CircBCAR3 accelerates esophageal cancer tumorigenesis and metastasis via sponging miR-27a-3p.

RATIONALE: Circular RNAs (circRNAs) have been demonstrated to contribute to esophageal cancer progression. CircBCAR3 (hsa_circ_0007624) is predicted to be differentially expressed in esophageal cancer by bioinformatics analysis. We investigated the oncogenic roles and biogenesis of circBCAR3 in esophageal carcinogenesis. METHODS: Functions of circBCAR3 on cancer cell proliferation, migration, invasion, and ferroptosis were explored using the loss-of-function assays. A xenograft mouse model was used to reveal effects of circBCAR3 on xenograft growth and lung metastasis. The upstream and downstream mechanisms of circBCAR3 were investigated by bioinformatics analysis and confirmed by RNA immunoprecipitation and luciferase reporter assays. The dysregulated genes in hypoxia-induced esophageal cancer cells were identified using RNA-seq. RESULTS: CircBCAR3 was highly expressed in esophageal cancer tissues and cells and its expression was increased by hypoxia in vitro. Silencing of circBCAR3 repressed the proliferation, migration, invasion, and ferroptosis of esophageal cancer cells in vitro, as well as inhibited the growth and metastasis of esophageal xenograft in mice in vivo. The hypoxia-induced promotive effects on esophageal cancer cell migration and ferroptosis were rescued by circBCAR3 knockdown. Mechanistically, circBCAR3 can interact with miR-27a-3p by the competitive endogenous RNA mechanism to upregulate transportin-1 (TNPO1). Furthermore, our investigation indicated that splicing factor quaking (QKI) is a positive regulator of circBCAR3 via targeting the introns flanking the hsa_circ_0007624-formed exons in BCAR3 pre-mRNA. Hypoxia upregulates E2F7 to transcriptionally activate QKI. CONCLUSION: Our research demonstrated that splicing factor QKI promotes circBCAR3 biogenesis, which accelerates esophageal cancer tumorigenesis via binding with miR-27a-3p to upregulate TNPO1. These data suggested circBCAR3 as a potential target in the treatment of esophageal cancer. Hypoxia induces the upregulation of E2F7, which transcriptionally activates QKI in esophageal cancer cells. QKI increases the formation of circBCAR3 by juxtaposing the circularized exons. CircBCAR3 binds with miR-27a-3p to promote TNPO1 expression. CircBCAR3 promoted the proliferation, migration, invasion, and ferroptosis of esophageal cancer cells by miR-27a-3p.

[Construction of a Prognostic Prediction Model of Patients with Pathologic N0 
in Resected Invasive Mucinous Adenocarcinoma of the Lung].

BACKGROUND: Invasive mucinous adenocarcinoma (IMA) was a rare and specific type of lung adenocarcinoma, which was often characterized by fewer lymphatic metastases. Therefore, it was difficult to evaluate the prognosis of these tumors based on the existing tumor-node-metastasis (TNM) staging. So, this study aimed to develop Nomograms to predict outcomes of patients with pathologic N0 in resected IMA. METHODS: According to the inclusion criteria and exclusion criteria, IMA patients with pathologic N0 in The Affiliated Lihuili Hospital of Ningbo University (training cohort, n=78) and Ningbo No.2 Hospital (validation cohort, n=66) were reviewed between July 2012 and May 2017. The prognostic value of the clinicopathological features in the training cohort was analyzed and prognostic prediction models were established, and the performances of models were evaluated. Finally, the validation cohort data was put in for external validation. RESULTS: Univariate analysis showed that pneumonic type, larger tumor size, mixed mucinous/non-mucinous component, and higher overall stage were significant influence factors of 5-year progression-free survival (PFS) and overall survival (OS). Multivariate analysis further indicated that type of imaging, tumor size, mucinous component were the independent prognostic factors for poor 5-year PFS and OS. Moreover, the 5-year PFS and OS rates were 62.82% and 75.64%, respectively. In subgroups, the survival analysis also showed that the pneumonic type and mixed mucinous/non-mucinous patients had significantly poorer 5-year PFS and OS compared with solitary type and pure mucinous patients, respectively. The C-index of Nomograms with 5-year PFS and OS were 0.815 (95%CI: 0.741-0.889) and 0.767 (95%CI: 0.669-0.865). The calibration curve and decision curve analysis (DCA) of both models showed good predictive performances in both cohorts. CONCLUSIONS: The Nomograms based on clinicopathological characteristics in a certain extent, can be used as an effective prognostic tool for patients with pathologic N0 after IMA resection.

An esophagectomy surgical Apgar score-based nomogram-a risk-based postoperative triage system: a phase II randomised trial.

Background: The esophagectomy surgical Apgar score (eSAS) has been found to be a predictor of postoperative complications in esophagectomy. In our previous study, we built a graphic nomogram based on eSAS and demonstrated that it can effectively predict the risk of major morbidity after esophagectomy. In this study, we aimed to assess the benefits of using an eSAS-based nomogram model as a postoperative risk-based triage system for patients undergoing esophagectomy. Methods: We enrolled 119 patients diagnosed with esophageal carcinoma and randomly assigned them to a nomogram group (NG) or control group (CG) from January 2019 to December 2020. Patients in the NG were assigned to a low-risk group and high-risk group based on the nomogram. Patients in the high-risk group were admitted to the intensive care unit (ICU) after esophagectomy. Risk estimation in the CG patients was based on the surgeon's clinical experience. Thirty-day major complications, postoperative hospital stay, hospital costs, and quality of life (QOL) during the follow-up were compared between the two groups. Results: Baseline clinicopathological characteristics were comparable between the NG (n=58) and CG (n=61). All patients underwent esophagectomy. Postoperative complications were significantly higher in the CG (30, 49.2%) than in the NG (14, 24.1%) (P=0.008), with pneumonia being the most common (CG: 23, 37.7%; NG: 12, 20.7%; P=0.042). There was no significant difference in anastomotic leakage (NG: 1, 1.7%; CG: 6, 9.8%; P=0.12). Postoperative median hospital stay was shorter in the NG (14 days) than in the CG (16 days) (P=0.041). Hospital costs (NG: ¥60,045.1; CG: ¥63,961.5; P=0.21) and postoperative QOL did not differ significantly between groups. Conclusions: An eSAS-based nomogram as a triage system can reduce the overall occurrence of postoperative complications and shorten postoperative hospital stay without increasing hospital costs. Trial Registration: Chinese Clinical Trial Registry ChiCTR1900021636.

Evaluating the predictive significance of systemic immune-inflammatory index and tumor markers in lung cancer patients with bone metastases.

Objective: This study aims to develop a predictive model for identifying lung cancer patients at elevated risk for bone metastases, utilizing the Unified Immunoinflammatory Index and various tumor markers. This model is expected to facilitate timely and effective therapeutic interventions, especially in the context of the growing significance of immunotherapy for lung cancer treatment. Methods: A retrospective analysis was conducted on 324 lung cancer patients treated between January 2019 and January 2021. After meeting the inclusion criteria, 241 patients were selected, with 56 exhibiting bone metastases. The cohort was divided into a training group (169 patients) and a validation group (72 patients) at a 7:3 ratio. Lasso regression was employed to identify critical variables, followed by logistic regression to construct a Nomogram model for predicting bone metastases. The model's validity was ascertained through internal and external evaluations using the Concordance Index (C-index) and Receiver Operating Characteristic (ROC) curve. Results: The study identified several factors influencing bone metastasis in lung cancer, such as the Systemic Immune-Inflammatory Index (SII), Carcinoembryonic Antigen (CEA), Neuron Specific Enolase (NSE), Cyfra21-1, and Neutrophil-to-Lymphocyte Ratio (NLR). These factors were incorporated into the Nomogram model, demonstrating high validation accuracy with C-index scores of 0.936 for internal and 0.924 for external validation. Conclusion: The research successfully developed an intuitive and accurate Nomogram prediction model utilizing clinical indicators to predict the risk of bone metastases in lung cancer patients. This tool can be instrumental in aiding clinicians in developing personalized treatment plans, thereby optimizing patient outcomes in lung cancer care.

Prognostic value of an immunohistochemical signature in patients with pulmonary squamous cell carcinoma undergoing complete surgical resection.

Background: The Tumor Node Metastasis (TNM) stage cannot accurately predict the prognosis of patients in pulmonary squamous cell carcinoma (SQCC). The aim of the present study was to evaluate the prognostic value of immunohistochemical (IHC)-based classifiers in patients with pulmonary SQCC who underwent complete surgery resection. Methods: From January 2010 to December 2014, a total of 556 patients with SQCC who underwent complete radical resection were included. The patients were grouped into a discovery group (n=334) and a validation group (n=222). Using the least absolute shrinkage and selection operator (LASSO) regression model, we extracted IHCs that were associated with progression-free survival (PFS) and then built classifiers. Clinicopathological variables and the IHC-based classifiers were analyzed using univariable and multivariable logistic regression analyses. A nomogram to predict PFS was constructed and validated using bootstrap resampling. Results: Following the LASSO regression model, 4 IHC markers associated with PFS were identified. We used the IHC-based classifiers to stratify patients in both groups into high- and low-risk groups. PFS was better in the low-risk group than in the high-risk group in both the discovery and validation groups. Multivariate analysis demonstrated that the IHC-based classifiers were independently prognostic in predicting the PFS of patients with SQCC. The performance of the nomogram was evaluated and proven to be clinically useful. Conclusions: By combining IHC-based classification and clinicopathology, we were able to have better insight into the prognostic assessment of patients with SQCC after surgery, which can inform postoperative patient management.

To explore the prognostic value of spread through air spaces and develop a nomogram combined with spread through air spaces in lung squamous cell carcinoma.

Background: Spread through air spaces (STAS), as a new pattern of invasion, was officially proposed by the World Health Organization (WHO) in 2015, and its importance was reiterated in 2021. Since it was proposed, numerous studies have confirmed that STAS is associated with poor prognosis, but there are relatively few studies related to the prognosis of postoperative patients with lung squamous cell carcinoma. By synthesizing different predictive variables, nomogram can obtain the individual digital probability of clinical events succinctly and intuitively, which can meet our needs for personalized medicine. In this study, our goal is to explore the prognostic value of STAS in lung squamous cell carcinoma and establish a prognostic prediction model. Methods: Under six inclusion criteria, 540 postoperative patients were enrolled in the study. STAS was re-evaluated by pathologists at Ningbo Clinicopathological Diagnosis Center. Progression-free survival (PFS) referred to the time from randomization to the first occurrence of disease progression or death of any cause. Recurrences were confirmed by clinical, radiological or pathological assessment. The survival information was collected by telephone and outpatient follow-up. A total of 540 patients were randomly divided into groups in a 6:4 ratio for survival analysis to determine the correlation between STAS and prognosis. Cox regression was used for univariate and multivariate analysis, so as to establish a predictive model. The assessment of the nomogram was carried out by receiver operating characteristic (ROC) curve analysis, calibration curve analysis and decision curve analysis (DCA). Results: The overall 5-year survival rate was 70.35% in STAS-negative patients and 42.35% in STAS-positive patients. In all cohorts, STAS-negative patients had longer 5-year PFS than STAS-positive patients (P<0.001). Multivariate analysis showed that stage [stage II P=0.008, hazard ratio (HR) =1.792; stage III P<0.001, HR =3.148], tumor differentiation (well-differentiation P=0.021, HR =0.436), and STAS (P=0.026, HR =1.470) were independent predictors of PFS. The area under the curve (AUC) of model 5 in discovery cohort is 0.720, while the AUC of model 5 in validation cohort is 0.693. Conclusions: The nomogram combined with STAS can provide a personalized visual survival probability prediction map for postoperative patients with lung squamous cell carcinoma, so as to aid in the pursuit of personalized medicine to a certain extent.

circRNA TCFL5 Promote Esophageal Cancer Progression by Modulating M2 Macrophage Polarization via the miR-543-FMNL2 Axis.

Objective: The mechanism of circRNA on M2 macrophage polarization, which contributes to esophageal cancer, remains unclear. This study is aimed at clarifying the mechanism of circRNA on esophageal cancer by regulating M2 macrophage polarization. Methods: The expression of circRNA TCFL5 and miR-543 was detected by qRT-PCR. Western blot was used to detect the expression of FMNL2 and CD163. CCK-8 and transwell assay was used to detect the proliferation, migration, and invasion of Eca109 and KYSE150, respectively. Flow cytometry was used to detect the CD163 positive cells. The contents of IL-10, TGF-ß, TNF-α, IL-6, and IL-1ß were detected by ELISA. A dual-luciferase reporter system was used to detect the regulation of miR-543 to circRNA TCFL5 and FMNL2. Results: 156 upregulated circRNAs and 91 downregulated circRNAs in esophageal cancer tissues were identified, and the expression of circRNA TCFL5 showed the most significant upregulation. Overexpression of circRNA TCFL5 promotes proliferation, invasion, and migration of Eca109 and KYSE150 and promotes tumor growth in vivo. circRNA TCFL5 served as a sponge of miR-543, and FMNL2 was a downstream target gene of miR-543. circRNA TCFL5 promotes cell proliferation, migration, and invasion of Eca109 and KYSE150 by modulating the miR-543/FMNL2 axis. Macrophage M2 polarization promoted proliferation, invasion, and migration of Eca109 and KYSE150 cells, and circRNA TCFL5 mediated macrophage M2 polarization by regulating the FMNL2/miR-543 axis. Conclusion: In the present study, we identified that circRNA TCFL5 was dramatically upregulated in esophageal cancer, and circRNA TCFL5 promotes esophageal cancer progression by modulating M2 macrophage polarization via the miR-543-FMNL2 axis, which provides a potential target for the treatment of esophageal cancer.

Expression of anti-apoptosis genes alters lactate metabolism of Chinese Hamster Ovary cells in culture.

In an effort to develop robust Chinese Hamster Ovary host cell lines, a variety of anti-apoptotic genes were over-expressed, either singly or in combination, followed by screening of transfectants for improved cell growth, extended longevity, reduced caspase 3/7 activity, and enhanced mitochondrial membrane potential (MMP). Two particular cell lines, one containing two anti-apoptotic genes, E1B-19K and Aven (EA167), and another containing three, E1B-19K, Aven, and a mutant of XIAP (EAX197), exhibited a reduction in caspase 3 activity of at least 60% and a 170% enhancement in mitochondrial membrane potential compared to controls when treated with staurosporine. In batch cell growth experiments, the peak viable cell densities and viabilities were higher resulting in a 186% increase in integrated viable cell densities. Analyses of metabolite utilization and formation of waste products indicated that the apoptotic resistant cell lines depleted all the lactate when grown in commercially available CD-CHO medium while significant levels (>1.8 g/L) accumulated in the host cell lines. When the lactate level was replenished daily in the apoptotic resistant cell lines, the cell lines consumed lactate and the culture longevity was extended up to four additional days compared to control cell lines. Furthermore, the anti-apoptosis cell lines also accumulated lower levels of ammonia. The ability of the apoptotic resistant cell lines to consume lactate was exploited by cultivating them in a "high" glucose medium containing 15 g/L (60 mM glucose) in which apoptotic resistant cell lines exhibited lower maximum lactate (1.8 g/L) compared to control cell lines which accumulated concentrations of lactate (2.2 g/L) that appeared to be deleterious for growth. The shaker flask titer of a therapeutic antibody product expressed in an apoptotic resistant cell line in "high" glucose medium reached 690 mg/L compared to 390 mg/L for a cell line derived from a control host cell line. These results represent to our knowledge the first example in the literature in which manipulation of the apoptosis pathway has altered the nutrient consumption profile of mammalian cells in culture; findings that underscore the interdependence of the apoptotic cellular machinery and metabolism and provide greater flexibility to mammalian bioreactor process development.

Caspase activation in equine influenza virus induced apoptotic cell death.

Equine influenza virus (EIV) is the leading cause of acute respiratory infection in horses worldwide. In recent years, the precise mechanism by which influenza infection kills host cells is being re-evaluated. In this report, we examined whether caspases, a group of intracellular proteases, are activated following EIV infection and contribute to EIV-mediated cell death. Western blotting analysis indicated that a nuclear target of caspase-3, poly(ADP-ribose) polymerase (PARP) was proteolytically cleaved in EIV-infected MDCK cells, but not in mock-infected cells. In comparison with caspase-3 specific inhibitor Ac-DEVD-CHO, a general caspase inhibitor Boc-D-FMK provided much stronger inhibition of EIV-induced cytopathic effect and apoptosis. Our results suggest that EIV may activate more than one caspase. Caspase activation and cleavage of its cellular targets may play a critical role in EIV-mediated cytotoxicity.

Infectivity of equine H3N8 influenza virus in bovine cells and calves.

BACKGROUND: Serological evidence for influenza A, subtype H1 and H3 virus infections of bovines, associated with respiratory disease and decreased milk production, has been reported. Equine H3N8 influenza virus circulates widely and was responsible for the introduction of H3N8 influenza into canines. OBJECTIVE: To explore the possibility that equine H3N8 influenza might also infect bovines. METHODS: To assess the incidence of seroconversion in the field, a retrospective survey of bovine serum samples was carried out. Also, primary cultures of bovine nasal turbinate cells, and live beef calves, were studied for their permissiveness to infection. RESULTS AND CONCLUSIONS: We found serological evidence of exposure of bovines in Kentucky to H3 influenza. We demonstrate that cultured bovine respiratory epithelium is permissive for the growth of equine H3N8 influenza virus in vitro, but this virus does not replicate extensively or produce disease in experimentally inoculated cattle.

Combining high-throughput screening of caspase activity with anti-apoptosis genes for development of robust CHO production cell lines.

A set of anti-apoptotic genes were over-expressed, either singly or in combination, in an effort to develop robust Chinese Hamster Ovary host cell lines suitable for manufacturing biotherapeutics. High-throughput screening of caspase 3/7 activity enabled a rapid selection of transfectants with reduced caspase activity relative to the host cell line. Transfectants with reduced caspase 3/7 activity were then tested for improved integrated viable cell count (IVCC), a function of peak viable cell density and longevity. The maximal level of improvement in IVCC could be achieved by over-expression of either single anti-apoptotic genes, e.g., Bcl-2Δ (a mutated variant of Bcl-2) or Bcl-XL, or a combination of two or three anti-apoptotic genes, e.g., E1B-19K, Aven, and XIAPΔ. These cell lines yielded higher transient antibody production and a greater number of stable clones with high antibody yields. In a 5 L fed-batch bioreactor system, BΔ31-1, a stable clone expressing Bcl-2Δ, had a product titer that was 180% as compared to an optimal clone (Con-1) from the control cell line. Although lactate accumulated to more than 5 g/L in the control culture, its concentration was reduced in the anti-apoptotic BΔ31-1 cultures to below 1 g/L, confirming our earlier findings that cells over-expressing anti-apoptotic genes consume the lactate that would otherwise accumulate as a by-product in the culture medium. To the best of our knowledge, this is the first study to use the high throughput caspase screening method to identify CHO host cell lines with superior anti-apoptotic characteristics.