NSCLC tumor tissues and adjacent non-tumor tissues (3 cm away from the tumor's edge) were collected from 237 patients from the Affiliated Huaian No.1 People’s Hospital of Nanjing Medical University between April 2013 and December 2014. The demographic and pathological characteristics were summarized in Table 1. All patients did not receive any chemotherapy or adjuvant therapy before the surgical section. The inclusion criteria were as follows: all patients were diagnosed with NSCLC by CT and pathologically diagnosed during surgery; all patients with other types of cancers other than NSCLC were excluded. This study obeyed the Declaration of Helsinki and written informed consent was provided by each patient. The Ethics Committee of the Affiliated Huaian No.1 People’s Hospital of Nanjing Medical University approved this study.
Table 1
Relationship of aberrant SFXN1 expression with clinicopathological features in all NSCLC patients
Clinicopathological features
|
N
|
SFXN1 expression
|
P value
|
High (n = 139)
|
Low (n = 98)
|
Gender
|
Female
|
104
|
56
|
48
|
0.1842
|
|
Male
|
133
|
83
|
50
|
Age (years)
|
≤ 60
|
96
|
57
|
39
|
0.8516
|
|
> 60
|
141
|
82
|
59
|
TNM stage
|
I ~ II
|
128
|
58
|
70
|
< 0.0001
|
|
III ~ IV
|
109
|
81
|
28
|
Lymph node metastasis
|
Yes
|
120
|
86
|
34
|
< 0.0001
|
|
No
|
117
|
53
|
64
|
Smoking
|
Yes
|
153
|
93
|
60
|
0.3878
|
|
No
|
84
|
46
|
38
|
Tumor size (cm)
|
≤ 3
|
142
|
69
|
73
|
0.0001
|
|
> 3
|
95
|
70
|
25
|
Differentiation
|
Low
|
138
|
78
|
60
|
0.4322
|
|
Moderate/High
|
99
|
61
|
38
|
Cell culture
Human NSCLC lines A549, HCC827, H1299, NCI-H460, and human normal lung epithelial cells BEAS-2B were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences and used for this experiment. RPMI-1640 medium containing FBS and penicillin-streptomycin was used as a nutrient source for the cells, and cells were continuously cultured at 37°C with 5% CO2 in a thermostatic cell incubator.
Cell transfection
In this experiment, si-SFXN1#, si-SFXN1* and their negative control si-NC, were synthesized by GenePharma and transfected into A549 cells using lipofectamine 2000 reagent to interfere with the expression of SFXN1. Briefly, A549 cells were inoculated into 96-well plates at a density of 5×103 cells/well and then incubated in an incubator containing 5% CO2 at 37°C for 24 hours. When cell growth reached 70%-80% confluence, cell transfection experiments were started under the manufacturer's instructions.
qRT-PCR assay
To detect whether there were differential expressions of SFXN1 in normal and lung cancer tissue or cells and whether si-SFXN1#, si-SFXN1*, and si-NC were successfully transfected into A549 cells, the relative expressions of SFXN1 at the RNA level in each corresponding group was detected by qRT-PCR, respectively. In brief, TRIzol reagent was used to lysed and extracted total RNA from cells or tissues. Then, a NanoDrop microspectrophotometer was applied to quantify the RNA concentrations extracted from samples. cDNA was synthesized from RNA using Multiscribe RT kit, and q RT-PCR was then conducted by Universal SYBR Green Master Kit on an ABI PRISM 7500 PCR machine (Applied Biosystems). The PCR thermal cycles were as follows: 95℃ for 1 minutes, 40 cycles of 95℃ for 15 seconds, and 62℃ for 45 seconds. Finally, solution curves were drawn, and the expressions of SFXN1 were quantified by equation 2-△△CT method.
Western blot assay
Total proteins were extracted from tissues or cells using RIPA buffer and quantified by the BCA method following the manufacturer's instructions. Then, 20 μg/lane protein was separated by 10% SDS-PAGE and transferred onto PVDF membranes. After blocking with 5% skimmed-milk at 37℃ for 50 minutes, membranes were incubated with primary antibodies at 4℃ overnight. Following the primary incubation, the membranes were washed with PBS/Tween-20 (0.03%) and further incubated with secondary antibody at 37℃ for 2 hours. Finally, protein bands were visualized with ECK and analyzed with ImageJ version 1.46 software (National Institutes of Health).
Cell viability measured by CCK-8 assay
To determine whether the cell viability of A549 cells was affected after interfering with si-SFXN1, the CCK-8 assay was measured accordingly. After transfection with si-SFXN1 or si-NC, cells were inoculated into 96-well plates, and the cell concentration was adjusted to the density of 5×103 cells/well. Then, cells were placed in a humidified incubator containing 5% CO2 at 37°C for 24 hours. Afterward, 10 μL CCK-8 solution was added into each well with a pipette gun and incubated for another 1 hour at room temperature. After the incubation, the absorbance value was measured at 450 nm using a fully automatic quantitative mapping enzyme scale. The cell viability of each group was calculated. Three parallel copies were set for each experimental group, and the experiment was repeated three times.
Cell apoptosis analysis
At 24 hours post-transfection, cells were inoculated in a six-well density of 1×105 cells/well and incubated for 24 hours at 37°C in a humidified environment containing 5% CO2. The cells were then collected for apoptosis detection. According to the Annexin V-FITC staining kit's instructions, cells were stained with ethylenediaminetetraacetic acid (EDTA)-free and digested with 0.25% trypsin. After washing the cells twice with PBS at 4℃, the resuspended sections were added 10 uL of AnnexinV-FITC and 5 uL of PI staining solution and cultured at room temperature for 15 minutes in the dark. The cells' apoptosis rate was measured by flow cytometry, and the apoptosis rate was calculated by CellQuest software (BD Biosciences, Franklin Lakes, NJ, USA). All experiments were set up in three parallel copies, and the experiments were repeated three times.
Cell migration and invasion capacity detected by Transwell
The migratory and invasive capacities of A549 cells after transfection with si-SFXN1 was measured using Transwell chambers (Corning, Inc.). In invasion assay, Transwell were pre-coated with 8 uM-pore Matrigel (Corning, Inc.). A 200 μL of the pipette was used to add cell suspension into the chambers' interior (upper chamber) with the serum-free medium; meanwhile, 600 μL of DMEM medium containing serum (DMEM complete medium) was added into the lower chamber. The chambers were placed in an incubator with 5% CO2 at 37°C for 48 hours. After the culture was completed, the cell culture medium in the upper and lower chambers was discarded and washed with PBS buffer twice. After absorbing the residual water with a dry cotton swab, the cells that have not migrated from the membrane's upper surface were removed with a wet cotton swab. Then, cells were stained with a crystalline violet solution for 10-20 minutes. After the staining was completed, the upper chamber was rinsed with PBS buffer, and the upper chamber was dehydrated with xylene for 2-3 minutes and rinsed again with PBS buffer. Finally, the membrane at the bottom of the chamber was cut off with a sterilized scalpel and affixed to a slide. The membrane was placed under a photomicroscope, and five randomly selected fields of view were used for cell counting and photographing.
Statistical analysis
All the data in this study were analyzed using SPSS 19.0 software (IMB Corp.). All experimental data results were expressed as mean ± standard deviation (SD) and analyzed by one-way analysis of variance (ANOVA) or student's t-test to calculate the Р values. The association between SFXN1 and clinicopathological features were determined using a χ2 test. 5-year survival rate and disease-free survival rate were analyzed by Kaplan-Meier survival analysis followed by log-rank. P<0.05 indicated that there was a statistically significant relationship among all results.