Aspirin inhibits proliferation of gastric cancer cells via IL 6/STAT3 signaling pathway

Purpose: To study the effect of aspirin on the proliferation and apoptosis of gastric cancer cells, and its key molecular mechanism of action. Methods: Gastric cancer SGC7901 cells were treated with aspirin at concentrations of 0, 1, 2 and 4 mmol/L. Cell proliferation was measured using cell counting kit (CCK)-8 assay, while messenger ribonucleic acid (mRNA) expressions of interleukin (IL)-6, B-cell lymphoma 2 (Bcl-2) and Bcl-2 associated X protein (Bax) were assessed by reverse transcription-polymerase chain reaction (RT-PCR). Cell apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Furthermore, the protein expression levels of the signal transducer and activator of transcription 3 (STAT3), phosphorylated STAT3 (p-STAT3), Bcl-2 and Bax were evaluated by Western blotting. Results: Compared with control group, 1, 2 and 4 mmol/L aspirin groups showed lower cell proliferation, and decreased mRNA expressions of Bcl-2 and Bax and IL-6 release at 24, 48 and 72 h (p < 0.05). Cell apoptosis in the aspirin groups was higher than in the control group. Also, compared with the control group, 1 mmol/L aspirin group did not exhibit significant changes in the expressions of STAT3 and p-STAT3 at 72 h. On the other hand, the 2 mmol/L aspirin group at 72 h and the 4 mmol/L aspirin group exhibited significant increases in the expressions of STAT3 and p-STAT3 (p < 0.05). Furthermore, the levels of Bcl-2 and Bax declined in the aspirin groups when compared with the control group (p < 0.05). Conclusion: Aspirin inhibits the proliferation of gastric cancer SGC7901 cells, and induces their apoptosis in vitro in IL-6/STAT3 signaling pathway. The results of the current study may provide new insight into the treatment of gastric cancer

Identification and validation of novel biomarkers associated with immune infiltration for the diagnosis of osteosarcoma based on machine learning

Objectives: Osteosarcoma is the most common primary malignant tumor in children and adolescents, and the 5-year survival of osteosarcoma patients gained no substantial improvement over the past decades. Effective biomarkers in diagnosing osteosarcoma are warranted to be developed. This study aims to explore novel biomarkers correlated with immune cell infiltration in the development and diagnosis of osteosarcoma.Methods: Three datasets (GSE19276, GSE36001, GSE126209) comprising osteosarcoma samples were extracted from Gene Expression Omnibus (GEO) database and merged to obtain the gene expression. Then, differentially expressed genes (DEGs) were identified by limma and potential biological functions and downstream pathways enrichment analysis of DEGs was performed. The machine learning algorithms LASSO regression model and SVM-RFE (support vector machine-recursive feature elimination) analysis were employed to identify candidate hub genes for diagnosing patients with osteosarcoma. Receiver operating characteristic (ROC) curves were developed to evaluate the discriminatory abilities of these candidates in both training and test sets. Furthermore, the characteristics of immune cell infiltration in osteosarcoma, and the correlations between these potential genes and immune cell abundance were illustrated using CIBERSORT. qRT-PCR and western blots were conducted to validate the expression of diagnostic candidates.Results: GEO datasets were divided into the training (merged GSE19276, GSE36001) and test (GSE126209) groups. A total of 71 DEGs were screened out in the training set, including 10 upregulated genes and 61 downregulated genes. These DEGs were primarily enriched in immune-related biological functions and signaling pathways. After machine learning by SVM-RFE and LASSO regression model, four biomarkers were chosen for the diagnostic nomogram for osteosarcoma, including ASNS, CD70, SRGN, and TRIB3. These diagnostic biomarkers all possessed high diagnostic values (AUC ranging from 0.900 to 0.955). Furthermore, these genes were significantly correlated with the infiltration of several immune cells, such as monocytes, macrophages M0, and neutrophils.Conclusion: Four immune-related candidate hub genes (ASNS, CD70, SRGN, TRIB3) with high diagnostic value were confirmed for osteosarcoma patients. These diagnostic genes were significantly connected with the immune cell abundance, suggesting their critical roles in the osteosarcoma tumor immune microenvironment. Our study provides highlights on novel diagnostic candidate genes with high accuracy for diagnosing osteosarcoma patients

Targeted Genome-Scale Gene Activation and Gene Editing in Human Cells to Understand Disease Models

Since the discovery of sequence directed DNA editing reagents such as CRISPR-Cas9 RNA-guided and TALEN DNA endonucleases, there has been a snowball of advances in the life sciences due to the ability to efficiently edit and control genomes within living cells. CRISPR-Cas9 based genomic tools, which facilitate the high-throughput precise manipulation of genes, allow for unbiased functional genomic screens. We used a human CRISPR-Cas9 Synergistic Activation Mediator pooled library which utilizes an engineered protein complex for transcriptional activation of 23,430 endogenous genes to investigate the development of novel resistance mechanisms to lung cancer targeted therapy, Erlotinib. We set out to identify genes that when activated cause resistance to Erlotinib, with the ultimate aim to develop parallel therapies to systematically inhibit the pathways that these genes control or their product so as to prevent the evolution of drug resistance. Unlike current methods, these genes, when targeted, should not affect cancer cell metabolism, thereby decreasing the chances for cytotoxic effects. We have identified at least six potential candidate genes that could be targeted to prevent resistance to tyrosine kinase inhibitor, Erlotinib. In a separate study, we attempted to develop an isogenic (same genetic background besides the disease mutation) Huntington’s Disease (HD) human cell lines through TALEN mediated gene editing. Multiple cellular pathways have been implicated in HD pathogenesis, but normal function of the gene, essential for embryogenesis in mouse, has remained controversial. Moreover, the effects of genetic variation at other loci on the abnormal Huntingtin protein toxicity have been indicated, yet remain poorly studied. An isogenic set of HD cell lines should allow for an unbiased look into these effects. HEK293 cells were co-transfected with TALEN expression constructs, a reporter plasmid, and donor DNA with part of the mutant (high-CAG) HTT gene. The reporter plasmid allowed for selection of transfected clones (RFP+) and confirmation of nucleolytic activity in clones (eGFP+). RFP+ and eGFP+ cells were FACS-sorted into individual wells and subcultured. Trinucleotide Repeat Sizing analysis indicated the presence of high CAG allele at an estimated targeting frequency (without the use of any selectable marker) of 38% for the FACS selected cell lines