Fibronectin 1: A Potential Biomarker for Ovarian Cancer

Methods OVCAR3 and A2780 are the two common cell lines that are used for ovarian cancer studies. The different invasion and migration abilities were observed by scratch tests and transwell experiments in our preliminary study. Gene chip was used to screen the expression gene in these two different cell lines, and then, the differentially expressed genes (at least 2-fold difference, P value < 0.05) were analyzed using KEGG. Result Fibronectin 1 (FN1) was found to be the most strongly correlated with the invasion and migration abilities of the OVCAR3 cells. Real-time PCR and FN1 knockout cell line was conducted and confirmed this finding. Based on the Oncomine database analysis, comparing with normal people, ovarian cancer patients exhibited high levels of FN1 expression. Additionally, higher FN1 expression was found in patients with higher FIGO stages of cancer. Conclusion FN1 could be a new biomarker for ovarian cancer detection and progress indicator.


Introduction
Ovarian cancer is the fifth leading cause of cancer-related deaths among women worldwide; unfortunately, early detection tests are relatively lacking. Furthermore, most women with ovarian cancer are diagnosed in the late stages of the disease, which portends a poor prognosis [1,2]. Current surveillance strategies, which are achieved by transvaginal ultrasound or serum tumor marker cancer antigen 125 (CA125) tests, are ineffective in detecting ovarian cancer at an early stage [3,4]. Therefore, the early detection and diagnosis of ovarian cancer have a very important role in the survival and prognoses of patients with epithelial ovarian cancer.
Fibronectin (FN) is a high-molecular-weight glycoprotein of the extracellular matrix that mediates a wide variety of cellular interactions with the extracellular matrix (ECM) and plays important roles in cell adhesion, migration, growth, and differentiation [5,6]. FN1 is a member of the FN family, which has different functions during a variety of biological processes including cell adhesion, cell migration, and cytoskeletal organization in many different diseases [7]. In a variety of tumors, such as nasopharyngeal carcinoma, osteosarcoma, esophageal cancer, and ovarian cancer, FN1 is an important tumor-related gene [8][9][10][11].
Currently, the cell lines A2780 and OVCAR3 are commonly used in many different studies of ovarian cancer [12,13], but no examinations of the differences between these two cell lines have been reported. In our study, greater migration and invasion abilities were found in the OVCAR3 cells compared with the A2780 cells. Therefore, we want to see if good protein candidates could be used as diagnostic markers for ovarian cancer based on these phenomena.

Cell Scratch Test.
Cells were seeded into six-well plates, and when the density reached 100%, a scratch was made through the cells. Photographs were taken at 48 h, 72 h, or 96 h after the scratch using an inverted tissue culture microscope (Nikon ECLIPSE Ti-U) at ×4 magnification.
2.3. Transwell Migration Assay. The two ovarian cancer cell lines, i.e., A2780 and OVCAR3 (5 × 10 4 cells/well), were seeded on transwell inserts (polycarbonate filters with 8 μm pores, Costor) filled with medium. Medium (600 μl) was added to the bottom chamber. After 48 h of culture at 37°C under humidified 5% CO 2 in air, the cells that remained inside the upper chamber were gently and completely removed with a cotton swab. The cells that migrated to the lower surface of the filter membrane were fixed with anhydrous methanol and stained with 0.1% crystal violet. The filters were air dried, and photographs were collected with an inverted phasecontrast microscope (Nikon ECLIPSE Ti-U). After collecting the photographs, the membrane was washed with 500 μl 50% aqueous acetic acid solution for approximately 5 min, 100 μl of an eluent was added to a clean 96-well plate, and the OD values were measured at 595 nm using an ELISA reader (Synergy 2, China).

Microarray
Analyses of the Nucleic Acids. Microarray analyses of the nucleic acids were performed using Affymetrix® Human Genome U219 Array Stripes (Affymetrix, USA) according to the manufacturer's instructions. The procedure included genomic DNA extraction fromA2780 or OVCAR3, digestion and ligation, PCR amplification, PCR product purification, quantification and fragmentation, labeling, array hybridization, washing, and scanning. Gene expression analyses were performed using the Affymetrix® Transcriptome Analysis Console (TCA) software, which evaluates the expressions of different genes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) was also used for subsequent related pathway analysis.
2.5. Fluorescence Quantitative Real-Time PCR. RNA was isolated from each sample using TRIzol reagent (Ambion, USA) according to the manufacturer's instructions. First-strand cDNA was synthesized from the total RNA using oligo (dT) primers from the FastQuant RT Kit. Real-time PCR was performed on a stratagene MX3005P. Glyceraldehyde-3phosphate dehydrogenase (GAPDH) was used as an internal control. The SYBR Green PCR Master Mix Reagent Kit was utilized for the PCRs, and the relative gene expressions were determined based on the threshold cycles (Ct) of the target gene and the internal reference gene. The average Ct value of the GAPDH gene was subtracted from the average Ct value of each target gene. The fold change (2 -ΔΔCt ) in expression was calculated for the gene of interest relative to the internal control gene (GAPDH) for each of the analyzed cancer cell lines. The primer sequences used in PCRs were as follows: FN1 sense: 5′-GTTCGGGAGGAGGTTGTTA CC-3 ′ and antisense: 5 ′ -GAGTCATCTGTAGGCTGGTTT AGG-3 ′ and GAPDH: sense: 5 ′ -CCTCCAAGGAGTAAGA CCCC-3 ′ and antisense: 5 ′ -AGGGGTCTACATGGCA ACTG-3 ′ . Each sample was run in triplicate.
2.6. FN1 KO Cell Construction. 293T cells were seeded on the plate, and then, the cells were cultured when the fusion degree was up to 80%. Lenti-CRISPR-V2-sgRNA-FN1 or lenti-CRISPR-V2, psPAX2, and pMD2.G were mixed into 1 ml Opti-MEM medium under a ratio of 4 : 3 : 1 and then incubated at room temperature for 5 min. 30 μl PEI was added into the mixture, and then, the mixture was incubated at room temperature for 30 min. After incubation, the mixture was added to 293T cells and incubated at 37°C for 6 h with 5% CO 2 . After 48 h, the cell culture medium was changed to low serum medium (4% FBS), and the virus packaging supernatant was collected and stored at -80°C.
OVCAR3 cells were incubated for 24 h before virus transfection, and then, virus packaging supernatant with the final concentration of 10 μg/ml polybrene mixed with complete medium under 1 : 1 ratio was performed into the OCVAR3 cells. After 36 hours of continuous culture, 0.2 μg/ml puromycin was added into the medium for FN1 KO cell screening.
2.9. Statistical Analysis. SPSS software was used for statistical analysis. The results are expressed as the mean ± SD or the mean ± SEM. Two-tailed unpaired Student's t-tests were used to determine the statistical significance, and P < 0:05 was considered statistically significant.

Detecting the Difference of Migration and Invasion
Abilities. To determine the differences of the migration and invasion abilities of these two ovarian cancer cell lines, scratch and transwell experiments were performed. The cells were seeded in 6-well plates, and the migration abilities were measured at 0 h, 48 h, 72 h, and 96 h. Compared with those in the A2780 group, the diameters of the scratches became narrow in the OVCAR3 group (Figure 1(a)). This difference indicated that the OVCAR3 cells exhibited stronger migration ability than A2780 cells. The invasion abilities were determined with the transwell experiments, and the numbers of invasive cells were counted at 48 h after the cells were seeded. The results revealed that a much greater number of invasive cells were observed in the OVCAR3 group ( Figure 1(b)). This conclusion was also confirmed by the 595 nm OD reading results after the invasive cells were dissolved in 50% aqueous acetic acid solution (Figure 1(c)). Based on these results, the migration and invasion abilities 2 Disease Markers of the OVCAR3 cells were much greater than those of the A2780 cells.

Determining the Different mRNA Expressions.
To determine the cause of the differences between these two cell lines, gene chip analysis was conducted with the OVCAR3 and A2780 cells. Many differentially expressed genes were found in these two ovarian cancer cell lines (Figures 2(a) and 2(b)) based on analyses of the volcano map results and the hierarchical clustering results. Additionally, the gene expressions of these two cell lines were analyzed in terms of the following four different gene ontology (GO) aspects: molecular function, biological processes, cellular components, and protein ( Figure 2(c), continued). The results indicated that the two types of cells, i.e., A2780 and OVCAR3, exhibited significant differences both in gene expression and cellular biology.

Potential Protein Candidate Analysis.
To identify different proteins, cell pathway analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG). The differentially expressed genes between the two cell lines are presented in Table 1. The pathway with the greatest number of differences was the focal adhesion pathway. Moreover, some candidates genes (P < 0:0001 and fold change ≥ 100) among the focal adhesion-related genes (i.e., CCND2, ITGB8, and FN1) were selected for further analysis (Figure 3(a)). Statistical analyses of the expressions of these three genes revealed that the difference in FN1 expression was the most statistically significant (Figure 3(b)). To confirm the array results, real-time PCR was used to verify the difference in FN1 expression between the OVCAR3 and A2780 cells. The results revealed that the OVCAR3 cells did express FN1 at a higher level than the A2780 cells (Figure 3(c)), which proved that FN1 plays an important role in cell migration and that a high level of FN1 expression indicates high tumor cell mobility.
To further confirm the results, FN1 KO cell line was constructed by CRISPR. Comparing the four different ovarian cancer cells, high FN1 expression was found in OVCAR3 at mRNA level (Figure 4 The differences in the migration and invasion abilities among the wild-type group, the control group (empty vector V2), and the KO group were detected by transwell experiments. The cells were seeded in 6-well plates, and the migration abilities were measured at 48 h. Compared with both the wild-type group and the control group, a significant less number of invasive cells were observed in the KO group (Figures 4(f) and 4(g)). Based on these results, FN1 should be a very important protein for the migration and invasion abilities of the ovarian cancer cells.

FN1 Clinical Feasibility Evaluation.
Clinical samples were used for a FN1 feasibility evaluation. According to the above results, stronger invasion and migration abilities are associated with greater FN1 expression. This relationship indicates that FN1 expression could be used as a good indicator for disease progress.
To examine this possibility, the FN1 expression levels in patients with clinical ovarian cancer were identified using the Oncomine database with set conditions of P < 0:05 and a fold change of >3 or <-3. This analysis revealed that different pathological types of ovarian tissue exhibited different FN1 expression levels; ovarian mucinous adenocarcinomas ( Figure 5(a)), ovarian clear cell adenocarcinomas (Figure 5(b)), ovarian endometrioid adenocarcinomas (Figure 5(c)), and ovarian

Discussion
Epithelial ovarian cancer (EOC) is one of the most common cancers among females and is the fifth leading cause of cancer death among women in the United States with 21,290 new cases and 14,180 deaths in 2015 [14]. Unfortunately, due to the lack of early detection markers and technical skills, most patients are diagnosed in the advanced stage of the disease, which is associated with a very poor survival rate and high levels of metastasis [15][16][17]. In our paper, we compared two different ovarian cancer cell lines with different migration and invasion abilities and found a new protein marker, FN1, which seems to be a strong candidate marker for the diagnosis of aggressive ovarian cancer. FN1 could also potentially be applied as an indicator of ovarian cancer progression or metastasis. OVCAR3 and A2780 are the cell lines that are most commonly used in ovarian cancer studies [18][19][20][21]. In our laboratory, we found that these two different cell lines exhibit strongly different cell migration and invasion abilities. Transwell and scratch experiments were conducted to confirm these differences. The results revealed that the OVCAR3 cell line exhibited significantly stronger invasion and migration abilities than the A2780 cell line. To understand the different abilities of these two cell lines, gene chips were used to analyze the differences in gene expression. All are differentially expressed if they met the condition of a fold change greater than 2 or less than -2 with a P value < 0.05. To narrow the set of differentially expressed genes, stricter conditions were applied for the gene screening. After applying these stricter conditions (cell adhesion related, P < 0:001, and a fold change of greater than 100 or less than -100), only three genes (i.e., CCND2, ITGB8, and FN1) were found. After the expressions of the CCND2, ITGB8, and FN1 genes in the gene chip were statistically analyzed, the gene that was most strongly correlated with the invasion and migration abilities was found to be FN1. Next, real-time PCR experiments were used to confirm that the OVCAR3 cells did exhibit higher levels of FN1 expression than the A2780 cells, which exhibited weaker invasion ability than the OVCAR3 cells. And the FN1 KO cell line was used for further confirmation.
FN1 is a core component of many extracellular matrices where it regulates a variety of cell activities through direct interactions with cell surface integrin receptors. FN1 is synthesized by many adherent cells and then assembled into a fibrillation network [2]. Moreover, FN1 expression has been demonstrated to be closely associated with various migration processes, including wound healing, embryogenesis, and   [23]. Another report also indicated that FN1 can significantly modulate the progression of glioma cells by preserving integrin β1 FN receptors in glioma cells [24]. Moreover, Franke et al. and Kujawa et al. also observed that fibronectin was an important prognostic factor in ovarian cancer and may be central to tumor progression [25,26]. Based on these results, we believe that FN1 could be involved in the progression of ovarian cancer and could be the main reason for the differences in the migration and invasion abilities of these two cell lines. Actually, it has been reported that FN1 can prevent the apoptosis of ovarian cancer cells caused by therapeutic agents. It was suggested that FN1 could be used as a marker to indicate tumor progression in ovarian cancer. This hypothesis was confirmed by the clinical samples.

Disease Markers
In our study, online database analysis (Oncomine) [10] was used to examine the expression levels in patients, and ovarian cancer patients were found to have higher FN1 expression levels than normal subjects. Additionally, there was a significant increase in FN1 expression in stage III cancer compared with lower FIGO stage cancers (stages I and II). Greater FN1 expression was associated with a higher FIGO stage. Both of the results indicated FN1 should be a good marker for ovarian cancer patients.
In conclusion, based on in vitro experiment results and the results from the online database analysis, we believe that FN1 could be used as a marker of ovarian cancer detection and could also be used as a progress indicator for ovarian cancer patients.

Data Availability
All the data has been presented in the paper.

Conflicts of Interest
The authors do not have any conflicts of interest.