Patients and tissue samples
In the present study, 25 paired NSCLC and adjacent normal lung tissue samples were collected from patients with pathologically diagnosed lung cancer, who were not subjected to chemotherapy or radiotherapy prior to surgery, between 2011 and 2015 at the Haidian Section of Peking University Third Hospital. The age of the 25 patients ranged from 35 to 86 years. Clinicopathological characteristics of the patients, including gender, age, histological grade, clinical stage, pathology type, pTNM status and tumor size were collected. Cancer stages were classified according to the American Joint Committee on Cancer criteria. Written informed consent was obtained from each recruited patient according to institutional guidelines. The present study was approved by the Human Research Ethical Committee of Haidian Section of Peking University Third Hospital, China.
SUSD2 expression was determined by qRT-PCR. Total RNA was extracted according to the manufacturer instructions (Stratagene). Reverse transcription was carried out on total RNA (5 μg) from tissues, and cDNA was generated using the random hexamer primer. SUSD2 forward primer (5-CTCCAATGACTGCCGCAACTA-3) and reverse primer (5- GAACATTCCTTTCAGGTCCATCC-3) and GAPDH forward primer (5-AGCCTCGCCTTTGCCGA-3) and reverse primer (5-CTGGTGCCTGGGGCG-3) were synthesized by Integrated DNA Technologies. qRT-PCR protocol was as follows: 94 ℃ for 3 min, 94 ℃ for 1 min (30 cycles), 65 ℃ for 1 min, and 72 ℃ for 1 min, with a final 5 min extension at 72 ℃. SUSD2 expression was calculated based on the following equation: F = 2 -ΔCt, where ΔCt = Ct (SUSD2) – Ct (GAPDH).
Total proteins from 25 pairs of fresh lung cancer and adjacent normal tissues were extracted using radioimmunoprecipitation assay buffer containing 1 mM phenylmethylsulfonyl fluoride (Beyotime Institute of Biotechnology, Haimen, China). Western blot was performed as follows: proteins were separated on SDS-PAGE gels and then transferred to PVDF membranes by electrophoresis (Sangon Biotech, China). The membranes were then incubated with a rabbit polyclonal anti-SUSD2 antibody, 1:1,000 dilution, for 12 h at 4 ℃ (Abcam, UK). Subsequently, horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Multisciences, China) was applied at a dilution of 1:3,000 for 1 h at room temperature. Signals were visualized using an enhanced chemiluminescence detection kit (GE Healthcare Life Sciences, UK).
A search for the CpG island of the SUSD2 gene was carried out using GENETYX-Mac Ver.12.2.0 software. The CpG island was determined using MethPrimer online software. The criteria used were the following: island size > 200 bp, GC % > 50.0, and observed/expected CpG ratio > 0.6.
Genomic DNA was extracted from lung cancer tissues and adjacent normal lung tissues using a QIAamp Fast DNA Tissue Kit (Qiagen, USA). After purification, the genomic DNA was treated with sodium bisulfite using a DNA Methylation Kit (Kangwei, China). The target region in the SUSD2 gene was amplified from the genomic DNA by PCR using a PCR Mix Kit (Kangwei, China) and primers were designed as follows: forward 1: 5-GTTGGGATTTAGGGTTTGAG-3; forward 2: GTTTGTTAAAGGTTTTGTGGT; reverse: 5-CCTATATCTACAACCAACAATAC-3. PCR was performed under the following conditions: 95 °C for 2 min; 40 cycles at 95 °C for 30 s, 53 °C for 30 s, 72 °C for 1 min and finally 72 °C for 5 min. To perform hot start PCR, the reverse primer was added to the reaction mix when the reaction mix first reached 95 °C. After being purified, the PCR products were cloned into a pGEM-T vector. Approximately each of the 5 clones obtained from three independent experiments was sequenced. CpG methylation status was analyzed by the web-based tool QUMA.
A database was established and SPSS 11.5 software was used for statistical analysis. Student’s t-test was used to analyze the statistical differences between two groups. The relationship between the expression of SUSD2 and clinicopathological characteristics was analyzed by the chi-squared test. Data were shown as mean ± standard deviation, and P < 0.05 was considered statistically significant.