Patients and specimens
A total of 125 patients with pathologically diagnosed NSCLC who suffered from surgical resection Cangzhou Central Hospital were recruited in this study, including 59 males and 66 females. None of them had received any radio- or chemo- therapy before surgery. Besides, 51 out of 125 patients were randomly selected to resect the adjacent normal tissues which were taken as internal controls. This study was approved by the Ethic Committee of the hospital and all participants signed the written informed consents in advance.
Tumor tissues and adjacent normal tissues were collected from patients with NSCLC and frozen in liquid nitrogen immediately. Then the tissues samples were severally stored at -80℃for RNA extraction. Detailed information of patients including gender, smoking status, histology, vascular invasion, distant metastasis, and TNM stage were summarized in a database. A 5-years’ follow-up was conducted with all patients and those who were died from unexpected events or other diseases were excluded from our study.
Cell lines and cell culture
The NSCLC cell line A549 was obtained from America Type Culture Collection and maintained in DMEM (Dulbecco’s modified Eagle’s medium), which was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37℃ under a 5% CO2 atmosphere.
Plasmid construction and cell transfection
The SFRP1 expressing plasmid pEF6/V5-SFRP1 and empty pEF6/V5 (negative control) were obtained from Yoshitaka Sekido (Nagoya University, Nagoya, Japan). Cells were transfected by these two types of vectors with FuGene HD Transfection Reagent (Roche, Mannheim, Germany) based on the instructions and maintained in normal medium.
RAN extraction and qRT-PCR analysis
RNA was first isolated from NSCLC tissues and adjacent normal tissues using Trizol reagent (Invitrogen) and then used to synthesize the first strand of cDNA through reverse transcription using a PrimeScript RT reagent Kit (Takara, Dalian, China) following the manufacture’s guides. RT-PCR reaction was performed with PRISM 7900 Sequence Detection System (Applied Biosystems). GAPDH served as the endogenous control. The relative mRNA expression of SFRP1 was calculated by -2∆∆CT method. Each sample was in triplicate.
IHC assay was performed to detect the expression of SFRP1 at protein level. In brief, 4-µm sections were prepared and then deparaffinized and rehydrated. Following, the sections were incubated with primary antibody and secondary antibody in order. Finally, all sections were stained with DAB and later counterstained by hematoxylin. Tissues were grouped according the percentages of stained cells. Those tissues with the percentage more than 30% belonged to the positive group and others were of the negative group.
Transwell assay was used to investigate the role of SFRP1 in migration and invasion of NSCLC cells. The transfected cells was first seeded into the upper chamber of the Transwell plates (BD Biosciences, San, Jose, CA, USA). Medium with 10% FBS was added into the lower chamber as a chemoattractant. After migration for 24h and invasion for for 48h, cells that migrated or invaded into the surface of the lower chamber were fixed by 90% alcohol, stained with 0.1% crystal violet and then counted in seven randomly selected fields. For invasion assay, the membrane should be per-coated by Matrigel (BD Biosciences, NJ, USA).
Statistical analyses were performed by SPSS 18.0 and Sigmaplot software. The differences between SFRP1 expression and clinical factors were analyzed by students’ t test. Chi-square test was adopted to estimate the relationship between SFRP1 expression and clinical factors of NSCLC patients. The overall survival of NSCLC patients were compared with the Kaplan-Meier analysis. The prognostic significance of SFRP1 was evaluated using the cox regression analysis. P<0.05 was regarded to be statistically significant.