Differently Expression Genes (DEGs) analysis
The cancer public database (Oncomine) was applied to download the original data of sequencing or expression profiling chip and analyzed the DEGs in the cancer tissue and the adjacent non-tumor tissue group. After that, the survival prognostic significance of DEGs in cancer was determined by the Kaplan-Meier plotter, and then Oncomine database was used to confirm the differential expression of DEGs between tumor tissues and normal tissues for the next step in research. In addition, a molecular signaling pathway network regulation map was constructed through using the genes co-expressed with differentially expressed genes in big data, and the related function enrichment map was drawn.
Cell Lines and Cell Culture
The human esophageal cancer cell lines (EC9706, TE-1 and ECA109) and the human esophageal epithelial cell line (HEEC) were purchased from the Shanghai Cell Bank Collection (Shanghai, China), and cultured in DMEM supplemented with 10% fetal bovine serum (Hangzhou Sijiqing Biological Engineering Materials, China) and maintained in a incubator at 37°C, 5% CO2.
The paraffin sections of esophageal cancer and adjacent non-tumor tissues (HEsoS030PG03, Shanghai Outdo Biotech Co., Ltd.) were deparaffinized with xylene and hydrated with absolute ethanol, 95% ethanol, 80% ethanol, 70% ethanol and distilled water, respectly. After 30min microwave heat recovery by citric acid antigen retrieval method, the sections were cooled to room temperature and washed with PBS (0.01M, pH 7.4), and then blocked with 2% BSA blocking solution at room temperature for 2 hours. After blocking, the sections were incubated with the primary MUC13 (1: 50, Santa Cruz, USA) antibody for 2 h at 37°C. Then rinsed three times in PBS, followed by incubation of secondary antibody (D110087,Sangon Biotech Co., Ltd) at 37°C for 2 h. DAB substrate kit was used to perform the chromogenic reaction. During which slices were observed under the microscope to confirm the color end point. Then, the nucleus is counter-stained with hematoxylin for 3 minutes. The slides were dehydrated in a series of graded ethanol solutions and transparented with the xylene, and then mounted with a neutral gum. Finally, semi-quantitative analysis of immunohistochemical results was conducted by Image ProPlus software, and positive cell percentages were calculated using IHC plug-in after color deconvulutionfor H&E DAB.
RNA Extraction and Quantitative Real-time PCR
Total RNA was extracted from the cells by using TRIzol® Plus RNA Purification Kit (Invitrogen, USA) and RNase-Free DNase Set (Qiagen, China) in accordance with the manufacturer’s protocol. GAPDH cDNA was used as an internal control for quantification. The following primers were used for qRT-PCR amplification: MUC13 (NM_033049.4) expression were
5’- TCAAGTGTCCTGATGCCTGC-3’ (forward)
and 5’- GCTCCCTTCTGCTCCAAGAT-3’ (reverse);
Human GAPDH (BC059110) expression were
5’- AGAAGGCTGGGGCTCATTTG-3’ (forward)
and 5’- AGGGGCCATCCACAGTCTTC-3’ (reverse);
MUC1 (NM_001018016.3) expression were
5’- TGCCGCCGAAAGAACTAC-3’ (forward)
and 5’- TGCCGCCGAAAGAACTAC-3’ (reverse);
MUC4 (NM_001322468.1) expression were
5’- CAGGGACGACGGGACTTA-3’ (forward)
and 5’- ACAGGGCACAGAGGTAGGG-3’ (reverse);
MUC12 (NM_001164462.2) expression were
5’- GAACGAAGTCGCAAATGA-3’ (forward)
and 5’- TGGGATAGGCTGAATAAGAT-3’ (reverse);
MUC3A (NM_005960.2) expression were
5’- TTTCGAGGACGACGGAACAG-3’ (forward)
and 5’- TCACACTGAGGACGAGGTCA-3’ (reverse);
GALNT14 (NM_001253826.2) expression were
5’- CCCCAGTGGTTCTTGTCC-3’ (forward)
and 5’- GGATGTCTGAGGTGGTTGC-3’ (reverse).
The conditions of 95°C for 60 s, 40 cycles of 95°C for 15 s, 63°C for 25 s were set for performing PCR.
Silencing of MUC13
Three small interfering RNA (siRNA) target sequence for MUC13 gene were designed as following:
5’- GGCAACUCAGCUGAUGCUGUATT-3’ (forward)
and 5’-UACAGCAUCAGCUGAGUUGCCTT-3’ (reverse);
5’- CCUGUGCAGAUAAUUCGUUAUTT-3’ (forward)
and 5’-AUAACGAAUUAUCUGCACAGGTT-3’ (reverse);
5’- CGACUGUAAGGACAAAUUUCATT-3’ (forward)
and 5’-UGAAAUUUGUCCUUACAGUCGTT-3’ (reverse).
The siRNA constructs were synthesized and cloned into GV115 plasmid vector. The MUC13-siRNA plasmid was then transfected into EC9706 and Eca109 cells with lentivirus following the manufacturer’s protocol.
The proteins were extracted from the cells harvested 72 hours after transfection using the total protein extraction kit (Thermo Pierce, USA) and Halt Protease and Phosphatase Inhibitor Cocktail kit (Thermo Pierce, USA), and then the BCA Protein Assay Kit (Biyuntian, Germany) was utilized to measure the consistency of the total proteins. After separating by SDS-PAGE gels, proteins were transferred onto a PVDF membrane (Millipore, USA) which was soaked in methanol for 20 sec in advance. The membrane was blocked with 5% BSA at room temperature for 1 h. The primary MUC13, MUC1, MUC4 and GALNT14 antibodies (1:1000, Abcam, UK), GAPDH antibody (1:10000, Abcam, UK), MUC12 antibody (1:500, Santa Cruz, USA) as well as the primary MUC3A antibody (1:200, Santa Cruz, USA) were incubated overnight at 4°C. After washingd with PBS for 5 min, the membrane was incubated at 37°C for 1 h with secondary antibody (1:5000, Thermo Pierce, USA). The ECL working solution was prepared following SuperSignal® West Dura Extended Duration Substrate kit instructions and used to develop the transfer film. Finally, the optical density values of the bands were analyzed and the GAPDH was adopted as control.
The amber dehydrogenase in living cells, especially in the mitochondria of proliferating cells, can reduce WST-8 which containing in Cell Counting Kit-8 (CCK8) reagent to yellow formazan. The cell proliferation could be evaluated by detecting the absorption value at 450nm wavelength. In brief, after siRNA transfection of EC9706 and ECA109 cells for 72 h, 100µL of cell suspension was added and incubated for 24 h at 37°C. Then, 10 µL CCK8 was supplemented and incubated for 2 h. At last, the absorbance was gauged at 450 nm on days 1, 2, 3, 4 and 5 respectively.
Clone formation assay
Clone formation is one of the effective methods for measuring cell proliferation ability. The cells in the logarithmic growth phasewere inoculated in a 6-well plate at a density of 500 cells/well after transfection and continued to culture until the number of cells in most single clones is greater than 50. After the culture is terminated, the cells were fixed with 1 mL of 4% paraformaldehyde for 30 min and stained with 1000 µL of crystal violet staining solution for 20 min. After cells were washed with deionized distilled water (ddH2O) for 3 times and images were captured with a digital camera.
Cell cycle analysis
Cells were harvested with trypsin when they have grown to a coverage rate of about 80%. After centrifuging at 1300 rpm for 5 min, the cells were washed by D-Hanks (pH 7.4) pre-cooled at 4°C and fixed in 75% ethanol pre-cooled at 4°C for 2 h. Then, the cells were washed with D-Hanks once again after discarding ethanol. Finally, cells were stained by working solution (PI: RNase stock solution : D-Hanks = 25: 10: 1000) for 30 min in the dark. The cells solution was collected for cell-cycle analysis by flow cytometry (Millipore, USA).
Cells were harvested with trypsin after transfection and centrifuged at 300 rpm for 5 min, and resuspended in 200 µL 1×binding buffer containing 2 µL Annexin V-FITC and 7AAD (PI) for 30 min at room temperature. According to the amount of cells, 200-300 µL 1×PBS was added to resuspend the cells for apoptosis analysis by flow cytometry (Millipore, USA).
All the animal experiments were carried out in strict accordance with the principles and procedures approved by the Committee on the Ethics of Animal Experiments of First People's Hospital of Xuzhou. 4 weeks old female BALB/c nude mice (Shanghai Lingchang Biotechnology Co. Ltd) were selected to xenograft studies. First, 7×106/200µL EC9706 cells transfected with siMUC13 or siCtrl were injected subcutaneously into the right hindlimbs of mice. We used the formula: π/6 × (length × width2) to calculate tumor size every 2 days and weighed the body weight of the mice (g). At end of experiment, the mice were euthanized with 2% sodium pentobarbital.
All experiments were repeated three times independently and two-tailed unpaired Student t test was applied to analyze data. P <0.05 was taken as statistically significant.