Sample collection
ESCC and distant matched non-malignant tissues were collected from patients undergoing endoscopy at the Department of Gastroenterology, All India Institute of Medical Sciences (AIIMS). The study has received the ethical approvalfrom the Guru Gobind Singh Indraprastha University (GGSIPU) and AIIMS research committee prior to its instigation and all the procedures involving human participants were performed in accordance with the ethical standards of both of the institutes. The whole research was carried out in accordance with “The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans”. All the individual participants included in the research plan were duly informed and their written informed consent was also obtained. For each patient, one pair of tissue samples was stored in 10% formalin vials to be embedded in paraffin and sectioned for immunohistochemical analysis. Information regarding various clinicopathological parameters was also obtained from each patient.
Immunohistochemistry (IHC)
Immunohistochemical analysis of MARCH7 in ESCC (n=25) and distant matched non-malignant tissues (n=24) was performed as described before[15]. Additionally, immunohistochemical analysis of CD8 (cluster of differentiation 8) and PD-1 (programmed cell death protein-1) expression in ESCC tissues was also carried out. Briefly, following deparaffinization and rehydration oftissue sections antigen retrieval was carried out using Tris-EDTA buffer (10 mM Tris-base, 1 mM EDTA, pH 9.0). Then, slides were treated with diluted rabbit polyclonal anti-MARCH7 (1:100; Novus Biologicals, USA), anti-CD8 (1:100; Proteintech, USA) and anti-PD-1 (1:100;Proteintech, USA) antibodies of human origin and incubated overnight at 4⁰C. Next day, the slides were treated with HRP conjugated anti-rabbit IgG [ImmPRESS Anti-Rabbit Ig (peroxidase) Polymer Detection Kit, Vector Laboratories Inc, USA] for further analysis.
Transfections of cultured cells
Esophageal carcinoma cell line of human origin, KYSE-410 (ECACC 94072023), was attained from Sigma-Aldrich and was also checked for mycoplasma contamination (Bangalore, India). The cells were transfected with 50nmol/l MARCH7 siRNA (Ambion, CA, USA) or scrambled sequence siRNA (Ambion) using Lipofectamine 3000 (Invitrogen, CA, USA) as transfecting agent in a serum- and antibiotics-free medium.
Quantitative Real time PCR (qRT-PCR)
RNA was isolated from KYSE-410 cells using the Qiagen miRNeasy Mini Kit as per the manufacturer’s protocol. The primers sequence details are given in Table I. To analyse the MARCH7 mRNA expression, real time PCR was carried out as described before [16].
Protein isolation and Western blotting
RIPA buffer (Sigma) was used for total protein isolation from the KYSE-410 cells. SDS-PAGE (sodium dodecyl sulphate-poly-acrylamide gel electrophoresis) and further analysis was performed as described before [15]. The immune-labelling was carried out using rabbit polyclonal anti-GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase, 1:200 dilution, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-MARCH7 (1:100), E-cadherin (1:1000) and β-catenin (1:1000) antibodies. The integrated density values (IDV) for each group were calculated using Image J software (https://imagej.nih.gov) and normalization was done by dividing the corrected IDV of proteins in each group by corrected IDV of GAPDH protein in the corresponding group.
MTT assay
Inhibition of cell growth was evaluated by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell growth inhibition rates were calculated at 24 and 48 hours using the equation (1-Absorbance570treatment/Absorbance570control) × 100 and results were expressed as percentage of inhibition relative to control cells.
Colony Formation Assay
Thousand transfected and untransfected KYSE-410 cells were cultured in 6-well plates for a week and the number of colonies formed was counted using 0.5% (w/v) crystal violet in distilled water.
Transwell assay
Transwell assay was also performed to check the effects of MARCH7 knockdown on KYSE-410 cell migration and invasion. The mean number of cells migrated/invaded was calculated as described before [15].
Statistical analysis
The statistical analysis was carried out using the Statistical Program for Social Sciences (SPSS) software, version 17.0 (SPSS Inc., Chicago, IL, USA). The correlation between clinic-pathological parameters of ESCC patients, such as age, gender, and stage and MARCH7/CD8/PD-1 protein expression were studied using the χ2 test. A p-value ≤0.05 was considered as indicative of statistical significance. The reported p-values are 2 tailed. To describe the discrimination between distant matched non-malignant and cancerous conditions based on MARCH7 expression, Receiver Operating Characteristic (ROC) curves were constructed and area under curve (AUC), sensitivity and specificity were calculated. The correlation between the expression of MARCH7 and CD8/PD-1 in tumor and distant matched non-malignant tissues was studied using Pearson correlation. Each experiment was performed in triplicate and the results obtained using either Student’s t-test or ANOVA, are presented as mean ± SD (standard deviation).