Molecular subtyping based on immune cell marker genes predicts prognosis and therapeutic response in patients with lung adenocarcinoma.

OBJECTIVE: Lung adenocarcinoma (LA) is one of the most common malignancies and is responsible for the greatest number of tumor-related deaths. Our research aimed to explore the molecular subtype signatures of LA to clarify the correlation among the immune microenvironment, clinical outcomes, and therapeutic response. METHODS: The LA immune cell marker genes (LICMGs) identified by single-cell RNA sequencing (scRNA-seq) analysis were used to discriminate the molecular subtypes and homologous immune and metabolic traits of GSE72094 LA cases. In addition, the model-building genes were identified from 1441 LICMGs by Cox-regression analysis, and a LA immune difference score (LIDscore) was developed to quantify individual differences in each patient, thereby predicting prognosis and susceptibility to immunotherapy and chemotherapy of LA patients. RESULTS: Patients of the GSE72094 cohort were divided into two distinct molecular subtypes based on LICMGs: immune activating subtype (Cluster-C1) and metabolically activating subtype (cluster-C2). The two molecular subtypes have distinct characteristics regarding prognosis, clinicopathology, genomics, immune microenvironment, and response to immunotherapy. Among the LICMGs, LGR4, GOLM1, CYP24A1, SFTPB, COL1A1, HLA-DQA1, MS4A7, PPARG, and IL7R were enrolled to construct a LIDscore model. Low-LIDscore patients had a higher survival rate due to abundant immune cell infiltration, activated immunity, and lower genetic variation, but probably the higher levels of Treg cells in the immune microenvironment lead to immune cell dysfunction and promote tumor immune escape, thus decreasing the responsiveness to immunotherapy compared with that of the high-LIDscore patients. Overall, high-LIDscore patients had a higher responsiveness to immunotherapy and a higher sensitivity to chemotherapy than the low-LIDscore group. CONCLUSIONS: Molecular subtypes based on LICMGs provided a promising strategy for predicting patient prognosis, biological characteristics, and immune microenvironment features. In addition, they helped identify the patients most likely to benefit from immunotherapy and chemotherapy.

[Expression of transcription factor SOX4 and its clinical significance in female lung cancer patients in Xuanwei area, Yunnan Province].

OBJECTIVE: To investigate the expression of transcription factor SOX4 in lung cancer tissues of female patients in Xuanwei area, Yunnan Province, and explore its correlation with clinicopathological characteristics and prognosis of the female patients. METHODS: Real-time PCR was applied on lung cancer specimens and their corresponding normal lung tissues from 96 female cases of Xuanwei area to assess the expression of SOX4 mRNA. Immunohistochemical staining was performed to investigate the SOX4 protein expression, and further to elucidate its correlation with clinicopathological characteristics and prognosis. RESULTS: The expression level of SOX4 mRNA in the cancer tissues (2.53 ± 1.65) was significantly higher than that of matched normal tissues (1.43 ± 1.14, P = 0.003). Immunohistochemical staining showed that there were 53.1% (51/96) positive expression of SOX4 protein in the cancer tissue and only 26.0% (25/96) in matched normal tissue (P < 0.001). The expression of SOX4 protein had a significant correlation with clinical stage, lymph node metastasis and differentiation of tumor (P < 0.05). The survival analysis by Kaplan-Meier method showed that patients with positive expression of SOX4 protein, lymph node metastasis and advanced tumor stage had a significantly shorter median survival time (P < 0.05). Cox regression survival analysis showed that pathological grade was a significant independent factor affecting prognosis. CONCLUSIONS: The expressions of SOX4 mRNA and protein are significantly up-regulated in Xuanwei female lung cancer patients. Patients with positive SOX4 expression have a shorter median survival time. SOX4 protein expression level combined with pathological grade can be used as a prognostic indicator of female lung cancer patients in Xuanwei area, Yunnan Province.

[Relationship between high incidence of lung cancer among non-smoking women and silica in C1 bituminous coal in Xuanwei, Yunnan Province, China].

OBJECTIVE: To measure the content of silica in C1 bituminous coal and its combustion products in the high-incidence area of lung cancer in Xuanwei, Yunnan Province, China and to investigate the relationship between high incidence of lung cancer among non-smoking women and silica produced naturally in C1 bituminous coal in Xuan Wei. METHODS: The C1 bituminous coal widely used in the high-incidence area of lung cancer in Xuanwei was selected as experiment group, while the C2+1, K7, and M30 bituminous coal that was mined and used in the low-incidence area of lung cancer in Xuanwei for more than 10 years were selected as control group. Fourteen paraffin-embedded cancer tissue samples from the non-smoking women with non-small cell lung cancer who were born in Xuanwei and were at least the 3rd generation of the family living there were collected from the department of pathology, the third affiliated hospital of kunming medical university (tumor hospital of yunnan province). Titrimetric potassium silicofluoride method was used to measure the content of silica in raw coal and its bottom ashes in 20 samples from the experimental group and control group. Scanning electron microscopy (SEM) was used to observe the morphology of silica particles in C1 bituminous coal and its bottom ashes, and scanning electron microscopy coupled with energy dispersive X-ray analyzer (SEM-EDX) was used to analyze the microscopic composition. Transmission electron microscope (TEM) was used to observe the morphology of silica particles in the bottom ashes and coal soot of C1 bituminous coal as well as the lung cancer tissue from the non-smoking women in Xuanwei, and transmission electron microscope coupled with energy dispersive X-ray analyzer (TEM-EDX) was used to analyze the microscopic composition. The silica particles were separated from the coal soot and bottom ashes and characterized by physical method. RESULTS: The silica content in C1 bituminous coal and its bottom ashes was significantly higher than that in C2+1, K7, and M30 bituminous coal (P < 0.05). The bottom ashes of C1 bituminous coal contained a large quantity of silica particles, mostly with microscale sizes. Silica particles were found in the soot of C1 bituminous coal and the lung cancer tissue from non-smoking women in Xuanwei. The silica particles in the bottom ashes were mostly 120 ∼ 500 nm in diameter, had various shapes, and contained such elements as iron, aluminium, calcium, and potassium; the silica particles in the coal soot were mostly nanoscale, ranging from 37 nm to 80 nm in diameter, had various shapes, with some in fibrous form, had non smooth surfaces, and contained such elements as iron, potassium, calcium, aluminium, and sulfur. CONCLUSION: In Xuanwei, the incidence of lung cancer among non-smoking women is high in the area where silica-rich C1 bituminous coal is produced. There are silica particles enriched in both the combustion products (coal soot and bottom ashes) of C1 bituminous coal and the cancer tissue from the non-smoking women with non-small cell lung cancer, with similar morphology and microscopic composition. We hypothesize that the silica particles from combusted C1 bituminous coal in Xuanwei are mixed with indoor air and inhaled along with other suspended particles.

The influences of TGF-ß1 upon the human adenocarcinoma cell of lung A549 and cellular immunity.

BACKGROUND: The cellular immunity of lung cancer patients is mainly the immune response of T cells, which plays an important role in tumour cell killing and immune surveillance. Transforming growth factor 1 (TGF-ß1) is secreted by tumour cells that can suppress the immune response and is an important group of immune down-regulation factors. Our study aims to investigate the effect of TGF-ß1 on the morphology and cellular immune function of A549 and peripheral blood mononuclear cells (PBMCs). METHODS: A549 cell line was cultured, PBMCs were cultured with different concentrations of TGF-ß1, and the morphology of A549 cells and PBMCs were seen. The levels of interleukin (IL)-2, IL-4, IL-6, IL-10, IFN-γ, and TNF and the numbers of CD3, CD4, CD8, CD4/CD8, and CD3 CD25 and CD4 CD25 in PBMCs were detected. RESULTS: During co-culture of A549 with PBMCs, TGF-ß1 can induced A549 showing epithelial-to-mesenchymal transition, enhanced its ability of migration and infiltration. Simultaneously, TGF-ß1 can depressing the growth and proliferation of PBMCs, inhibiting T-cell activation, and accelerating the PBMCs apoptosis. TGF-ß1 can inhibits A549 Th1 related-cytokines, enhance Th2 related-cytokines, cause the disorder of Th1/Th2, resulting in the Th1 cellular dominate immunity decline. CONCLUSIONS: TGF-ß1 may affect the secretion of related cytokines, hinder the activation of T lymphocytes, destroy the immune surveillance and killing effect of the body, and thus inhibit the cellular immunity.

3-(4-Amino-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)pyridinium chloride.

In the title compound, C(7)H(8)N(5)S(+)·Cl(-), the dihedral angle formed by the pyridine ring with the triazole ring is 10.0 (1)°. There are weak inter-molecular hydrogen-bond inter-actions in the crystal structure, involving the NH and NH(2) groups as donors, and the chloride anion, the S atom in the thio-ketone group and the unsubstituted ring N atom as acceptors.

[Expression profile of miR-501-5p in lung adenocarcinoma patients from Xuanwei area].

OBJECTIVE: To investigate the relationship between miR-501-5p expression and the clinicopathological factors in patients with lung adenocarcinoma in Xuanwei area. METHODS: Surgical specimens of lung adenocarcinoma and paired adjacent tissues from 24 patients with lung adenocarcinoma from Xuanwei area were examined for miR-501-5p expression using microRNA microarray technique and qPCR. Chi-square test was used to analyze the association of miR-501-5P expression with the clinicopathological characteristics of the patients. Multiple regression analysis was performed to analyze the association of miR-501-5p expression with the patients' gender, age, tumor stage, and preoperative CEA level. RESULTS: MicroRNA microarray analysis and qPCR validation results revealed significantly upregulated expressions of miR-501-5p in patients with lung adenocarcinoma from Xuanwei area (Plt;0.01). The microarray data showed an up-regulation of miR-501-5p by 3.17 folds in lung adenocarcinoma tissue compared with the adjacent tissue (P=0.22376, FDR=0.071395). Chi-square test indicated that miR-501-5p expression level was associated with the patients' age (f=7.168, P=0.014), TNM stage (f=36.627, P<0.01), and preoperative serum CEA level (f=30.045, Plt;0.01), but not with the patients' gender (f=3.612, P=0.071). Multiple regression analysis revealed that miR-501-5p expression was positively correlated with the patients' age, TNM stage of the tumor, and serum CEA (Plt;0.05). CONCLUSION: miR-501-5p expression is up-regulated in lung adenocarcinoma with significant associations with the patients' age, TNM stages and serum CEA level in patients from Xuanwei area, suggesting its potential role in the tumorigenesis and progression of lung adenocarcinoma in Xuanwei area.

Effect of miR-1244 on cisplatin-treated non-small cell lung cancer via MEF2D expression.

The aim of this study was to investigate the function of miR-1244 in cisplatin-treated non-small cell lung cancer (NSCLC). The results of quantitative PCR analysis revealed that the expression levels of miR-1244 in cisplatin­treated A549 and NCI-H522 human lung cancer cell lines were lower than those in untreated A549 and NCI-H522 cells. Similarly, the expression level of miR-1244 in NSCLC tissue samples from cisplatin-treated patients was also lower than that in non-cisplatin-treated NSCLC patients. Notably, the overall survival times of cisplatin-treated NSCLC patients with high miR-1244 expression were superior to those patients with low miR-1244 expression. We found that overexpression of miR-1244 suppressed cell viability and increased LDH toxicity in cisplatin-treated A549 and NCI-H522 cells. Additionally, overexpression of miR-1244 induced the apoptosis of cisplatin-treated A549 and NCI-H522 cells. Furthermore, overexpression of miR-1244 promoted caspase-3 activity and p53 and Bax protein expression, and suppressed myocyte enhancer factor 2D (MEF2D) and cyclin D1 protein expression in cisplatin­treated A549 and NCI-H522 cells. Small interfering RNA (siRNA) targeting MEF2D suppressed the protein expression of MEF2D, and was able to decrease the proliferation, promote caspase-3 activity, p53 and Bax protein expression and inhibit cyclin D1 protein expression in cisplatin-treated A549 and NCI-H522 cells following the overexpression of miR-1244. In summary, we found that miR-1244 affected cisplatin-treated NSCLC via MEF2D expression.

[Specific microRNA expression profiles of lung adenocarcinoma in Xuanwei region and bioinformatic analysis for predicting their target genes and related signaling pathways].

OBJECTIVE: To identify differentially expressed microRNAs (miRNAs) related to lung adenocarcinoma in Xuanwei region and predict their target genes and related signaling pathways based on bioinformatic analysis. METHODS: High-throughput microarray assay was performed to detect miRNA expression profiles in 34 paired human lung adenocarcinoma and adjacent normal tissues (including 24 cases in Xuanwei region and 10 in other regions). Gene ontology and KEGG pathway analyses were used to predict the target genes and the regulatory signaling pathways. RESULTS: Thirty-four miRNAs were differentially expressed in lung adenocarcinoma tissues in cases in Xuanwei region as compared with cases in other regions, including 23 upregulated and 11 downregulated miRNAs. The predicted target genes included GF, RTK, SOS, IRS1, BCAP, CYTOKINSR, ECM, ITGB, FAK and Gbeta;Y involving the PI3K/Alt, WNT and MAPK pathways. CONCLUSION: The specific microRNA expression profiles of lung adenocarcinoma in cases found in Xuanwei region allow for a better understanding of the pathogenesis of lung adenocarcinoma in Xuanwei. The predicted target genes may involve the PI3K/Alt, WNT and MAPK pathways.

Study on the Structure of Candida Albicans-Staphylococcus Epidermidis Mixed Species Biofilm on Polyvinyl Chloride Biomaterial.

The objective of the study was to establish an in vitro model of Candida albicans-Staphylococcus epidermidis mixed species biofilm (BF) on polyvinyl chloride (PVC) material, and to investigate the formation and the structure of mixed species BF formation using a combined approach of confocal laser scanning microscope (CLSM), scanning electron microscope (SEM), and 3D image reconstruction technique. Mixed species BF is achieved by co-incubating Staphylococcus epidermidis bacteria (ATCC35984) and Candida albicans fungal (ATCC10231) with PVC pieces in Tris-buffered saline. BF formation was examined at 2, 6, 12, 24, 48, and 72 h of co-culture. Thickness of these BFs and the number, and percentage of viable cells in BFs were measured. CT scan images of BFs were obtained using CLSM and SEM and reconstructed 3D images of mixed species BF were acquired, in an effort to examine structure of the BF. Staphylococcus epidermidis attached to various forms of candida albicans (spores, pseudohyphae, and hyphae), formed complex and dense mesh arrays. The BF is constituted of a large number of viable and dead pathogens, the surface of mixed species BF is uneven, with living pathogens predominating protrusive portions and dead pathogens aggregating in concaves. Mixed species BF formation on the surface of PVC material was found to be a dynamic process, with rapid growth being at 24 h of co-culture, maximal thickness peaked at 48 h. These mixed species BF matured at 48-72 h. Significant differences were observed in the proportion of viable cells between interior, middle, and outer layers of BFs (p < 0.05). Mixed species BF Candida albicans-Staphylococcus epidermidis is sophisticated in structure. The combined approach involving CLSM, SEM, and 3D image reconstruction technique is ideal for the investigation of mixed species BF on PVC material.

Establishment of rat model of central venous catheter (CVC): associated infection and evaluation of the virulence of bacterial biofilms.

In this study, a central venous catheter (CVC)-associated infection model was established in rats to investigate and evaluate the effect of biofilms on the virulence of the pathogens. Twenty-four adult SD rats were randomly divided into biofilm positive (BF+) and biofilm negative (BF-) groups to be challenged with strains of S.epidermidis. Serum levels of inflammatory cytokines were measured and the infection rate and counts of bacteria cells were studied. Compared to rats of BF- group, the serum level of TNF and IL-6 significantly increased in rats of BF+ group (P < 0.01) and the level of IL-10 and IFN-γ significantly decreased (P < 0.01), striking the balance of pro-inflammatory/anti-inflammatory cytokines. The infection rate and bacterial counts in tissues and blood of rats of BF + group were significantly higher than those of rats of BF- group (P < 0.05).Inflammatory cell infiltration in vital organs (heart, lung, liver and kidneys) was more significant in rats of BF+ group than that of rats of BF- group. CVC-associated infection model can be successfully reproduced in rats by injecting 5 × 10(6) CFU of S.epidermidis. Biofilm formation can significantly enhance the virulence of the bacteria, leading to uncontrolled infection. The serum level of inflammatory cytokines, infection rate and the extent of inflammatory cell infiltration are important markers for evaluating the virulence of biofilm.

In vitro model of bacterial biofilm formation on polyvinyl chloride biomaterial.

The aim of the study was to establish an in vitro model of Staphylococcus epidermidis biofilms on polyvinyl chloride (PVC) material, and to investigate bacterial biofilm formation and its structure using the combined approach of confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM). Staphylococcus epidermidis bacteria (stain RP62A) were incubated with PVC pieces in Tris buffered saline to form biofilms. Biofilm formation was examined at 6, 12, 18, 24, 30, and 48 h. Thicknesses of these biofilms and the number, and percentage of viable cells in biofilms were measured. CT scan images of biofilms were obtained using CLSM and environmental SEM. The results of this study showed that Staphylococcus epidermidis biofilm is a highly organized multi-cellular structure. The biofilm is constituted of large number of viable and dead bacterial cells. Bacterial biofilm formation on the surface of PVC material was found to be a dynamic process with maximal thickness being attained at 12-18 h. These biofilms became mature by 24 h. There was significant difference in the percentage of viable cells along with interior, middle, and outer layers of biofilms (P < 0.05). Staphylococcus epidermidis biofilm is sophisticated in structure and the combination method involving CLSM and SEM was ideal for investigation of biofilms on PVC material.

WITHDRAWN: Fem1b interacts with Ankrd37 in mouse testis and induces its degradation by ubiquitin-mediated proteolysis pathway.

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