Background
Esophageal cancer is the eighth most common cancer with an increasing morbidity and mortality rates worldwide [
1,
2]. About 90% of esophageal cancers are esophageal squamous cell carcinoma (ESCC) on pathological examination [
3]. Despite the progress in diagnosis and treatment, the overall five-year survival rate of ESCC is 15 to 25% due to diagnosis in later stages [
4,
5]. Therefore further elucidation of the molecular events in ESCC development may yield alternative means for the management of ESCC [
6,
7].
Prenylated Rab acceptor 1 domain family, member 2 (PRAF2, also called JM4), a 178 amino acid endoplasmic reticulum (ER)-resident protein, comprise a prenylated Rab acceptor motif and four transmembrane domains [
8,
9]. PRAF2, which is strongly expressed in human normal tissues [
10], has been identified as a novel protein involved in ER-to-Golgi transport and vesicular traffic [
11‐
13]. Unlike the PRAF1 and PRAF3 (also belong to the PRA family) well characterized in tumorigenesis [
14‐
17], PRAF2 has not been investigated until quite recently. Now, PRAF2 has been considered as oncogene since it is highly expressed in multiple tumor tissues of the breast, colon, lung and ovary cancer [
10]. It has been reported that overexpression of PRAF2 shows a significantly correlation with malignant clinical characteristics and predicts poor prognostic outcome in malignant glioma, neuroblastoma and hepatocellular carcinoma [
18‐
20]. Moreover, PRAF2 may play important roles in the malignant transformation of above tumor types [
18‐
21]. In molecular mechanism, monomeric/dimeric state of PRAF2 may influence its subcellular localization and distribution that leads to functional differences in malignant glioma cells [
18]. Conversely, Vento et al. reported that PRAF2 may exert as a tumor suppressor gene for its function in inducing apoptotic cell death through interacting with Bcl-xL/Bcl-2 and translocating Bax to mitochondria [
22]. However, the expression profile and biological role of PRAF2 in ESCC have not been explored.
In present study, we find that PRAF2 expression was an independent prognostic biomarker for poor survival in patients with ESCC. Furthermore, knockdown of PRAF2 expression in ESCC cells blocked cell growth and migration and stimulated cell apoptosis.
Methods
Patients and sample collection
A total of 77 ESCC patients and relevant clinical data (including age, gender, tumor location, clinical stage, and differentiation grade) were obtained from The Affiliated Huaian No.1 People’s Hospital of Nanjing Medical University (Huai’an, Jiangsu Province, China) from May 2010 to June 2014. Overall survival (OS) was calculated from the date of diagnosis to the date of last follow-up or mortality from any cause. The surgically resected tumor tissues and matched surrounding non-tumor tissues were obtained from patients for further analysis of gene expression. Written informed consent for the use of tissue specimens was obtained from all patients, and the protocol for the present study was approved by the Ethics Committee of The Affiliated Huaian No.1 People’s Hospital of Nanjing Medical University.
Cell culture and transfection
The TE1 cell line (no. TCHu 89; cell line was last tested and authenticated on January 8, 2018 by STR Profiling) was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) in 2016. Cells were cultured in DMEM media (KeyGEN, Nanjing, China) supplemented with 10% fetal bovine serum (Gibco, Grand Island, USA), and cultured at 37 °C in a humidified incubator containing 5% CO2. PRAF2 silencing was achieved using small interfering RNA (siRNA). Transfection of siRNA was performed using Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, USA). The PRAF2 siRNA (siPRAF2) sequence was 5′-UCAACAACCUCCUCUACUA-3′ and the negative control sequence was 5′-UAGUAGAGGAGGUUGUUGA-3′ (RiboBio, Guangzhou, China).
Cell proliferation analysis
For the Cell Counting Kit-8 (CCK-8; Dojindo, Japan) assay, transfected cells were plated in a 96-well plate at a density of 2000 cells/100 μL and cultured for 24, 48, and 72 h, respectively. Then the absorbance was measured at 450 nm using a microplate reader (BioTek, Winooski, USA). For colony formation assay, a total of 200 cells were placed in a fresh 6-well plate and cultured in medium containing 10% fetal bovine serum, with medium replacement every 3–4 days. After 2 weeks, cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Visible colonies were manually counted.
Cell cycle analysis
After transfection, the TE1 cells were collected and subjected to PI staining using the CycleTEST™ PLUS DNA Reagent Kit (BD Biosciences, Franklin Lakes, USA). Next, Flow cytometry (BD Biosciences, Franklin Lakes, USA) analysis was performed to detect cell cycle distribution. Finally, the percentage of cells in G1, S, and G2-M phases were analyzed by the ModFit program.
Invasion assays
Cell invasion were examined using transwell assay. Briefly, transfected cells were resuspended in 100 μl serum-free medium and added to the upper chamber of transwell filter, containing a Matrigel-coated membrane (BD Biosciences, Franklin Lakes, USA). The lower chamber was filled with 500 μl medium containing 20% fetal bovine serum. After incubation, cells that migrated to the lower compartment were fixed with methanol, stained with crystal violet in each well. Imaging and cell counting were performed at × 10 objective lens under a light microscope.
Apoptotic analysis
The Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences, Franklin Lakes, USA) was used to detect apoptotic cells. Cells were collected after transfection and mixed with Annexin V-FITC/PI buffer. Flow cytometry (BD Biosciences, San Diego, USA) was used to identify cells of normal status, early apoptosis, late apoptosis and death. The relative ratios of cells in the initial stages of apoptosis were analyzed.
Quantitative real-time polymerase chain reaction (qPCR)
After frozen specimens (~ 10 mg/per) were ground in liquid nitrogen and TE1 cells collected, the total RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, USA) and reverse with transcripted with FastQuant RT Kit (Tiangen, Beijing, China). Then, PCR reaction was performed in triplicate with SuperReal PreMix Plus (Tiangen, Beijing, China) using the Real-Time PCR Detection System (Roche, California, USA). The specific primers for PRAF2 were forward: 5′ -GCCGGTGCTTCTGATCCTG- 3′ and reverse: 5′ -GATCCAGCCTCCTGCTCTTG- 3′. The forward and reverse primers for GAPDH were 5′ -ACCAGCCTCAAGATCATCAGC- 3′ and 5′ -TGCTAAGCAGTTGGTGGTGC- 3′, respectively. The qPCR results were analyzed and expressed as relative expression of threshold cycle (Ct) value, which was then converted to fold changes.
Western blot
Transfected cells were harvested and treated with RIPA lysis buffer (KeyGEN, Nanjing, China) on ice, and protein concentration was determined using a BCA Kit (KeyGEN, Nanjing, China). Equal amounts of total protein were loaded on SDS-PAGE gels and subjected to electrophoresis. After separation on the gel, proteins were transferred to a PVDF membrane. The primary antibodies against PRAF2, BCL-XL, BAX, caspase3, cleaved-caspase3, caspase9, cleaved-caspase9 and GAPDH (Abcam, Cambridge, UK) were used according to the manufacturers’ recommendations. Then membranes were incubated with HRP-conjugated secondary antibody (Abcam, Cambridge, UK) and visualized by using ECL detection (KeyGEN, Nanjing, China).
TCGA verification of hub genes
UALCAN (
http://ualcan.path.uab.edu) was applied to construct an algorithm on the basis of The Cancer Genome Atlas (TCGA) database [
23]. The expression level of PRAF2 was verified in UALCAN esophageal cancer database.
Statistical analysis
All results are presented as the mean ± SD. Pearson’s chi-squared test, Fisher’s exact test, Kaplan-Meier method (log-rank test), multivariate Cox regression analysis, Student’s t-test and Kruskal-Wallis test were used to analyze the data using SPSS Statistics software (version 19.0, Chicago, USA). Relative PRAF2 expression in each tumor was calculated by normalizing to matched normal tissue, and the mean fold-change was employed as the cut-off points for case grouping. All cell experiments were performed in triplicate. P < 0.05 was considered statistically significant. Graphs were made using the GraphPad Prism 6.0 software package (La Jolla, CA, USA).
Discussion
ESCC is the most common histopathological subtype of esophageal cancer, and it has a high mortality rate largely due to late diagnosis [
2,
5]. Therefore, there is a compelling need to screen for new early biomarkers and therapeutic targets. Here, for the first time, we demonstrated the prognostic significance and the biological action of PRAF2 in ESCC.
PRAF2 mRNA expression was remarkable increased in ESCC samples compared with associated non-tumor tissues. The finding was in agreement with the results reported in several human cancers [
18‐
20]. Further, the prognostic value of PRAF2 was investigated and PRAF2 was recognized as a novel independent prognostic marker for poor survival in ESCC patients. Similar to our results, Wang et al. [
20] discovered that PRAF2 overexpression was strongly associated with unfavorable prognosis in hepatocellular carcinoma. These results indicate that PRAF2 may be a good prognostic marker for patients with ESCC.
As we known, a critical mechanism underlying tumorigenesis is the disequilibrium in cell survival and death [
24]. PRAF2 has been reported to be involved in the occurrence and progression of several solid cancers [
18‐
20]. In the present study, PRAF2 silencing impaired ESCC cell proliferation and cell cycle. The result was consistent with studies in hepatocellular carcinoma and neuroblastoma [
19,
20]. Though the function role of PRAF2 on cell apoptosis was not observed in tumors mentioned above, we found that knockdown of PRAF2 induced apoptosis of ESCC cells, and influenced the expression of apoptotic biomarkers which include BCL-XL, BAX, caspase3 and caspase9. These results indicated that PRAF2 silencing impaired ESCC cell growth via mitotic arrest and apoptosis. Further studies should be performed to clarify how PRAF2 involved in these functional roles.
Aggressive characteristic lead to tumor metastasis and then cause the poor prognosis. Our study has confirmed that high PRAF2 expression was associated with lower tumor differentiation grade and later TNM stage in ESCC. The results might suggest that PRAF2 was essential for ESCC progression. Also, PRAF2 silencing decreased the invasiveness of ESCC cells. Previous study shows PRAF2 interacts with the CCR5 and thereby contributes to cancer cell migration [
8,
25], which may partially explain the mechanism of metastasis. Unlike the PRAF2, PRAF1, another member of PRA family, is known as a potential inhibitory factor of tumor cell invasion by regulating the MAPK and integrin αvβ3 pathways [
26,
27]. Therefore, we speculated whether PRAF2 could function in reverse to regulate these pathways.
In summary, PRAF2 was an independent prognostic marker for poor survival in patients with ESCC. Further, we demonstrated that PRAF2 is a novel oncogene that facilitates cell growth and invasion, and inhibits apoptosis in ESCC cells.
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