The aim of this study is to investigate the expression levels and clinical significance of ILF2 in gastric cancer. The mRNA and protein expression levels of ILF2 were, respectively, examined by quantitative real-time PCR (qRT-PCR) and Western blot from 21 paired fresh frozen GC tissues and corresponding normal gastric tissues. In order to analyze the expression pattern of ILF2 in GC, 60 paired paraffin-embedded GC slides and corresponding normal gastric slides were detected by immunohistochemistry (IHC) assay. The correlation between ILF2 protein expression levels and clinicopathological parameters, overall survival (OS), disease-free survival (DFS), and clinical prognosis were analyzed by statistical methods. Significantly higher levels of ILF2 were detected in GC tissues compared with normal controls at both mRNA and protein level. High expression of ILF2 was tightly correlated with depth of invasion, lymph node metastasis, pathological stage, and histological differentiation. Log-rank test showed that high expression of ILF2 was positively associated with poor clinical prognosis. Multivariate analysis identified that ILF2 was an independent prognostic factor for OS and DFS. Our findings suggest that ILF2 may be a valuable biomarker and a novel potential prognosis predictor for GC patients.
Gastric cancer (GC) is one of the most common digestive tract cancers, and nearly one million new cases occur every year around the world [
Currently, surgical treatment is regarded as a preferred procedure to cure GC [
With the rapid development of molecular medicine, many biomarkers have been discovered to be associated with carcinogenesis, progression, and prognosis of GC. For example, many researchers observed that HER2 was overexpressed and related to poor prognosis in GC patients. The biotherapy of trastuzumab, which can specifically target HER2-positive GC, obtained a perfect curative effect [
Interleukin enhancer-binding factor 2 (ILF2), which also known as nuclear factor 45 (NF-45), a subunit of NF-AT (nuclear factor of activated T cells), is encoded by a gene located on human chromosome 1 (1q21.3) [
The mRNA and protein expression of ILF2 in gastric cancer were examined for the first time in this research. The possible relationship between high expression of ILF2 and clinicopathological parameters was further analyzed by statistical methods. Then, survival analysis was calculated to evaluate the prognostic value of ILF2 in GC. Finally, our data imply that ILF2 may play a pivotal role in the clinical prognosis of GC.
In this retrospective study, a total of 60 paired formalin-fixed, paraffin-embedded GC specimens and corresponding normal gastric tissues were recruited from patients who were diagnosed with primary GC and have undergone partial or total gastrectomy at the Department of General Surgery in the First Affiliated Hospital of Anhui Medical University (Hefei, China) from December 2010 to January 2011. All patients, enrolled in our study, had no history of preoperative radiotherapy or chemotherapy. The main clinicopathological parameters of the patients were listed in Table
Relationship between ILF2 expression and clinicopathological parameters (
Parameters | ILF2 expression | |||
---|---|---|---|---|
High ( |
Low ( |
|
| |
Gender | ||||
Male | 21 | 13 | 0.000 | 0.986 |
Female | 16 | 10 | ||
Age (years) | ||||
>60 | 19 | 14 | 0.519 | 0.471 |
≤60 | 18 | 9 | ||
Location | ||||
Cardia | 19 | 12 | 0.004 | 0.951 |
Body/antrum | 18 | 11 | ||
Size (cm) | ||||
>5 | 13 | 9 | 0.097 | 0.755 |
≤5 | 24 | 14 | ||
Histological differentiation | ||||
Well/moderate | 7 | 15 | 13.092 | 0.000 |
Poor/not | 30 | 8 | ||
Depth of invasion (T classification) | ||||
T1/T2 | 6 | 9 | 3.972 | 0.046 |
T3/T4 | 31 | 14 | ||
Lymph node metastasis | ||||
No | 10 | 14 | 6.769 | 0.009 |
Yes | 27 | 9 | ||
Surgical resection | ||||
Partial | 10 | 7 | 0.081 | 0.776 |
Total | 27 | 16 | ||
TNM stage | ||||
I/II | 9 | 16 | 11.944 | 0.001 |
III/IV | 28 | 7 | ||
CA19-9 | ||||
Positive | 13 | 6 | 0.537 | 0.464 |
Negative | 24 | 17 | ||
CEA | ||||
Positive | 13 | 7 | 0.141 | 0.707 |
Negative | 24 | 16 | ||
CA125 | ||||
Positive | 12 | 6 | 0.272 | 0.602 |
Negative | 25 | 17 |
TNM: tumor node metastasis;
Total RNA was extracted from 21 paired fresh specimens, and corresponding normal specimens with TRIzol reagent (Invitrogen, USA) and RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA) were used for reverse transcription according to the manufacturer’s protocol. Quantification of ILF2 mRNA was detected by quantitative reverse transcriptase PCR using Perfectstart SYBR Green qPCR Master Mix (Omega Bio-Tek, USA). The primers used for amplifying ILF2 are as follows: ILF2, forward primer: 5′-CGCCTCTTCAGTTGTCTGC-3′ and reverse primer: 5′-GACCACGGCCTCTGTCAC-3′; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an internal control, forward primer: 5′-AGCCACATCGCTCAGACAC-3′ and reverse primer: 5′-GCCCAATACGACCAAATCC-3′. The amplification protocol was done with the following steps: denaturation at 95°C for 10 min, followed by 40 cycles of degeneration at 95°C for 15 s, annealing at 60°C for 20 s, and extension at 72°C for 40 s. The reaction was performed on the Stratagene Mx3000p Sequence Detection System (Applied Agilent, USA). All assays were done in triplicates. The 2−ΔΔCt method was used to quantify the relative expression levels of ILF2 mRNA of each specimen.
Total proteins were extracted from 21 paired fresh frozen GC specimens and corresponding adjacent normal gastric specimens using RIPA lysis buffer and PMSF (Beyotime, China). The protein concentration was detected by BCA protein assay kit (Beyotime, China). Subsequently, an equivalent amount of protein of each paired specimen was separated by SDS-PAGE on 10% polyacrylamide gels and then was electrotransferred to 0.45 mm polyvinylidene fluoride membranes (Millipore, USA) for 1 h at 200 ma. After blocking in 5% nonfat milk diluted with TBST (tris-buffered saline/Tween-20) for 1 h at room temperature, the membranes were separately incubated with rabbit anti-ILF2 monoclonal antibody (1 : 3000; Abcam) and rabbit anti-GAPDH antibody (1 : 3000; Bioss) at 4°C overnight. On the second day, after washing 3 times with TBST per 10 min, the membranes were incubated with peroxidase-conjugated AffiniPure goat anti-rabbit IgG (1 : 6000; zsgb) for 1 h at room temperature. Finally, after washing 3 times with TBST per 10 min, the targeted protein was detected with the enhanced chemiluminescence system according to the manufacture’s instruction. The intensity of ILF2 protein band was quantified by ImageJ software and normalized with GAPDH.
ILF2 protein expression was measured by IHC staining in 4
All statistical analyses were performed with the SPSS 17.0 software (SPSS, Chicago, IL, United States). The Pearson
As far as we know, high expression of ILF2 has been reported in several malignant tumors. However, the expression level of ILF2 in gastric cancer is not yet completely understood. In this study, qRT-PCR assay and Western blot assay were performed in 21 paired fresh GC tissues and corresponding adjacent normal gastric tissues. The expression levels of ILF2 mRNA in GC tissues were significantly higher than those in adjacent normal gastric tissues (Figure
The expression levels of ILF2 mRNA in 21 paired samples by qRT-PCR. Scatter plots of the relative expression of ILF2 between GC tissues (tumor) and normal controls (normal) to GAPDH, GAPDH as an endogenous control (
The expression levels of ILF2 protein in six representative paired clinical samples by Western blot analysis. The levels of ILF2 protein were higher in GC tissues (T) compared with adjacent normal tissues (N), GAPDH as an endogenous control (a). Relative quantification results of the intensity of ILF2 bands of GC tissues (tumor) and normal controls (normal) to GAPDH were quantified by gray analysis (b).
Correlation between ILF2 mRNA and ILF2 protein expression levels in 21 GC tissues was analyzed by Pearson correlation analysis (
Our study illuminated that ILF2 mRNA and protein were overexpressed in GC. To evaluate the association of ILF2 protein expression levels with clinicopathological parameters, a further IHC staining experiment was conducted consisting of 120 tissues slides (60 GC and 60 normal controls). According to the IHC results, ILF2 protein was mainly expressed in the nucleus of GC cells (Figures
Representative microphotographs for ILF2 protein expression by IHC staining in GC tissues and corresponding normal gastric tissues. ILF2 protein in GC tissues in strong staining (sepia) (a), moderate staining (claybank) (b), weak staining (light yellow) (c), no staining (d), and negative controls (e). Weak staining (light yellow) (f) and barely stained (g) adjacent normal gastric tissues. The comparison of ILF2 immunostaining score between GC tissues (
The characteristics of the patients were listed in Table
In this retrospective study, postoperative follow-up was conducted: the median time was 45 months (range 2–62 months) and the mean time was 42.950 ± 2.398 (mean ± SE) months. Kaplan-Meier survival analysis was calculated to evaluate the prognostic values of ILF2 protein in GC patients. The median and mean survival times of GC patients with high ILF2 expression, respectively, were 38 months and 36.519 ± 2.985 months, which were shorter than those with low expression groups (median 60 months, mean 53.043 ± 2.916 months) (
Kaplan-Meier survival analysis and log-rank test for OS and DFS of GC patients. The OS of GC patients with ILF2 high expression and low expression (a). The DFS of GC patients with ILF2 high expression and low expression (b).
To explore the independent prognostic factor for DFS and OS of GC patients, univariate and multivariate Cox regression analyses were performed in this study. Univariate Cox regression analysis suggested that ILF2 expression (
Univariate and multivariate Cox regression analyses for OS and DFS of GC patients.
Variables | OS | DFS | ||
---|---|---|---|---|
RR (95% CI) |
|
RR (95% CI) |
| |
|
||||
Gender (male versus female) | 1.856 (0.976–3.529) | 0.059 | 1.579 (0.884–2822) | 0.123 |
Age (years) (>60 versus ≤60) | 1.799 (0.944–3.429) | 0.074 | 1.701 (0.948–3.050) | 0.075 |
Location (cardia versus body/antrum) | 1.095 (0.589–2.035) | 0.774 | 0.794 (0.448–1.406) | 0.429 |
Size (cm) (>5 versus ≤5) | 1.073 (0.560–2.056) | 0.831 | 1.344 (0.748–2.416) | 0.323 |
Histological differentiation (well/moderate versus poor/not) | 2.403 (1.173–4.926) | 0.017 |
2.040 (1.089–3.823) | 0.026 |
Depth of invasion (T1/T2 versus T3/T4) | 3.135 (1.311–7.495) | 0.010 |
2.741 (1.319–5.695) | 0.007 |
Lymph node metastasis (no versus yes) | 1.698 (0.865–3.257) | 0.111 | 1.578 (0.878–2.837) | 0.128 |
TNM stage (I/II versus III/IV) | 4.727 (2.218–10.074) | <0.001 |
2.897 (1.565–5.365) | 0.001 |
ILF2 expression (high versus low) | 4.496 (2.045–9.883) | <0.001 |
3.251 (1.699–6.224) | <0.001 |
CA19-9 (positive versus negative) | 2.254 (1.191–4.264) | 0.012 |
2.223 (1.219–4.054) | 0.009 |
CA125 (positive versus negative) | 2.677 (1.399–5.122) | 0.003 |
2.340 (1.268–4.319) | 0.007 |
CEA (positive versus negative) | 2.133 (1.117–3.995) | 0.021 |
2.103 (1.105–3.669) | 0.022 |
|
||||
TNM stage (I/II versus III/IV) | 3.462 (1.451–8.260) | 0.005 |
— | — |
ILF2 expression (low versus high) | 2.996 (1.173–7.654) | 0.022 |
3.464 (1.727–6.947) | <0.001 |
Depth of invasion (T1/T2 versus T3/T4) | 3.849 (1.452–10.206) | 0.007 |
3.669 (1.613–8.343) | 0.002 |
CA19-9 (positive versus negative) | 2.911 (1.414–5.994) | 0.004 |
3.210 (1.538–6.701) | 0.002 |
CA125 (positive versus negative) | 3.632 (1.695–7.786) | 0.001 |
2.391 (1.193–4.791) | 0.014 |
95% CI: 95% confidence interval; RR: relative risk; TNM: tumor node metastasis;
It is generally known that GC is one of the most common causes of cancer-related deaths around the world [
ILF2 combines with ILF3 to participate in the process of mitosis, transcription regulation, DNA repair, microRNA processing, and virus replication. In addition, the role of ILF2 as a tumor promoter has also been recognized [
It is worth noting that this is the first research to investigate the expression and clinical significance of ILF2 in GC. It was found that ILF2 was overexpressed at both mRNA and protein levels in GC tissues compared with corresponding normal controls. This result indicated that ILF2 might function as a tumor promoter in GC. Next, the relationship between ILF2-positive expression and clinicopathological characteristics of GC patients was assessed. The result revealed that the high expression of ILF2 was significantly related to histological differentiation, TNM stage, depth of invasion (T classification), and lymph node metastasis; moreover, the more advanced the tumor was, the higher possibility the overexpression of ILF2 existed. In short, ILF2 may play a vital role in the occurrence and progression of GC.
It had been reported in some previous literature that the high expression of ILF2 increased the risk of poor clinical prognosis in certain tumors [
There are some limitations in this study. Firstly, although the expression and prognostic significance of ILF2 in GC have been evaluated, the specific functions and molecular mechanisms of ILF2 in GC need to be further investigated based on in vivo and in vitro experiments. Secondly, because of the source of tissue specimens, there is only a small number of tissue samples. Thus, a larger number of tissue samples are needed to exhibit the clinical significance of ILF2 in gastric cancer.
In summary, this study for the first time confirms that ILF2 is overexpressed and is an independent prognostic factor for OS and DFS of GC. ILF2 may be a potential novel prognostic indicator for GC. At the same time, it also provides a new idea for the treatment of GC.
The research protocols were approved by the Ethics Committee of the First Affiliated Hospital of Anhui Medical University, Hefei, China. All specimens were handled and made anonymous according to the ethical and legal standards.
Informed consent was obtained from all individual participants included in the study.
The authors declare no conflict of interest in regard to this study.
The authors thank Li Yang, Ting Du, Lixiang Zhang, Huan Wang, and Professor Wensen Jin for the help. The authors would like to thank the Development Center for Medical Science and Technology of Heath Ministry of China (W2013FZ03) for supporting this research.