Clinical Signicance and Prognostic Value of the Expression of Fibronectin Type III Domain Containing 3B in Pancreatic Carcinoma

Background: Pancreatic carcinoma (PC) is one of the most aggressive cancers affecting human health. Identifying candidate biomarkers is essential for the early diagnosis and good prognosis of PC. Fibronectin type III domain containing 3B (FNDC3B) promotes many types of cancer, but its role in pancreatic cancer is not clear. The purpose of this study was to investigate the relationship between the expression of FNDC3B and incidence of PC. Methods: We downloaded data related to the levels of FNDC3B mRNA in patients of PC from the Cancer Genome Atlas (TCGA) database, and conducted in-depth research on this data and its clinical signicance through the R tools. We used GO and KEGG enrichment analyses to study the genes and signaling pathways that may be regulated by FNDC3B. Results: The results showed that the level of FNDC3B mRNA in PC tissues was higher than that in the normal tissues, and this was associated with proliferation and lymph node metastases of PC. Increased levels of FNDC3B mRNA also predict poor prognosis for the patients with PC. In addition, enhanced levels of FNDC3B mRNA were involved in the regulation of cell junction, tissue and epithelial cell migration, and several signal transduction pathways, including notch, TGF-β, VEGF, and Wnt. Conclusions: In short, high levels of FNDC3B mRNA may be associated with progression of PC and indicate a poor prognosis in patients with PC.


Background
Pancreatic carcinoma (PC) is a common gastrointestinal tumor with a high degree of malignancy and a poor prognosis. The 5-year survival rate is less than 6% [1], and its morbidity and mortality have been increasing. It is estimated that by the year 2020, the number of new cases of PC in the world will be approximately 420,000 and the number of deaths due to it will be approximately 410,000 [2]. By 2030, PC will become the second most common malignant tumor, with high mortality rate [3]. Genes and environment are two important causes of PC. Many unhealthy lifestyle choices, such as smoking, unbalanced diet, and obesity are also important risk factors for PC. Previous epidemiological studies have shown that chronic pancreatitis increases the risk of pancreatic cancer [4]. Due to the lack of identi able clinical symptoms and speci c markers at the early stages, the tumors escape early detection, grow rapidly and metastasize early. Because of this, more than 90% of patients are clinically diagnosed only when the pancreatic cancer is in the middle and advanced stages, missing the best time for treatment [1]. Therefore, early detection, diagnosis and treatment, are the key to reduce the mortality rate of the PC, and to improve the quality of life of the patients. Identi cation of speci c markers to facilitate early diagnosis of PC is an active area of research at present [5][6][7].
Fibronectin type III domain containing 3B (FNDC3B), is a protein of the FNDC3 family that includes FNDC3A, FNDC3B and FNDC3C, also known as factor for adipocyte differentiation 104 (FAD104), it contains a proline-rich region, and 9 bronectin type III structures domain and a transmembrane region, and is a regulator of adipocyte and osteoblast differentiation [8][9][10][11][12]. Fibronectin type III (FNIII) domain of FNDC3B serves as a tail sheath, integrates with different proteins, and plays an important role in cell adhesion and growth signaling [9,13,14]. Homozygous disruption of FNDC3B causes mice to die at birth from abnormalities in the lungs [14,15], and has a signi cant effect on the adhesion, proliferation and migration of mouse embryonic broblasts [9,16]. Cell adhesion is important in tumorigenesis because it provides tumor cells with the necessary contacts and cell-matrix interactions needed for cell signaling, proliferation, and migration [17]. FNDC3B is one of the most common genes up-regulated in cancer tissues, regardless of the cancer type and tumor origin [18]. Recent studies have found that FNDC3B is highly expressed in breast cancer, esophageal cancer, glioblastoma, and hepatocellular carcinoma, and is associated with cancer cell proliferation, invasion and metastasis [19][20][21][22][23]. Therefore, we hypothesized that FNDC3B promotes cell migration and tumorigenesis in PC. In this study, our bioinformatics analyses explored the role of FNDC3B in pancreatic cancer, as well as the processes and signaling pathways it may regulate.

Methods
Evaluation of transcription level of FNDC3B in PC patients RNA-sequence data of PC patients along with individual clinical information were downloaded from the Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov/). An in-depth analysis of FNDC3B expression at the transcriptional level in unpaired PC samples was performed by R version 3.6.2 (https://cran.r-project.org/src/base/R-3/) using the limma package version 3.8 (https://bioconductor.org/packages/release/bioc/html/limma.html) and Statistical Computing version 3.5.1 software.

Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis
According to the median value of FNDC3B mRNA expression, PC patients from the TCGA database were divided into high FNDC3B mRNA group and low FNDC3B mRNA group. Signaling pathways and molecules related to FNDC3B expression in PC patients were studied by GO and KEGG enrichment analysis (http://www.broadinstitute.org/gsea/index.jsp).

Evaluation of overall survival rate by KM curves
The overall survival rate in PC patients from both high and FNDC3B mRNA groups was assessed by KM (Kaplan-Meier) curves and the log-rank test. Patients of PC who had over ve years of survival were considered as with a long overall survival (OS) had, whereas those who died or experienced recurrence in less than 2 years were classi ed as with a short OS. A value of P < 0.05 was considered statistically signi cant.

Statistical analysis
The mRNA levels of FNDC3B in patients of PC are expressed as the mean ± standard deviation. Student's t-test (unpaired) was used for comparison between two groups, and analysis of variance (ANOVA) was used to identify signi cant differences between multiple groups. All statistical analyses were performed with SPSS 21.0 software (IBM, Armonk, NY, USA). A P value < 0.05 was considered statistically signi cant.

AUC value determination for the ROC curve
The area under the curve (AUC) value was computed for the ROC curve to assess the discriminatory ability of FNDC3B expression in patients of PC, with distinct clinical parameters.

Results
Gene set enrichment analysis of FNDC3B expression in patients of PC To investigate the effect of upregulated FNDC3B expression in PC, we performed gene set enrichment analysis of FNDC3B expression in patients, with corresponding clinical information from TCGA datasets. Our results showed that the expression of FNDC3B was signi cantly upregulated in patients with advanced PC and distant metastasis. We also found that the mRNA level of FNDC3B was higher in patients of PC with good OS than in those with poor prognosis, whereas no signi cant difference in the expression of FNDC3B was observed in patients according to age, sex and lymph node metastasis. These results suggest that upregulation of FNDC3B is associated with distant metastasis of PC and predicts a poor prognosis for patients ( Fig. 1).

Clinical and molecular characteristics of PC with high FNDC3B expression
We further investigated the mRNA expression of FNDC3B in patients with PC from the TCGA database, and found that the mRNA level of FNDC3B was signi cantly higher in the cancer cells than in corresponding noncancerous normal tissues ( Fig. 2A). In grade (G stage) G2 and G3, FNDC3B mRNA levels were higher compared to G1 (Fig. 2D). In the TNM stage II, the level of FNDC3B mRNA was higher than in stage I (Fig. 2E). In patients older than 65 years, FNDC3B mRNA levels were higher than in younger patients (Fig. 2C). In the advanced tumor stage (T stage) T3, the FNDC3B mRNA level was higher than in T2 (Fig. 2F). Gender-wise, the FNDC3B mRNA level in male patients was comparable to that in female patients (Fig. 2B). These results showed a signi cant association of higher expression of FNDC3B with advanced age, G stage, tumor stage, and TNM stage, but not with the sex of the patient.
Upregulation of FNDC3B mRNA levels indicates poor prognosis for patients of PC PC patients from the TCGA data set were divided into two groups, based on the median expression of FNDC3B mRNA, to investigate the prognostic role of FNDC3B in PC. We found that level of FNDC3B mRNA was higher in patients with lymph node metastasis, and poor OS, and patients who had died ( Fig. 3A, 3C, 3D), whereas it was lower in patients with distant metastasis (Fig. 3B). Overall, these data suggested that upregulation of FNDC3B mRNA levels indicated poor prognosis for patients with PC. This may serve as a potential indicator of prognosis in patients with PC.
The ROC curve shows that high levels of FNDC3B mRNA is associated with PC Using ROC curves, we analyzed whether expression levels of FNDC3B mRNA can effectively distinguish PC patients with different pathological parameters, to con rm that FNDC3B mRNA expression is related to cancer cell proliferation and poor prognosis. FNDC3B mRNA expression could be used to distinguish PC tissues from normal tissues, with an AUC of 0.9510 (95% CI: 0.9253-0.9767; P < 0.0001) (Fig. 4A). We also found a signi cant difference in FNDC3B expression in PC subgroups, including subgroups of G stage ( Fig. 4B-4I). These results con rm that higher levels of FNDC3B mRNA is associated with cancer proliferation, metastasis and poor prognosis in PC.
High FNDC3B mRNA level correlates with short OS in PC patients KM survival analysis was used to determine the correlation of level of FNDC3B mRNA with OS of the patients with PC. Patients with higher levels of FNDC3B mRNA had shorter OS than those with lower levels (Fig. 5A, P = 0.0025). Moreover, we analyzed the association between OS and FNDC3B mRNA level in various clinical subgroups of PC. As shown in Fig. 5B GO enrichment analysis shows that FNDC3B mRNA level is associated with genes involved in regulation of cell junction, tissue migration and epithelial cell migration To identify the molecules regulated by FNDC3B, we performed GO enrichment analysis. We found that higher level of FNDC3B mRNA was associated with regulation of cell junction, tissue migration and epithelial cell migration (Fig. 6A). Furthermore, the it was also strongly associated with HIF1A, ADAM17, CORO1C, ITGB1, LIMS1, MAP4K4, PDLIM5 and PIK3CA (Fig. 6B). These results indicate that high level of FNDC3B mRNA is associated with junction and migration of PC cells.

KEGG enrichment analysis shows that FNDC3B is associated with pathways related to signal transduction
To investigate the association between FNDC3B and signaling pathways, we performed KEGG enrichment analysis. The results showed that FNDC3B expression was positively associated with PC. We also found that FNDC3B expression was signi cantly enriched in four main well-studied signaling pathways in PC, including the notch, TGF-β, VEGF and Wnt signaling pathways (Fig. 7A). Moreover, the expression of genes in these four signaling pathways, including ACVR1, BMPR2, CTNNB1, ITGAV, MAPK1, NOTCH2, PIK3CA and ROCK1, was associated with the level of FNDC3B mRNA (Fig. 7B). All these data suggest that level of FNDC3B mRNA is associated with four main signaling pathways in PC.

Discussion
PC is an aggressive disease with almost no symptoms until the cancer is well established [24,25]. Carbohydrate antigen 19 − 9 is the only diagnostic marker approved by the FDA, but its diagnostic potential is limited due to its limited sensitivity and speci city [26,27]. Therefore, screening new tumor biomarkers is important for the early diagnosis and targeted treatment of PC. In this study, by analyzing the TCGA database, it was found that the level of FNDC3B mRNA was increased in tissues involved in PC, which indicates that FNDC3B may be involved in PC progression.
Many studies have con rmed that FNDC3B promotes tumor progression. Lin et al. found that FNDC3B enhances the migration and metastasis of hepatocellular carcinoma cells by binding to annexin A2 (ANXA2) through the FNIII1-4 domain [14]. Cai et al. con rmed that FNDC3B increases the proliferation of liver cancer cells by inducing epithelial to mesenchymal transition (EMT) to activate the PI3K / AKT, Rb1 and TGF-β pathways [28]. Studies have found that miR-129-5p binds to the 3'UTR of FNDC3B and downregulates its expression, leading to inhibition of the proliferation, invasion and migration of glioma cells [21]. Silencing FNDC3B expression in tongue squamous cell carcinoma cells can inhibit the occurrence of EMT, suggesting that FNDC3B plays an important role in promoting the proliferation, invasion and migration of tongue squamous cell carcinoma cells [29]. Present study found that FNDC3B mRNA levels had increased in PC tissues with lymph node metastasis. In addition, high levels of FNDC3B mRNA in PC promote cell junction, tissue migration, epithelial cell migration, and positively regulate several signaling pathways, including notch, TGF-β, VEGF and Wnt. This indicates that FNDC3B may promote migration of PC cells. However, Zhang et al. found that miR-143, upregulated by NF-κB transcription, inhibited the expression of FNDC3B, which promoted the metastasis of HBV-related liver cancer [30]. Fan et al. also found that miR-143 was signi cantly up-regulated during the differentiation of prostate cancer stem cells, and that the increased miR-143 could inhibit the expression of FNDC3B and promote the metastasis of prostate cancer [31]. Studies have shown that tumor cells can promote tumor invasion and metastasis by inducing EMT. During this process, the expression of β-catenin is up-regulated, thereby inhibiting the transcription of E-cadherin, weakening the cell-to-cell connection and spindle-shaped cell transformation, which further enhances its ability to migrate, accompanied by down-regulation of the expression of epithelial markers and up-regulation of interstitial markers [32,33].
Overall, our results highlight the involvement of FNDC3B mRNA in tumorigenesis and metastasis. These results need to be further veri ed by testing PC tissues and in vivo and in vitro studies.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Availability of data and material
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
This study was funded by Fujian Province Natural Science Foundation (grant number 2018J01266) and Joint Funds for the Innovation of Science and Technology, Fujian Province (grant number 2017Y9074).
Authors' contributions LQM, XHL, KXD and XQK designed the study, downloaded data from TCGA, performed KEGG enrichment analysis and KM survival analysis, and drafted the manuscript. JLL and HHL were responsible for GO enrichment analysis and OS analysis. XYL and JG were responsible for ROC curve analysis. All authors read and approved the nal manuscript for publication.  TNM stage and (F) T stage. TCGA-CRC, The Cancer Genome Atlas colorectal carcinoma; *p < .05, **p < .01, ***p < .001, and ****p < .0001; ns denotes not signi cant; data were analyzed by Student's t-test or ANOVA. G stage: grade stage; G1: grade 1; G2: grade 2; G3: grade 3; G4: grade 4.

Figure 3
High levels of FNDC3B mRNA were correlated with poor prognosis in patients of PC based on the data from TCGA datasets. (A) Comparison of the levels of FNDC3B mRNA in non-metastatic PCs and PC with lymph node metastasis. (B) Comparison of FNDC3B mRNA levels between non-metastatic PC and PC with distant metastasis. (C) Levels of FNDC3B mRNA in PC with good and poor OS. (D) Comparison of levels of FNDC3B mRNA between survival and death of patients with PC. *p < 0.05, **p < 0.01; ns denotes not signi cant; data were analyzed by Student's t-test. OS-good referred to patients who survived or are disease-free for ≥5 years. OS-poor referred to patients who succumbed to the disease or relapsed within two years.