ß1 in ankle fractures.

Mechanical Properties Study of Fe-Mn-Si Shape Memory Alloys Welding Seam Formed by Laser Welding with Filler Powder.

To reduce the residual stress and improve the fatigue property of the laser weldment by using the stress self-accommodation characteristic of Fe-Mn-Si shape memory alloys (SMAs), a Fe15Mn5Si12Cr6Ni memory alloy welding seam was formed inside 304 stainless steel by laser welding with filler powder. The combination of the hole-drilling method and the ANSYS software was used to research the distribution law of residual stress inside the laser welding specimen. The fatigue strength of the laser welded specimens with the Fe-Mn-Si SMAs welding seam (experimental materials) and 304 stainless steel welding seam (comparative materials) was measured by cycle bending fatigue test. The microhardness of the welding specimens was measured by the microhardness tester. The thermodynamic model of the laser welding process and the phase transition crystallography of Fe-Mn-Si SMAs were evaluated to analyze the strengthening mechanism of the mechanical properties in the experimental materials. The results show that the distribution law for residual stress in the experiment and simulation are consistent. The experimental materials possess low residual stress, high fatigue strength and high microhardness. The strengthening mechanism for mechanical properties is the welding residual stress-induced γ→ε martensitic transformation inside the experimental materials, which causes the tensile plastic strain of the welding seam to resist residual compression strain, and the residual stress, as the transition driving force, is released in shear processing.

Study on effect of peptide-conjugated near-infrared fluorescent quantum dots on the clone formation, proliferation, apoptosis, and tumorigenicity ability of human buccal squamous cell carcinoma cell line BcaCD885.

Quantum dots (QDs) have shown great development potential in noninvasive imaging and monitoring of cancer cells in vivo because of their unique optical properties. However, the key issue of whether or not QDs-labeled cancer cells affect the proliferation, apoptosis and in vivo tumorigenicity ability has not been reported. The primary issue is if the results obtained from the noninvasive visualization of QDs-labeled tumors are scientific. Here, we applied peptide-linked near-conjugated fluorescent QDs to label human buccal squamous cell carcinoma cell line (BcaCD885). We performed in vivo tumorigenicity ability assays, tumorigenic cells proliferation, and apoptotic capability assays detected by flow cytometry and plate clone formation experiment, and found that peptide-linked near-conjugated fluorescent QDs labeling did not affect the growth, proliferation, apoptosis, and tumorigenicity ability of those cancer cells. Our study provides scientific foundation to support the application of near-infrared fluorescent QDs in noninvasive imaging and monitoring of cancer cells in vivo.

[Effect of peptide-conjugated quantum dots on the tumorigenicity and lymph node metastasis of human tongue squamous cell carcinoma cell line Tca8113 and mouse uterine cervix carcinoma U14 in vivo].

OBJECTIVE: To observe the effect of peptide-conjugated quantum dots with a maximal emission of 655 nm (QD655) on growth, proliferation, apoptosis and lymphatic metastasis of human tongue squamous cell carcinoma cell line Tca8113 and mouse uterine cervix carcinoma U14 in vivo. METHODS: Tca8113 and U14 cells were labeled by QD655 (Tca8113-QD655, U14-QD655). Tca8113-QD655 and U14-QD655 were inoculated subcutaneously into nude mice and Kunming mice. The tumor formation of Tca8113-QD655 and Tca8113, U14-QD655 and U14 was observed and compared in vivo. The proliferation and apoptosis of Tca8113-QD655 and Tca8113, U14-QD655 and U14 cells from tumors formed in vivo were analyzed by flow cytometry. U14-QD655 and U14 were inoculated into the buccal mucosa of Kunming mice to establish the cervical lymph node metastasis model of buccal cancer. The cervical lymph node metastatic ability of U14-QD655 and U14 was compared. RESULTS: The tumor weight and volume of Tca8113-QD655 and Tca8113, U14-QD655 and U14 in vivo were not significantly different (P>0.05), the cell proliferation index and apoptosis index of Tca8113-QD655 and Tca8113, U14-QD655 and U14 in vivo were not significantly different (P>0.05). The cervical lymph node metastasis rate of U14-QD655 and U14 buccal cancer were not significantly different (P>0.05). CONCLUSIONS: QD showed no effects on tumorigenicity and lymph node metastasis of Tca8113 and U14 cells. These results provide the scientific basis for noninvasive imaging and long-term tracing study.

In vivo study of the effects of peptide-conjugated near-infrared fluorescent quantum dots on the tumorigenic and lymphatic metastatic capacities of squamous cell carcinoma cell line Tca8113 and U14.

Quantum dots (QDs) have great potential in non-invasive monitoring and imaging of tumor cells in vivo, but it is unknown if QDs affect their tumorigenesis and metastasis. Here, we applied peptide-conjugated near-infrared fluorescent QDs (NIRF-QDs) to label the squamous cell carcinoma cells Tca8113 and U14. We tested the proliferation and apoptotic capacities of both cells, and the capacity of cervical lymph node metastasis after tumorigenesis in U14 cells'. We find that QDs do not affect the tumor cells' capacities to grow, proliferate, and metastasize. Our study provides critical data to support the application of NIRF-QDs in non-invasive monitoring and imaging of tumor cells in vivo.

[Preparing of semiconductor quantum dots-Smad4 monoclonal antibody fluorescent probes and identification of their ability of specific immuno-recognition].

OBJECTIVE: To prepare semiconductor quantum dots (SQD)-Smad4 monoclonal antibody fluorescent probes and to detect the optical qualities and the ability of specific recognition of the probes. METHODS: SQD were chemically modified with Smad4 monoclonal antibody proteins to prepare water soluble probes, and the fluorescence intensity, photostability, absorption spectra and emission spectra of the probes were studied. The location of Smad4 in rat dental papillae cells (RDPC) was examined by SP anti-Smad4 immunocytochemical method and SQD-Smad4 direct immunofluorescent imaging. RESULTS: SQD and monoclonal antibody covalently bonded to form the fluorescent probes which could specifically recognize Smad4 in RDPC. These fluorescent probes still had properties, including broad absorption band, narrow emission band, high fluorescence intensity and photostability. CONCLUSIONS: SQD and monoclonal antibody could covalently bond to form the fluorescent probes with distinct optics character and ability of specific recognization, which provides the scientific evidence that SQD trace the molecular movement in living cells in long-term, in situ and in real time.

[Preparing of semiconductor quantum dots-Smad2 monoclonal antibody fluorescent probes and testing of its related properties].

OBJECTIVE: To prepare semiconductor quantum dots (QDs)-Smad2 monoclonal antibody fluorescent probes, to detect the optical qualities and the ability to specific recognition of Smad2 in rat dental papillae cells (RDPC) of quantum dots-Smad2 monoclonal antibody fluorescent probes. METHODS: (1) QDs were chemically modified with Smad2 proteins to prepare water soluble QDs-Smad2 monoclonal antibody fluorescent probes which were purified after preparation. (2) The absorption band and emission band of these probes were obtained through ultraviolet spectrophotometer and fluorospectrophotometer, the shape, fluorescence intensity and photochemistry stability of these probes were studied through confocal laser scanning fluorescence microscopy. (3) Before the location of Smad2 proteins in RDPC was studied with anti-Smad2 immunocytochemical method and direct immunofluorescence imaging, RDPC were incubated with transforming growth factor-beta1 (TGF-beta1), and the related optical qualities of quantum dots-Smad2 monoclonal antibody fluorescent probes in RDPC were detected. RESULTS: QDs and monoclonal antibody linked together through covalent bond to form the fluorescent probes which could specifically and effectively recognize Smad2 proteins in RDPC. These fluorescent probes still had good properties, including broad excite spectra, narrow emission spectra, high fluorescence intensity and photostability. CONCLUSION: QDs and monoclonal antibody could link together through covalent bond to form the nanometer molecular probes with distinct optics character and photostability, which provides the scientific evidence that QDs can visualize the molecular movement in living cells in long-term, in situ and in real time.

[Effect of semiconductor quantum dots on cyto-biological behavior of tongue squamous cell carcinoma cell line Tca8113].

OBJECTIVE: To study the effect of semiconductor quantum dots (QD), a kind of new luminescent inorganic non-crystals on biological behavior of tongue squamous cell carcinoma cell line Tca8113. METHODS: Different concentrations of QD were cocultured with Tca8113 cells, and the status of cancer cell growth in three experimental groups and control was compared respectively. Tca8113 cells were labeled by QD (Tca8113-QD), and then transwell chambers and washing away method were used to detect the difference of invasion and metastatic ability between Tca8113-QD and Tca8113 cells. RESULTS: The different concentrations of QD showed no negative effects on growth of Tca8113 cells. The ability of invasion, attachment and chemotaxis movement of Tca8113 cells were not significantly different between the experimental groups and control. CONCLUSIONS: QD showed no effects on growth, invasion and metastatic ability of Tca8113 cells and may serve as a new fluorescence probe in living tumor cell study.