Ethics statement
This study was approved by the Academic Committee of Wuxi 9Th People's Hospital Affiliated to Soochow University and conducted according to the ethical standards formulated in the Helsinki Declaration. It was confirmed that all written informed consents were obtained as all data sets were retrieved from published literatures.
Classification and gene structure of TBX2 subfamily
The HUGO Gene Nomenclature Committee (HGNC) assigns unique symbols and names to human genes based on the European Bioinformatics Institute (EMBL-EBI). In current, the HGNC database not only integrates more than 40000 approved gene symbols, but also curates genes into family sets based on shared characteristics such as homology, function or phenotype. Herein, we retrieved basic information of Tbx2 subfamily from the HGNC database (http://www.genenames.org/cgi-bin/genefamilies/set/766). TBX2 subfamily contains four members, including TBX2, TBX3, TBX4 and TBX5. Then, we retrieved detailed information about Tbx2 subfamily through the search terms of TBX2/homo, TBX3/homo, TBX4/homo and TBX5/homo from the NCBI (National Center for Biotechnology Information) database. Finally, graphic mode of structure of Tbx2 subfamily transcription products including coding region, untranslated coding region (UTR) and T-BOX domain was drawn with software.
UALCAN Analysis
UALCAN gene expression array datasets is a cancer microarray database and online data-mining platform which aimed at facilitating discovery from genome-wide expression analysis . Herein, we use this database to analyze the transcriptional level of TBX2 subfamily genes in different types of cancers. We compared the expression level of TBX2 subfamily in clinical cancer specimens with that in normal controls and used Student’s t test for result analysis. The cutoff of P value and fold change were defined as 0.01 and 1.5, respectively. Gene rank: 10%, data type: mRNA. We also performed protein expression analysis of the TBX2 subfamily in lung squamous cell carcinoma and lung adenocarcinoma compared to normal lung tissue using the protein expression module of the UALCAN database.
Immunohistochemistry (IHC) Staining
To evaluate differences in TBX2 subfamily expression at the protein level, IHC images of TBX2 subfamily protein expression in normal tissues and Lung Squamous Cell Carcinoma were downloaded from the The Human Protein Atlas (http://www.proteinatlas.org/) and analyzed.
Cell culture
H-226, H-1792, Beas-2B, and HepG2 cell lines are gifts donated by the Clinical Medical College of Suzhou University. All the above-mentioned cells were cultured in an environment with a temperature of 37℃ and a gas environment of 5% CO2. All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture (HyClone, USA).
Western blotting analysis
To prepare protein lysate, cells were lysed with RIPA lysis buffer supplemented with protease inhibitors to reduce protein degradation. The lysate was maintained at an icy temperature during the entire protein extraction process to uphold sample integrity. The proteins were then extracted by centrifugation of the lysate at 1400g for 15 minutes under 4℃. The protein concentration of the extracted proteins was quantified with the BCA protein assay kit. After protein extraction, SDS-PAGE gel electrophoresis was performed to separate the protein, which was then transferred onto a PVDF membrane. The PVDF membrane was shaken in 5% skim milk, and then blocked overnight at 4℃ in a wet box containing primary antibodies against TBX2, TBX3, TBX4 TBX5 and GAPDH.On the following day, after 1 hour of incubation at room temperature with secondary antibodies, analysis was conducted using an AFP imaging system.
Kaplan-Meier plotter
The Kaplan-Meier plotter (http://kmplot.com/analysis/) enables to assess the effect of any gene or gene combination on survival in various types of tumors using over 50,000 samples measured with gene arrays, RNA-seq or next generation sequencing . The Kaplan-Meier plotter (K-M plotter) is capable of assessing the effect of up to 54k genes on survival of 21 cancer types and the largest datasets contain breast (n=6,234), lung (n=3,452), ovarian (n=2,190), and gastric (n=1,440) cancer. Herein, we used the K-M plotter to evaluate the prognostic value of mRNA expression of distinct TBX2 subfamily genes in lung cancers. In K-M plotter, we divided cancer participants into low and high expression group according to median values of TBX2 subfamily mRNA expression. The prognostic value of TBX2 expression was validated by K-M survival curves. Information about the number-at-risk cases, median values of mRNA expression levels, HRs, 95% CIs and p-values is all available on the K-M plotter website. P-value <0.05 was considered statically significant.
DNA methylation database
MethHC was a database for human cancer gene expression and methylation . The correlation between TBX2 subfamily expression and its methylation were confirmed by the Pearson correlation coefficients. P<0.05 has statistical significance. DiseaseMeth 2.0 is a database that aims to provide the most complete collection and annotation of abnormal DNA methylation in human diseases, especially in various cancers. The database includes 175 large-scale methylation datasets of 88 diseases and more than 14000 accurate experimental information extracted from PubMed .