Identification of immune-associated biomarker for predicting lung adenocarcinoma: bioinformatics analysis and experiment verification of PTK6.

BACKGROUND: Abnormal expression of protein tyrosine kinase 6 (PTK6) has been proven to be involved in the development of gynecological tumors. However, its immune-related carcinogenic mechanism in other tumors remains unclear. OBJECTIVE: The aim of this study was to identify PTK6 as a novel prognostic biomarker in pan-cancer, especially in lung adenocarcinoma (LUAD), which is correlated with immune infiltration, and to clarify its clinicopathological and prognostic significance. METHODS: The prognostic value and immune relevance of PTK6 were investigated by using bio-informatics in this study. PTK6 expression was validated in vitro experiments (lung cancer cell lines PC9, NCI-H1975, and HCC827; human normal lung epithelial cells BEAS-2B). Western blot (WB) revealed the PTK6 protein expression in lung cancer cell lines. PTK6 expression was inhibited by Tilfrinib. Colony formation and the Cell Counting Kit-8 (CCK-8) assay were used to detect cell proliferation. The wound healing and trans-well were performed to analyze the cell migration capacity. Then flow cytometry was conducted to evaluate the cell apoptosis. Eventually, the relationship between PTK6 and immune checkpoints was examined. WB was used to estimate the PD-L1 expression at different Tilfrinib doses. RESULTS: PTK6 was an independent predictive factor for LUAD and was substantially expressed in LUAD. Pathological stage was significantly correlated with increased PTK6 expression. In accordance with survival analysis, poor survival rate in LUAD was associated with a high expression level of PTK6. Functional enrichment of the cell cycle and TGF-ß signaling pathway was demonstrated by KEGG and GSEA analysis. Moreover, PTK6 expression considerably associated with immune infiltration in LUAD, as determined by immune analysis. Thus, the result of vitro experiments indicated that cell proliferation and migration were inhibited by the elimination of PTK6. Additionally, PTK6 suppression induced cell apoptosis. Obviously, PD-L1 protein expression level up-regulated while PTK6 was suppressed. CONCLUSION: PTK6 has predictive value for LUAD prognosis, and could up regulated PD-L1.

CD4+CD25+ regulatory T cells ex vivo generated from autologous naïve CD4+ T cells suppress EAE progression.

CD4+CD25+ regulatory T cells (Tregs) play an important role in maintaining immune homeostasis in multiple sclerosis (MS). Hence, we aimed to explore the therapeutic efficacy and safety of adoptive cell therapy (ACT) utilizing induced antigen-specific Tregs in an animal model of MS, that is, in an experimental autoimmune encephalomyelitis (EAE) model. B cells from EAE model that were activated with soluble CD40L were used as antigen-presenting cells (APCs) to induce the differentiation of antigen-specific Tregs from naïve CD4 precursors, and then, a stepwise isolation of CD4+CD25highCD127low Tregs was performed using a flow sorter. All EAE mice were divided into Treg-treated group (2 × 104 cells in 0.2 mL per mouse, n = 14) and sham-treated group (0.2 mL normal saline (NS), n = 20), which were observed daily for clinical assessment, and for abnormal appearance for 6 weeks. Afterward, histological analysis, immunofluorescence and real-time PCR were performed. Compared to sham-treated mice, Treg-treated mice exhibited a significant decrease in disease severity scores and reduced inflammatory infiltration and demyelination in the spinal cord. Additionally, Tregs-treated mice demonstrated higher CCN3 protein and mRNA levels than sham-treated mice. The results of this preclinical study further support the therapeutic potential of this ACT approach in the treatment of MS.

Analysis between macrophage-related genes with prognosis and tumor microenvironment in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a highly morbid and fatal disease that exhibits individualized differences in prognosis and drug efficacy. Therefore, understanding the molecular mechanism of the occurrence and progression of lung cancer can improve early diagnosis, treatment and prognosis. Macrophages are a crucial component of the tumor microenvironment (TME) due to their high plasticity and heterogeneity. They play a multifaceted role in tumor initiation and progression. In order to elucidate the pathogenesis of tumor-associated macrophages (TAMs) related genes in NSCLC, transcriptomic sequencing, univariate COX regression, LASSO regression and multivariate COX regression analyses were conducted to identify the 11 genes that have the most significant association with prognosis. These genes include FCRLA, LDHA, LMOD3, MAP3K8, NT5E, PDGFB, S100P, SFXN1, TDRD1, TFAP2A and TUBB6. The risk score (RS) was computed, and all samples were split into high- and low-risk groups based on the median RS. The correlation of RS and 11 genes with macrophages was verified by the CIBERSORT deconvolution algorithm. These above results suggest that the risk score developed in this study can be utilized for predicting patients' prognosis and evaluating their immune infiltration status. This study can serve as a guide for subsequent tumor immunotherapy and gene targeting therapy.

K63 Ubiquitination of P21 Can Facilitate Pellino-1 in the Context of Chronic Obstructive Pulmonary Disease and Lung Cellular Senescence.

Chronic obstructive pulmonary diseases (COPD) is a kind of age-related, airflow-obstruction disease mostly caused by cigarette smoke. However, the relationship between COPD and lung cellular senescence is still not fully understood. Here, we found silencing Pellino-1 could inhibit the protein level of P21. Then, through constructing cell lines expressed ubiquitin-HA, we found that the E3 ubiquitin ligase Pellino-1 could bind to senescence marker p21 and modify p21 by K63-site ubiquitination by co-IP assays. Furthermore, we found that p21-mediated lung cellular senescence could be inhibited by silencing Pellino-1 in a D-galactose senescence mice model. Moreover, by constructing a COPD mouse model with shPellino-1 adenovirus, we found that silencing Pellino-1 could inhibit COPD and inflammation via reduction of SASPs regulated by p21. Taken together, our study findings elucidated that silencing E3 ligase Pellino-1 exhibits therapeutic potential for treatment to attenuate the progression of lung cellular senescence and COPD.