Nuclear overexpression of DNA damage-inducible transcript 4 (DDIT4) is associated with aggressive tumor behavior in patients with pancreatic tumors

DNA damage-inducible transcript 4 (DDIT4) is induced in various cellular stress conditions. Several studies showed that the dysregulation of DDIT4 is involved in different malignancies with paradoxical expressions and roles. Therefore, this study investigated the clinical significance, prognostic, and diagnostic value of DDIT4 in different types of pancreatic tumors (PT). The expression of DDIT4 and long non-coding RNA (TPTEP1) in mRNA level was examined in 27 fresh PT samples using Real-time quantitative PCR (RT-qPCR). Moreover, 200 formalin-fixed paraffin-embedded PT tissues, as well as 27 adjacent normal tissues, were collected to evaluate the clinical significance, prognostic, and diagnosis value of DDIT4 expression by immunohistochemistry (IHC) on tissue microarrays (TMA) slides. The results of RT-qPCR showed that the expression of DDIT4 in tumor samples was higher than in normal samples which was associated with high tumor grade (P = 0.015) and lymphovascular invasion (P = 0.048). Similar to this, IHC findings for nucleus, cytoplasm, and membrane localization showed higher expression of DDIT4 protein in PT samples rather than in nearby normal tissues. A statistically significant association was detected between a high level of nuclear expression of DDIT4 protein, and lymphovascular invasion (P = 0.025), as well as advanced TNM stage (P = 0.034) pancreatic ductal adenocarcinoma (PDAC) and in pancreatic neuroendocrine tumor (PNET), respectively. In contrast, a low level of membranous expression of DDIT4 protein showed a significant association with advanced histological grade (P = 0.011), margin involvement (P = 0.007), perineural invasion (P = 0.023), as well as lymphovascular invasion (P = 0.005) in PDAC. No significant association was found between survival outcomes and expression of DDIT4 in both types. It was found that DDIT4 has rational accuracy and high sensitivity as a diagnostic marker. Our results revealed a paradoxical role of DDIT4 expression protein based on the site of nuclear and membranous expression. The findings of this research indicated that there is a correlation between elevated nuclear expression of DDIT4 and the advancement and progression of disease in patients with PT. Conversely, high membranous expression of DDIT4 was associated with less aggressive tumor behavior in patients with PDAC. However, further studies into the prognostic value and biological function of DDIT4 are needed in future studies.


Evaluation of DDIT4 and TPTEP1 mRNA expression level in fresh tumor tissues of pancreatic tumor and their adjacent normal tissues
The analysis of RT-qPCR data using the Mann-Whitney U test indicated that the mRNA expression levels of DDIT4 were significantly increased in PT patients compared to the healthy group (P < 0.05) (Fig. 2A).However, there was no significant difference between TPTEP1 mRNA expression in tumors and normal samples (Fig. 2B).

Expression of DDIT4 in pancreatic tumor compared with adjacent normal samples
The protein expression levels of DDIT4 were evaluated using the IHC technique on TMA sections by three distinct scoring methods that comprise the intensity of staining, percentage of positive tumor cells, and H-score.DDIT4 was expressed at different intensities in the nucleus, cytoplasm, and membranous in two types of PT as well as adjacent normal samples (Fig. 3).The mean expression level (based on H-score) of DDIT4 was 80 in tumor samples (in all expression); however, the mean expression of DDIT4 was 20 in the nucleus, 30 in the cytoplasm, and 32 in the membranous in adjacent normal tissue, indicating that the expression of DDIT4 in PTs was high compared to adjacent normal tissues (Table 2 and Fig. 4).Besides, Mann-Whitney U tests showed a significant difference between the median nuclear, cytoplasmic, and membranous expression of DDIT4, and the median expression of adjacent normal tissues (P = 0.012, P = 0.001, and P = 0.026, respectively) (Table 2).

Associations between DDIT4 expression (nucleus, cytoplasm, and membrane) and clinicopathological characteristics of PDAC tissue samples
The association between the expression of DDIT4 protein and the clinicopathological parameters of PT was investigated using Pearson's χ2 test.Our analysis demonstrated a significant association between the nuclear expression of DDIT4 protein and lymphovascular invasion (P = 0.025).We also found a statistically significant association between a membranous expression of DDIT4 protein and histological grade (P = 0.011), margin involvement (P = 0.007), perineural invasion (P = 0.023), as well as a lymphovascular invasion (P = 0.005).There was no significant association between cytoplasmic expression of DDIT4 and clinicopathological features (Table 3).Besides, the correlation between DDIT4 protein expression and clinicopathological characteristics was examined using spearman's correlation tests.Our results indicated a significant direct correlation between a Figure 1.Box plot and overall survival (OS) results of DDIT4 expression for cancer compared to normal tissues through the Gene Expression Profiling Interactive Analysis (GEPIA2).The data through GEPIA2 indicated a few changes in the mRNA expression level of DDIT4 in pancreatic adenocarcinoma tissue samples compared to the normal tissues (P < 0.05, Fig. 1A).A high expression level of DDIT4 between the worse OS (HR (high) = 1.5, P (HR) = 0.037) and in PAAD patients (Fig. 1B).

Associations between DDIT4 expression (nucleus, cytoplasm, and membrane) and clinicopathological characteristics of PNET tissue samples
Pearson's χ2 test exhibited a significant association between the nuclear expression of DDIT4 protein and histological grade (P = 0.034).Moreover, our Spearman's correlation tests showed a direct correlation between the nuclear expression of DDIT4, and the high grade (P = 0.014).We did not find any association or correlation between cytoplasmic and membranous DDIT4 protein expressions and the clinicopathological parameters (Table 4).

Information on clinical outcomes in types of PT
In the current study, from 200 patients, follow-up data for 10 patients were not available.Of them, 7 patients had PDAC, and 3 patients had PNET.www.nature.com/scientificreports/high cytoplasmic and membranous expression of DDIT4 was related to longer survival.The main characteristics of survival outcomes are exhibited in Table 5.

Pancreatic neuroendocrine tumor
In this type of PT, tumor recurrence and metastasis occurred in 2 (10.0) and 4 (20.0)patients, respectively.Throughout follow-up, all death cases died of pancreatic tumor.The median and mean OS and DSS time were 27 months (Q1 = 18 and Q3 = 37) and 27 (SD = 12.92), respectively.Additionally, the median and mean follow-up periods for PFS were 25 (Q1 = 17, Q3 = 35) and 24 (SD = 14.03) months, respectively.Patients with high nuclear DDIT4 expression have been reported to have lower mean OS, DSS and PFS survival, which is consistent with PDAC survival data.However, there is not any difference between the mean OS/DSS/PFS and high/low cytoplasmic and membranous expression.

Survival analysis based on the expression of DDIT4 in PT types
Pancreatic ductal adenocarcinoma cancer Kaplan-Meier survival analysis exhibited no statistically significant differences between OS/DSS/PFS and the patients with high /low expression of DDIT4 (Fig. 5).
Univariate and multivariate Cox regression analyses were performed to evaluate the clinical significance of potential prognostic factors for OS, DSS, and PFS.On univariate analyses, tumor size, TNM stage, and distant metastasis were detected as potential prognostic factors for OS, DSS, and PFS.In addition, age and lymphovascular invasion have a prognostic role for OS/PFS and DSS, respectively.Statistically significant univariate analyses were incorporated into multivariate Cox regression analysis, in which the results indicated that distant metastasis was an independent risk factor affecting OS (HR: 187; 95% CI 1.479-3.233;P < 0.001), DSS (HR: 2.286; 95% CI 1.528-3.421;P < 0.001), and PFS (HR: 2.680; 95% CI 1.819-3.949;P < 0.001, respectively) (Table 6).

Diagnostic value of the DDIT4 in PT versus adjacent normal tissues
ROC curves, and the AUC of the expression level of DDIT4 protein (nuclear, cytoplasmic, and membranous) were performed to explore the diagnostic value of the marker in discriminating pancreatic tumor from adjacent normal tissues (Fig. 6).The results of ROC curves demonstrated an AUC of 0.64 (95% CI 0.53-0.75),with 68% sensitivity, and 57% specificity for nuclear expression of DDIT4 in PTs versus adjacent normal tissues.For cytoplasmic expression of DDIT4, an AUC, sensitivity, as well as specificity were 0.70 (95% CI 0.59-0.80),66%, and 75%, respectively.The membranous expression of DDIT4 also had an AUC, sensitivity, and specificity of 0.62 (95% CI 0.53-0.71),63%, and 50%, respectively.Table 7 displays the overall diagnostic results for DDIT4 expression.

Discussion
Pancreatic tumor is recognized as the most fatal and aggressive malignancy among other gastrointestinal cancers, which is estimated to become the second leading cause of cancer-related deaths by 2030 1,46 .PT still has a high death rate, with a 5-year survival rate of around 10%, despite significant advancements and notable improvements in the detection and treatment of many malignancies 7 .By the time of diagnosis, PT often presents at an www.nature.com/scientificreports/advanced stage of the disease, and has often metastasized to other organs 47,48 .As a result, it seems sensible to discover innovative biomarkers for early detection, monitoring of recurrence and metastasis precisely, and the reduction of death related to the cancer.www.nature.com/scientificreports/DDIT4 is a main coding RNA whose expressions are altered under the effect of stressor factors, such as endoplasmic reticulum stress, hypoxia, heat shock, and ionizing radiation 20,49 .Research evidence showed that DDIT4 expression was dysregulated in various cancers 50,51 .The multifaceted involvement of DDIT4 in angiogenesis, cancer medication therapy, and the regulation of intricate intercellular signaling networks has led to the proposition www.nature.com/scientificreports/ that this molecule may possess a dualistic function in the initiation and advancement of cancer 27,35,38,39,52 .Notably, previous studies revealed that DDIT4 has cancer stem cell (CSC) traits, which include self-renewal properties, quiescence, and dysregulated DNA repair 31,38,[53][54][55] .CSCs, which are a subpopulation of cancer cells with selfrenewal properties, lead to the maintenance of tumors by generating differentiated tumor cells [56][57][58][59] .These features or characteristics of CSCs play crucial roles in heterogeneity, resistance to treatment, recurrence, and metastasis of tumors [56][57][58] .In addition, the elevated levels of DDIT4 expression increase the survival and growth of tumor cells through stabilizing HIF1α, phosphorylation of P53, and the inhibition of apoptosis pathways, and could also be related to the expression of stem cell markers 27,29,30 .
There was merely one study addressing the clinical significance of DDIT4 in pancreatic tumor, which showed that mutations in the 3'-UTR region of DDIT4 mRNA may affect autophagy by regulating the expression level of DDIT4 in PDAC tissues 49 .Therefore, the expression and function of DDIT4 in PT still need to be clarified.Thus, this is the first study that evaluates the expression, prognostic, and diagnostic value of DDIT4 in both mRNA and protein levels in either PDAC or PNET forms of pancreatic tumor.
Consistent with the result of the bioinformatics analysis, which demonstrated increased mRNA expression of DDIT4, our results in fresh tissue samples showed that mRNA levels of DDIT4 significantly increased in the pancreatic cancer patients when compared to adjacent normal tissues and had a positive correlation with the high grade of the cancer and also with invasion of the lymphovascular.These findings align with the study conducted by Fattahi et al., which showed that the upregulation of DDIT4 in colorectal cancer stem cell-enriched spheroids contributed to tumor advancement and metastasis 31 .Also, upregulation of DDIT4 was observed in GC tissues, which promotes proliferation and tumorigenesis 50 .These data show that increased mRNA expression of DDIT4 is substantially related to more aggressive tumor behavior.
In continuation, our research results indicate a considerable upregulation of DDIT4 protein expression in malignant cells as compared to the adjacent normal samples.Our findings are in agreement with prior studies showing the increased expression of DDIT4 protein in the nucleus, cytoplasm, and membranous tissues in various types of cancer 22,28,31,32,41,50,60,61 .It has also been reported that DDIT4 is expressed mainly in the cell membrane and cytoplasm of normal cells, while it is more expressed in the nucleus of tumor cells 20,62 , suggesting that translocation of the molecule from the membrane into the nucleus may be a risk factor for DDIT4 function changes that lead to oncogenes and may be associated with poor differentiation 22 .Our results confirmed this hypothesis showed that the increased nuclear level of DDIT4 has a positive correlation with invasion to the lymphovascular tissues in PDAC and a high grade of the disease in the PNET samples.Both results revealed that DDIT4 can be considered a risk factor for the progression of pancreatic tumor and plays an oncogenic role in the pancreas.In parallel with the findings, Wei et al. reported that the DDIT4 gene has a p53 transcription-factor binding site, thus it may play a critical role in the p53-dependent tumorigenesis in the nucleus 63 .Similarly, other studies have stated this fact that the high nuclear expression of DDIT4 protein is associated with advanced histological grade and enhances cancer proliferation and tumorigenesis 32,33,64 .To confirm the results, a review article by Ding, F., and colleagues collected the recent information regarding the roles played by DDIT4 in the progression of tumors and proved that the nucleus expression of DDIT4 is associated with tumor deterioration 42 .Therefore, DDIT4's functionality may be dependent on its precise subcellular location inside pancreatic cells.Notably, higher levels of DDIT4 nuclear expression have been associated with more aggressive tumor behavior.
Moreover, although there were no significant differences between the expression of DDIT4 and survival in either PDAC or PNET patients, the pancreatic cancer patients with the high nuclear expression of DDIT4 had shorter survival duration compared to those with the low nuclear expression of DDIT4, which was consistent with our clinicopathological findings.Our findings are parallel to the previous studies, concluding that DDIT4 expression is not a prognostic factor for tumors 32,50 .Besides, our survival analysis demonstrated that the patients with high cytoplasmic and membranous expression of DDIT4 had a higher survival rate in PDAC cases.Besides, our survival analysis showed that the patients with high cytoplasmic and membranous expression of DDIT4 had a higher survival rate in PDAC cases.However, there was no difference in DSS or PFS follow-up months between PNET patients with high cytoplasmic or membranous expression of DDIT4 and low cytoplasmic or www.nature.com/scientificreports/membranous expression.While cytoplasmic overexpression of DDIT4 protein was shown in several studies to be a poor prognosis factor for tumor progression in ovarian carcinoma, acute myeloid leukemia, OSCC, and BUC 22,28,33,65 .Moreover, in this study, distant metastasis was indicated as an independent prognostic factor for OS, DSS, and RFS in PDAC patients.Previous studies reported that metastasis has been significantly associated with disease progression as well as unfavorable outcomes 66,67 .Distant metastasis has been considered as the major cause of the high death rate in patients with PT 68 .As well, in terms of diagnosis, it has been shown that less than twenty percent of pancreatic cancer patients can be surgically removed at the time of their initial diagnosis 69 .Currently, carbohydrate antigen 19-9 (CA19- 19), which is extensively used in clinical practice, has been authorized by the US Food and Drug Administration (FDA) as the only biomarker for the detection of pancreatic tumor.Nevertheless, the specificity of the current biomarker is limited, thus necessitating the urgent exploration and creation of new biomarkers 70 .Hence, we aimed to assess the diagnostic accuracy of DDIT4 and its sensitivity and specificity in discriminating PT from adjacent normal tissue samples.Our results revealed that the expression levels of DDIT4 protein proved that the molecule may be considered the potential marker for diagnosis of pancreatic cancer patients with high sensitivity.Of note, these data support the direct role of DDIT4 in pancreatic tumors, which can be used to diagnose the PT.Table 6.Univariate and multivariate cox regression analyses of potential prognostic factors for Overall Survival (OS), Disease-Specific survival (DSS), and Progression-Free Survival (PFS) in patients with pancreatic ductal adenocarcinoma (PDAC).HR; hazard ratio, CI; confidence interval.The variables with P value less than 0.05 were included in multivariable analyses.Values in bold and italic are statistically significant.

Multivariate analysis HR (95% CI) P-value HR (95% CI) P-value HR (95% CI) P-value HR (95% CI) P-value HR (95% CI) P-value HR (95% CI) P-value
Age (years) www.nature.com/scientificreports/However, we suggest assessing the concentration of DDIT4 in the serum of PT patients, a noninvasive detection method, as a potential diagnostic index, which might be an innovative approach.
Our study presents persuasive and consistent evidence regarding the clinical significance of DDIT4 in PT patients.However, we must recognize some limitations.These include the relatively small number of PDAC and PNET samples utilized in the study, as well as the short duration of follow-up.These limitations may cast doubt on the prognostic role of DDIT4.Therefore, it is crucial to evaluate the expression of DDIT4 in a larger sample size and extend the duration of follow-up to confirm the diagnostic and prognostic role of this marker.
Furthermore, we also acknowledge that we did not explore the detection mechanism and signaling pathways of DDIT4 in PT.To gain better insight into the mechanisms and functions of DDIT4 in PT patients, it is advisable to determine the signaling pathways involved.

Conclusion
In summary, bioinformatics analysis confirmed that DDIT4 in mRNA level has increased in PT and can be considered as a potential prognostic marker.Our findings showed that DDIT4 at the level of mRNA and protein was upregulated in the PT compared to the adjacent normal tissues.In contrast to its cytoplasmic and membranous expression, higher nuclear level of DDIT4 is associated with more aggressive behavior, advanced disease, and a worse survival rate.Therefore, the nuclear expression of DDIT4 could serve as a promising marker and a potential therapeutic target for PT patients.However, further investigations should be conducted regarding the molecular mechanisms of DDIT4 in the progression of pancreatic tumor.

Data mining through bioinformatics tools (Investigation of DDIT4 in patients with pancreatic adenocarcinoma)
Gene Expression Profiling Interactive Analysis (GEPIA2) is an online bioinformatics tool for expression data from RNA sequence for The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx), tumor and normal samples 71 .In the primary search for the investigation of DDIT4 expression in sample tissues from patients with pancreatic adenocarcinoma, GEPIA2 was performed.Moreover, GEPIA2 was applied to evaluate the prognostic potential with the overall survival curve of the DDIT4 expression in these samples which was stratified according to the median expression of DDIT4 in GEPIA2.

Patient's characteristics and sample collection
In this cross-sectional study, 27 PDAC fresh tumor tissues and their adjacent normal tissue samples from 2020 to 2021 were collected from Firoozgar Hospital, a university-based hospital in Tehran, Iran.Patients who had undergone surgery without any prior chemotherapy/radiotherapy were enrolled in this study.The samples were transferred to the laboratory, and frozen in liquid nitrogen.Patient information, including gender, age, TNM stage, histological grade, lymphovascular invasion, perineural invasion, and lymph node metastasis were recorded.
Moreover, 200 formalin-fixed paraffin-embedded (FFPE) tissue samples from PT patients were collected from two university-based referral hospitals (Firoozgar and Imam Khomeini) in Tehran, Iran, between 2010 and 2021.These samples comprised two primary classifications of PT (PDAC and PNET) that we encounter within our hospitals.None of the surgical participants received adjuvant therapy.Hematoxylin and Eosin (H & E) stained slides and the medical archival documents were acquired to extract clinical and pathological parameters, comprising age, gender, tumor size (maximum tumor diameter), histological grade, TNM stage, distant metastasis, tumor recurrence, margin involvement, macroscopic tumor extension, lymphovascular invasion, perineural invasion, and lymph node metastasis.However, it is important to note that chemotherapy was virtually always given following surgery to all of our patients in accordance with recommendations, thus we did not include it as a variable in our analysis.Furthermore, 27 adjacent normal tissues were utilized to determine the expression of DDIT4 compared to cancerous samples.OS represents the duration starting from the surgery date until the date of either death or the last follow-up visit.DSS calculated from the date of surgery to the date of death related to the patient's tumor and PFS defined as the date of the primary surgery and the last follow-up visit for the patient with no evidence of disease, metastasis or recurrence, was also collected.Tumor staging was performed according to the pTNM classification for pancreatic tumor 72 .This study received its ethical approval (Code: IR.IUMS.REC.1400.350.) from the Research Ethics Committee of Iran University of Medical Sciences.All procedures,

RNA extraction and cDNA synthesis
Total RNA was extracted from fresh tumor tissue samples, as well as normal adjacent tissues, using RNA Minipreps Super Kit (Bio Basic, Canada) according to the manufacturer's instructions.All extracted RNAs were measured in quantity and quality by Nanodrop (ThermoFisher Scientific, USA).Then RNA was reverse transcript into cDNA using the cDNA Synthesis Kit (Yekta Tajhiz Azma, YT4500, Iran).

Real-time-quantitative polymerase chain reaction (RT-qPCR)
The specific primers (DDIT4, TPTEP1) for amplification with real-time-quantitative PCR (RT-qPCR) were designed as described previously 31 .RT-qPCR was performed using the Rotor-Gene Q Light Cycler (Qiagen, Germany) by the SYBR Green qPCR master mix (Yekta Tajhiz Azma, YT2551, Iran).After that, the mRNA level of the reference gene, GAPDH, was used as an internal control in each sample to normalize the relative expression levels of mRNAs.RT-qPCR data were analyzed using the comparative Ct method (known as the 2 -ΔΔCt method) to present relative expression levels of the genes 73 .The sequence-specific primers are shown in Table 8.

Tissue microarray (TMA) construction
Pancreatic tissue TMA blocks were constructed as described previously 74,75 .In brief, two experienced pathologists (B.B. and P.B) marked the three most representative tumor areas in different parts of each block after matching them with corresponding H&E slides.The chosen tumor sections of each block were then punched out with a 0.6 mm diameter and placed into a fresh recipient paraffin block using a precision arraying tool (Tissue Arrayer Minicore; ALPHELYS, Plaisir, France).Next, completed TMA blocks were cut at 4-μm sections and transferred to adhesive slides.To avoid tumor heterogeneity as a significant concern during the TMA method and increase the accuracy and validity of the data analysis, TMA blocks were constructed in three copies from each tumor specimen, and the mean scores of the three cores were calculated as the final score [76][77][78] .Notably, to compare the expression patterns of DDIT4 with tumor tissue specimens, adjacent normal tissue samples were also included in each TMA block 79 .

Immunohistochemistry (IHC) staining
DDIT4 expression was evaluated using the IHC method as previously described 80,81 .First, all TMA sections were deparaffinized at 60 °C for 40 min and then rehydrated with xylene and graded ethylic alcohol.To block endogenous peroxides and non-reactive staining, slides were treated with 3% H2O2 for 20 min at room temperature.
After washing the slides in Tris Buffered Saline (TBS), antigens were retrieved by immersing the tissue slides in citrate buffer (pH 6.0) for 10 min in an autoclave.Next, the slides were washed in TBS three times again and were stained by the primary antibody of anti-DDIT4 (Biorbyt, Cambridge, UK) with a dilution of 1:80 overnight at 4 °C.Besides, rabbit immunoglobulin IgG (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) was utilized at the same dilution of primary antibody for the isotype control test.The next day, following three times washing in TBS, TMA sections were treated with secondary antibody, including EnVision TM Mouse/Rabbit PolyVue HRP reagent (Dako; code K5007; Denmark), for 30 min at room temperature.Slides were incubated by 3,3′-diaminobenzidine (DAB) (Dako, Glostrup, Denmark) as a chromogen and, after washing in TBS, counterstained by Mayer's hematoxylin dye (Dako, Glostrup, Denmark) for 15 min to visualize the antigen.Eventually, the slides were dehydrated the same in graded ethylic alcohol, cleared in xylene, and mounted for examination.Furthermore, TBS was utilized instead of the primary antibody as a negative control and normal liver tissue as a positive control.

Evaluation of immunostaining and scoring score
The staining of DDIT4 protein marker on TMA slides was scored by two independent pathologists (B.B and P.B.) using a semiquantitative scoring system, blinded to the clinicopathological and survival parameters of the patients.To evaluate the expression of DDIT4, three scoring systems were used as follows: staining intensity, the percentage of positive tumor cells, and the H-score.The intensity of staining was scored based on a 4-point scale (negative or non-staining = 0, weak = 1, moderate = 2, and strong = 3), and the percentage of positive tumor cells was assessed and categorized into four groups (< 25, 25-50%, 51-75%, and > 75%).Then, the overall score was attained through the Histochemical score (H-score) by multiplying the staining intensity and the percentage of Table 8.Primer sequence of genes for RT-qPCR.

Figure 2 .
Figure 2. Boxplot of RT-qPCR data presented as median (min-max) for comparing expression levels of the (A) DDIT4 and (B) TPTEP1 genes between pancreatic tumor (PT) tissues and adjacent normal tissues; (C) Relationship between the expression of DDIT4 and histological grade; statistical significantly expression level of DDIT4 and increased with moderate/poor tumor histological grade.(D) Statistically increased expression level of DDIT4 was associated with lymphovascular invasion.Relationship between expression of DDIT4 gene and (E) stage, as well as (F) perineural invasion and (G) lymph node metastasis was not statistically significant.

Figure 3 .
Figure 3. Immunohistochemical analysis of DDIT4 protein expression in pancreatic samples.DDIT4 expression in pancreatic ductal adenocarcinoma: A low expression, B moderate expression, C high expression.In pancreatic neuroendocrine tumor: D low expression, E moderate expression F high expression.IHC staining of (G) adjacent normal tissue, normal liver tissues as (H) positive and (I) negative controls, and also (J) isotype controls (Figures shown with a magnification of 400 ×).

Figure 4 .
Figure 4. Box plot analysis of DDIT4 expression levels in tumor and normal tissue samples.The result of data analysis showed a statistically significant association between the (A) nuclear, (B) cytoplasmic, and (C) membranous expression of tumor and normal samples.
TGG GAC CGC TTC TCG TC R: GGT AAG CCG TGT CTT CCT CCG TPTEP1 F: AGC CGC AGA CAA AAG ACC TCGG R: CCA CCA AAC AGG CTT CGT GTGA Housekeeping gene GAPDH F: GCA CCG TCA AGG CTG AGA AC R: TGG TGA AGA CGC CAG TGG A

Table 1 .
Patients and tumor clinicopathological characteristic of pancreatic tumor and histological types of PTs.

Table 3 .
The association between expression of DDIT4 and clinicopathological features of pancreatic ductal adenocarcinoma carcinoma (PDAC) (P value; Pearson's χ2 test).P value; Pearson's chi-square.H-score; histological score.Values in bold and italic are statistically significant.

Table 4 .
The association between expression of DDIT4 and clinicopathological features of pancreatic neuroendocrine tumor (PNET) (P value; Pearson's χ2 test).P value; Pearson's chi-square.H-score; histological score.Values in bold and italic are statistically significant.

Table 5 .
The main characteristics of patients' survival analysis based on types of pancreatic tumor (PT).

Table 7 .
Diagnostic evaluation of the DDIT4 in pancreatic tumor patients.Values in bold and italic are statistically significant.includingobtaining informed consent from each human participant before surgery, were by the above-mentioned ethical standards.Thus, we confirm that all research was performed by relevant guidelines and regulations.