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The aim of this study was to determine the relationship and underlying mechanisms between ectopic expression of phosphatidylethanolamine-binding protein 4 (PEBP4) and cisplatin (DDP)-induced cytotoxicity in the lung cancer cell line A549 to provide an experimental basis for future chemotherapeutic applications involving PEBP4 in human lung cancer. A recombinant plasmid, pcDNA3-PEBP4, and a PEBP4-targeting small hairpin RNA (shRNA) were transfected into the lung cancer cell line A549. The PEBP4 protein expression levels were determined for each group by Western blot, and after 48 h of cisplatin (DDP) treatment, the viability of cells in the treatment and control groups was determined by 3-[4,5-dimethylthiazol-2-yl]-3,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis in each treatment group was determined using flow cytometry. Western blotting was used to examine expression of the p53 protein in A549 cells from each group. We employed a luciferase reporter-gene assay to confirm PEBP4 as a target gene of miR-34a. Western blotting was used to determine the effects of miR-34a on PEBP4 protein expression in A549 cells. Following transfection of A549 cells with either the recombinant plasmid pcDNA3-PEBP4 or a PEBP4-targeting shRNA, Western blotting analyses showed PEBP4 protein expression was significantly higher in the pcDNA3-PEBP4-transfected group compared with the control or PEBP4-shRNA-transfected groups (p < 0.01). Furthermore, PEBP4 protein expression was significantly reduced in the PEBP4-shRNA-transfected group (p < 0.01). After 48 h of DDP treatment, MTT assays indicated that A549 cell viability was significantly lower in the DDP-treated group compared with the control group (p < 0.01). The viability of A549 cells in the pcDNA3-PEBP4-transfected group was lower than that in the control group (p < 0.05) but higher than that in either the DDP-treated or PEBP4-shRNA-transfected groups (p < 0.05). Moreover, the viability of A549 cells in the PEBP4-shRNA-transfected group was significantly lower than that in either the control (p < 0.01) or DDP-treated (p < 0.05) groups. Flow cytometry and Western blotting analyses indicated that the number of apoptotic cells and p53 protein expression were significantly higher in the DDP-treated group compared with the control group (p < 0.01). In the pcDNA3-PEBP4-transfected group, the number of apoptotic cells and p53 protein expression level were higher than those in the control group (p < 0.05) but lower than those in the DDP-treated and PEBP4-shRNA-transfected groups (p < 0.05). The number of apoptotic cells and p53 protein expression level in the PEBP4-shRNA-transfected group were higher than those in the control (p < 0.01) and DDP-treated (p < 0.05) groups. The luciferase reporter-gene assay showed that the relative luciferase activity after transfection with a miR-34a mimic was significantly reduced compared with the control group (p < 0.01). Western blotting analysis demonstrated that PEBP4 protein expression was significantly decreased in A549 cells 48 h after transfe...

The phosphatidylethanolamine-binding protein 4 (PEBP4) is a member of the PEBP family. It not only plays a role in the inhibition of the MAPK signaling pathway but also is involved in the inhibition of the JNK pathway that promotes the activation of AKT. Recent research has also shown that overexpression of PEBP4 was related to the development, invasion, and metastasis of a variety of tumors. This study aimed to investigate the correlation between PEBP4 protein expression in lung squamous cell carcinoma tissue and the clinical pathology of lung squamous cell carcinoma. Immunohistochemistry was used to detect PEBP4 expression in lung squamous cell carcinoma tissue and adjacent normal tissue from 61 patients. Western blotting was used to detect changes in the expression of PEBP4 protein between lung squamous cell carcinoma tissue and adjacent normal tissues. The correlation of PEBP4 expression and the occurrence, development, and clinical pathology of lung squamous cell carcinoma was analyzed. Of 61 patients, four patients were PEBP4 negative (-; 6.6%) and 57 patients were positive (+ to +++; 93.4%). Of those positive for PEBP4 expression, 7 patients were weakly positive (+; 11.5%), 21 patients were positive (++; 34.4%), and 29 patients were strongly positive (+++; 47.5%). PEBP4 protein was more highly expressed in lung squamous cell carcinoma tissue than in the adjacent normal lung tissue (p < 0.05). In PEBP4-positive patients, PEBP4 protein expression was significantly greater in those with lymph node metastases than in those without (p < 0.05). PEBP4 expression was significantly lower in patients at early (I and II) stages than in patients at advanced (III and IV) stages (p < 0.05). In less differentiated lung squamous cell carcinomas, PEBP4 protein expression was greater (p < 0.05); however, this was unrelated to the gender, age, or tumor size of the patient (p > 0.05). PEBP4 protein overexpression was associated with the occurrence, invasion, and metastasis of lung squamous cell carcinoma.

Background Hypoxia is the hallmark of the tumor microenvironment (TME) and plays a critical role during the progress of tumor development. A variety of microRNAs (miRNAs) transmitted by tumor-derived exosomes were involved in intercellular communication. We aimed to elucidate the precise mechanism by which tumor cell-derived exosomes promote lung cancer development by affecting macrophage polarization under hypoxic conditions. Methods CD163 signal in tumor tissue from lung cancer patients was detected by immunohistochemical (IHC). The M2 polarization-related markers were assessed by flow cytometry and western blot. Exosomes were isolated from normoxic and hypoxic lung cancer cell culture and characterized by transmission electron microscope (TEM), dynamic light scattering (DLS), and western blot. RNA sequencing was performed to show the abnormally expressed miRNAs in exosomes from normoxic and hypoxic lung cancer cell culture. In addition, CCK-8 and clone formation assays were used to assess cell proliferation. Dual luciferase reporter assay was used to evaluate the relationship between miR-21 and IRF1. For in vivo experiment, the male nude mice were injected with H1299 cells with exosomes and miR-21 mimic treatment. Results Firstly, we found a strong CD163 signal in tumor tissue from lung cancer patients by IHC. Subsequently, we co-cultured lung cancer cell line H1299 with M0 macrophage THP-1 and found that H1299 in a hypoxic environment promoted THP-1 M2 polarization. PKH67 fluorescence staining experiments confirmed that exosomes of H1299 origin were able to enter THP-1 and induced M2 polarization. RNA sequencing of exosomes showed that miR-21 level was significantly higher in the hypoxic culture group compared to the normoxic group. Subsequent cellular assays showed that miR-21 inhibited the expression of IRF1 by targeting it. In addition, the overexpression of IRF1 reversed the role of miR-21 on macrophage M2 polarization. Finally, we have confirmed through animal experiments that either hypoxic environment or high miR-21 level promoted tumor progression. Conclusions High miR-21 level in hypoxic environments promoted macrophage M2 polarization and induced lung cancer progression through targeting IRF1.

Background: The current National Comprehensive Cancer Network (NCCN) guidelines for non-small cell lung cancer (NSCLC) recommend that surgeons sample is not clear. We aimed to define a minimal number of examined lymph nodes for removal or sampling for optimized nodal staging recommendation, with a focus on T 1-3 N 0 M 0 patients. Methods: A total of 55,101 consecutive patients were selected, including 52,099 patients with US Surveillance, Epidemiology, and End Results (SEER) data and 3,002 patients in a Chinese multicenter database from 11 thoracic referral centers, who underwent complete resection plus lymph node dissection or sampling for stage T 1-3 N 0 M 0 NSCLC. Propensity score-matching analysis was performed with R software, and a cut-off value was calculated using X-tile software. Survival was evaluated using the Kaplan-Meier method and Cox proportional hazard models.Results: Five-year survival rates with respect to total examined lymph nodes numbers (examined lymph nodes <10 vs. examined lymph nodes ≥10) were 69% and 64% (group A), 66% and 63% (group B), 62% and 58% (group C), 81% and 75% (group D). There were significant differences between examined lymph nodes <10 and examined lymph nodes >10 in each group (P<0.001).Conclusions: A minimum of 10 examined lymph nodes would significantly improve T 1-3 N 0 M 0 NSCLC prognosis and patients' survival rates if implemented as a minimum standard for lymphadenectomy.Therefore, we recommended a minimum of 10 examined lymph nodes for T 1-3 N 0 M 0 patients.