Differential expression of Mad2 gene is consequential to the patterns of histone H3 post-translational modifications in its promoter region in human esophageal cancer samples

Raw areca nut (AN) consumption increases esophageal squamous cell carcinoma (ESCC) due to overexpression of securin (pituitary tumor transforming gene1), causing chromosomal instability. Mitotic arrest deficient protein 2 (Mad2), a crucial spindle assembly checkpoint protein, is at risk of aneuploidy and tumor development when overexpressed or underexpressed. This study evaluates Mad2 status in human ESCC with AN consumption habits, revealing unclear molecular mechanisms. Human ESCC samples (n = 99) were used for loss of heterozygosity analysis at 4q25-28, while 32 samples were used for expression analysis of Mad2, E2F1 genes, and Rb-phosphorylation. Blood samples were used for metaphase preparation. The Mad2 deregulation was assessed using chromatin immunoprecipitation-qPCR assay in the core promoter region, establishing its association with the pRb-E2F1 circuit for the first time. The study revealed overexpression and underexpression of Mad2, premature anaphase, and chromosome missegregation in all the samples. LOH pattern identified a deletion in D4S2975 in 40% of ESCC samples. The study reveals the deregulation of pRb-E2F1 circuit in all samples. 4q27 disruption could be a factor for Mad2 underexpression in AN-induced esophageal carcinogenesis, while overexpression may be due to the deregulation of the Rb-E2F1 circuit and consequently elevation of H3K4me3 and H3K9ac. Mad2 expression levels with chromosomal abnormalities can be a clinical biomarker, but further research is needed to understand pRb’s role in Mad2 down-regulation.


Patients and tissue samples
The detail information on patients from whom the samples were collected is given in the Supplementary Table 1.

Quantitative real-time PCR
The primers of target genes used for this analysis were Mad2, and GAPDH (as the reference gene).The primer sequences are listed in Supplementary Table 2.

Immunohistochemistry (IHC)
Formalin fixed, paraffin embedded tissues were sectioned (5 μm thickness) for IHC staining.Slides were deparaffinized, rehydrated and treated with PBS at 4°C for 5 min, with 3% hydrogen peroxide added to inhibit intrinsic peroxidase activities.Antigen retrieval was done with 0.01% sodium-citrate buffer followed by blocking in PBST containing 0.1% BSA and 10% FBS.The samples were reacted with primary antibody (1:100) for overnight and rinsed with PBS.Slides were incubated with appropriate biotinylated secondary antibody for 1hr at room temperature.Following washing, slides were treated with streptavidin-HRP (1:1000) and subsequently washed (PBS containing 0.1% Tween-20) and color was developed with DAB+H 2 O 2 .Slides were counterstained with haematoxylin, washed and mounted in DPX (Sigma-Aldrich, USA).
Two investigators assessed staining intensity in histologic sections, categorizing chromogenic immunolabeling into four groups: 0 (no labelling), 1+ (weak labelling), 2+ (moderate labelling), and 3+ (strong staining; observable with 10× objective).Semiquantitative staining analysis was done by H-score by counting 500 cells from ten different fields in the slides considering low, moderate and higher intensity of Mad2, pRb-phosphorylation and E2F1 expression.The percentage of positive cells with a given intensity for each sample was determined independently by a pathologist and a trained reader.A single manual H-score based on a scale of 0 to 180 was generated for each labelled section by taking the sum of the percentage of cells labelling 1+, double the percentage of cells labelling 2+, and triple the percentage of cells labelling 3+ (H-Score = ((%3+) × 3) + ((%2+) × 2) + (%1+)).

Analysis of LOH
The sequences of each microsatellite primer for 4q are given in the Supplementary Table 3.

Quantitative Real-time PCR
cDNA systhesis was performed from 1 μg of total RNA from each sample using Quantiscript Reverse Transcriptase, Quantiscript RT-buffer and RT Primermix of the QuantiTect Reverse Transcription kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's protocol.Quantitative real-time PCR was performed on 96-well optical reaction plates (Applied Biosystems, Darmstadt, Germany) using a StepOnePlus amplification and detection system (Applied Biosystems).The realtime RT-qPCR reactions were performed with reagents containing SYBR green and primer sets and the following conditions were used: 95°C for 5min, 40 cycles of 95°C for 30 s, 60°C for 30 s and 72°C for 30 s.The gene copy numbers of Mad2 were calculated using a standard curve that was constructed using the OE33 cell line (obtained from European Collection of Authenticated Cell Cultures, Cat No. 96070808; maintained in RPMI 1640 medium with 2 mM Glutamine and 10% Foetal calf serum).The 2−ΔΔCT method was used as a relative quantification strategy for qPCR data analysis.