Int J Clin Exp Med 2019;12(11):12825-12832 www.ijcem.com /ISSN:1940-5901/IJCEM0099329. Original Article The expression of FOXC1 in non-small cell lung cancer and its effect on the biological behavior of carcinoma cells. Rong Chen, Yahui Shen, Huiyu Lu. Department of Respiratory and Critical Care Medicine, Taizhou People’s Hospital of Jiangsu Province, Taizhou 225300, Jiangsu, China. Received July 7, 2019; Accepted October 3, 2019; Epub November 15, 2019; Published November 30, 2019. Abstract: Objective: To investigate the impact of FOXC1 expression on the clinical prognosis of non-small cell lung cancer (NSCLC) patients and the biological behavior of NSCLC cells. Methods: We analyzed the correlations of the expression pattern of FOXC1 with patients’ clinicopathological features and prognosis. Immunohistochemical staining was carried out to determine the expressions of FOXC1 in the NSCLC tissues and the corresponding tumor- adjacent normal tissues in 97 patients. Additionally, A549-shFOXC1 cell lines were constructed, with the A549-shNC cell line and the A549 cell line as the negative control and blank, respectively. FOXC1 expressions were determined using Western blot in the cell lines and the normal human lung cell line MRC-5. Furthermore, the proliferation and invasion abilities of these NSCLC lines were determined using a plate colony formation assay and a Transwell assay. Results: Compared with the tumor-adjacent normal tissues, the positive expression rate of FOXC1 was significantly higher in the NSCLC tissues (P < 0.05). Tumor malignancy and the positive expression rate of FOXC1 are positively correlated with TNM stage, number and size of lymphatic metastatic regions, tumor diameter, and tissue infiltra- tion. Patients with positive expressions of FOXC1 had a 5-year survival rate of 11.11%, which is 28.00% lower than those with negative expressions of FOXC1 (P < 0.05). Compared with the MRC-5 cell line, FOXC1 was observably up-regulated in the A549 cell line (P < 0.05), and the knockdown of FOXC1 decreased the proliferation and invasion of the A549 cells (P < 0.05). Conclusion: FOXC1 is highly expressed in NSCLC, and its expression is significantly cor- related with patients’ prognosis. Furthermore, FOXC1 promotes the proliferation and invasion of A549 cells.. Keywords: Lung cancer, clinical prognosis, FOXC1, E-Cadherin, proliferation, invasion. Introduction. As air pollution becomes increasingly serious and more people start to smoke, lung cancer has ranked first in prevalence and mortality among all malignancies in Europe, the Americas , and China . Non-small cell lung cancer (NSCLC), is a different kind of lung cancer from small cell lung cancer (SCLC), accounting for nearly 80% of all lung cancer cases, including adenocarcinoma, squamous carcinoma, and adenosquamous carcinoma . Lung cancer is also known for its extremely low 5-year survival rate, despite the tremendous efforts in research for an efficient way to treat it. In addi- tion, almost 30% of patients with lung cancer usually have distant metastasis at the time of diagnosis due to the lack of specific symptoms. in the disease’s early stages. Thus, it is quite urgent to determine the risk factors affecting the prognosis of NSCLC [4, 5].. Forkhead box (FOX) proteins are a highly con- servative transcription factor family of proteins with a high structural similarity, which are impli- cated in a series of biological behaviors involv- ing cell proliferation, differentiation, apoptosis, and metastasis [6, 7]. FOXC1, as a member of the FOX family, has the most conservative DNA- binding and transcription regulation regions. The conservative DNA-binding region, consist- ing of 100 amino acid residues in a helical structure, is located in chromosome 6p25 with a length of 3500 bp. FOXC1 encodes a protein containing 553-amino acid residues encoded by a 1600-bp exon . From N-terminal to. http://www.ijcem.com. The expression of FOXC1 in non-small cell lung cancer. 12826 Int J Clin Exp Med 2019;12(11):12825-12832. C-terminal, FOXC1 includes the following re- gions: AD1, FHD, AD2, and NLS in each end of FHD. Recent evidence has shown that FOXC1 expression is associated with the development and progression of laryngeal carcinoma and breast cancer.. Currently, there is little information regarding the correlation of FOXC1 with the clinical prog- nosis of NSCLC and its effect on the biological behavior of carcinoma cells. Thus, we analyzed the correlation of FOXC1 expression with the clinical prognosis of NSCLC and explored the effect and mechanism of FOXC1 on the prolif- eration and invasion of NSCLC cells.. Material and methods. Subjects. Between March 2012 and March 2014, a total of 194 samples were collected from the NSCLC tissues and tumor-adjacent normal tissues of 97 patients who underwent surgical resection in this hospital. Inclusion criteria: a) Patholo- gical finding of tumor tissues showing no com- plications of other tumors; b) Pathological find- ing of the region at 2 cm outside the tumor showing no cancerization; c) Patients with no history of radiotherapy, chemotherapy, or other treatment prior to the surgery, but with the inte- grated follow-up data. Among the patients, 50 were males and 47 were females, between 38 and 80 years old, with an average age of (55.16±10.43) years old. There were 41 patients under 50 years old and 56 patients over 50 years old. As for the TNM staging, there were 33 patients in stages I and II, and 64 patients in stages III and IV. The patients were all diagnosed with adenocarcinoma, including low-differentiation adenocarcinoma (65 cases), moderate-differentiation adenocarcinoma (21 cases), and high-differentiation adenocarcino- ma (11 cases). Considering metastasis, 74 patients were found with lymphatic metastasis, and 23 with no metastasis. As for the tumor diameter, there were 69 patients with tumors having a diameter ≥ 3 cm, and 28 patients with tumors having a diameter < 3 cm. For the infil- tration depth, the tumors in 24 patients did not reach the serous membrane, but the tumors broke through the serous membrane in 73 patients. All the patients accepted the 5-year follow-up through regular clinic visit or tele- phone, and the overall survival (OS) were evalu-. ated from the surgery to the death or the end- ing date of the follow-up. Patients and their families were informed of the study protocol, and this study was approved by the Ethics Committee of Taizhou People’s Hospital of Jiangsu Province.. Cells and reagents. We obtained: 293FT cells, A549 cells (NSCLC cells), and MRC-5 cells (normal human lung cell line) (Cellbank, Shanghai Institute of Biochemistry and Cell Biology, CAS); pLVTHM- shFOXC1 and pLVTHM-shNC, as the negative control (Shanghai GenePharma Co., Ltd.); Anti-FOXC1 antibody-ChiP Grade (Polyclonal antibody of anti-goat to FOXC1-ChiP-Grade; ab5079) and goat anti-rabbit IgG H&L (HRP) (ab205718; Abcam, US). We also obtained a two-step detection kit (PV9000) and a DAB kit (ZLI-9018) (Beijing Zhongshan Golden Bridge Co., Ltd).. FOXC1 expression determined by immunohis- tochemistry. The NSCLC samples and the normal samples were regularly fixed and embedded in paraffin for sectioning. The sections were then subject- ed to the regular dewaxing and hydration proce- dures, then they were placed in a citrate buffer for antigen retrieval under high pressure. Thereafter, the sections were immersed in 3% H2O2 to block the endogenous peroxidase, fol- lowed by blocking. Then, on the sections, pre- diluted anti-FOXC1 antibody was added for incubation at 4°C overnight, and then the sec- tions were washed in the PBS at room tempera- ture. The sections were then incubated in the presence of goat anti-rabbit IgG for 30 min at room temperature, and the reaction was stopped by rinsing with PBS. Immunoblots on the sections were stained in DAB for 5 min and then deionized water was dropped on the sur- face to terminate the reaction. The sections were placed in hematoxylin for counterstain- ing, and washed in tap water several times and dehydrated in gradient ethanol.. Evaluation of the results: Two experienced pathologists were blinded to the pathological sections. On each section, 5 high-power fields were randomly selected, and 200 cells were randomly counted, and the cells with yellow or brown stains were regarded as having a posi-. The expression of FOXC1 in non-small cell lung cancer. 12827 Int J Clin Exp Med 2019;12(11):12825-12832. tive expression. Scores were given according to the staining strength and the rate of positive cells. The staining strength was evaluated in light of the following standard: 0 points for no staining; 1 point for mild yellow; 2 points for brown; 3 points for deep brown. The scores for the rate of positive cells: 0 points for no posi- tive cells; 1 point for fewer than 25%; 2 points for between 25% and 50%; 3 points for be- tween 51% and 75%; 4 points for higher than 75%. The cells with the sum of these two sco- res higher than 3 points were considered as deemed positive.. Construction of the stably interfered cell lines. After digestion, 293FT cells in the logarithmic phase were cultured in a high-glucose medium supplemented with 10% fetal bovine serum (FBS). 293FT cells were spread evenly on a 6-well plate, and cultured at 37°C and 5% CO2. At the confluence of 70%, the original medium was refreshed by the serum-free medi- um. According to the instructions of the Lipofectamine 2000 kit, pLVTHM-shFOXC1 and pLVTHM-shNC were transfected into the 293FT cells. The lentivirus solution was har- vested to transfect the A549 cells after 48 hours. Fluorescence of GFP was observed under the fluorescent microscope, and the suc- cessfully constructed cell lines were labeled as A549-shFOXC1 and A549-shNC. A549 cells without treatment were considered the blank control.. The expression of FOXC1 in cells determined by Western blot. From the MRC-5 cells, A549 cells, A549- shFOXC1 cells, and A549-shNC cells, the total proteins were extracted to measure the protein levels using a bicinchoninic acid (BCA) kit. The proteins were boiled in the loading buffer, and together, they were added into the SDS-PAGE wells. The separated proteins in the gel were transferred on the PVDF membrane which was later blocked in 5% skimmed milk for 2 h at room temperature. The primary antibodies of FOXC1, after being diluted (1:500) in the block- ing buffer, were added on the surface of the membrane for incubation overnight at 4°C, and the incubation was ended by rinsing with PBST. Then, the immunoblots were further probed by incubating them with goat anti-rabbit IgG at room temperature for 2 h on a shaker. After. several rinses with PBST, the immunoblots were treated with enhanced chemilumines- cence (ECL) for 5 min in the dark. The intensity of the immunoblots was normalized to that of the GAPDH.. Cell proliferation by plate colony formation assay. After digestion, the A549 cells, the A549- shFOXC1 cells, and the A549-shNC cells were inoculated on a 6-well plate at density of 5 × 103/well. The plate was then shaken slightly, and placed at 37°C and 5% CO2, with the medi- um being refreshed every 7 days. Two weeks later, the cells were stained with Coomassie Brilliant Blue in order to count the number of colonies under the microscope. This experi- ment was conducted in triplicate and the results were averaged.. Cell invasion by Transwell experiment. Matrigel was diluted in a serum-free 1640 medium at a ratio of 1:5, and then it was added to the Transwell chamber (50 μL/chamber) for coagulation. At 12 h prior to the experiment, the cell medium was refreshed using the serum-free medium, followed by digestion and rinses with PBS for counting. Then, the cells were resuspended in serum-free medium to attain a density of 5 × 105/mL, from which 250 μL was added into the Transwell chamber. In the lower chamber, 600 μL 1640 medium sup- plemented with 10% FBS was carefully added to avoid the generation of bubbles. Following 24 hours of culture at 37°C and 5% CO2, the medium in the Transwell chamber was dis- carded, and the chamber was washed in PBS. Cells in the lower chamber were fixed in 4% paraformaldehyde, and then stained in 0.1% crystal violet for 15 min after being rinsed with PBS. The cells that failed to pass into the lower chamber were scrubbed using a cotton swab, and the chamber was inverted to be dried. The chamber was then placed on an inverted fluo- rescent microscope in order to count the cells in the lower chamber at 5 high-power fields. This experiment was conducted in triplicate, and the results were averaged.. Statistical analysis. The statistical analysis was carried out using SPSS 18.0 software. The measurement data. The expression of FOXC1 in non-small cell lung cancer. 12828 Int J Clin Exp Med 2019;12(11):12825-12832. Figure 1. The expressions of FOXC1 in the NSCLC tissues and tumor-adja- cent normal tissues (× 400). Differences in the expression of FOXC1 in the different clinicopathological groups.. Table 1. Expression of FOXC1 in the NSCLC tissues and tumor- adjacent normal tissues. Source of tissue FOXC1. - + Positive rate (%) NSCLC tissue (n = 97) 25 72 74.23 Tumor-adjacent normal tissue (n = 97) 56 41 42.27 χ2 20.369 P 0.000. are presented as the means ± standard deviation and com- pared among groups using a One-way analysis of variance (One-way ANOVA) The signifi- cant differences between gr- oups were determined by an independent t test. The enu- meration data were expressed as percentages and analyzed using a chi-squared test. A statistical significant differ- ence was accepted when P < 0.05.. Results. The expression of FOXC1 in the NSCLC tissues and tumor- adjacent normal tissues. The positive expression rate of FOXC1 in NSCLC was 74.23%, which was signifi- cantly higher than the 42.27% rate in the tumor-adjacent nor- mal tissues (P < 0.05), sug- gesting that FOXC1 possibly plays a positive role in the growth of cancer, with a high expression in NSCLC (Figure 1 and Table 1).. No correlation was detected between the expression pro- file of FOXC1 and the gender or age of the NSCLC patients (P > 0.05), but a strong corre- lation was found with the cl- inicopathological features of NSCLC (P < 0.05). Tumor ma- lignancy and the positive expression rate of FOXC1 are positively correlated with TNM stage, number and size of lymphatic metastatic regions, tumor diameter, and tissue infiltration (P < 0.05; Table 2).. FOXC1 expression and the survival analysis of NSCLC patients. Patients with a positive ex- pression of FOXC1 had a. Table 2. Expression of FOXC1 in the different clinicopathological groups. Clinicopathological features Cases. (n) FOXC1. - + χ2 P Gender 0.003 0.958 Male 50 13 37 Female 47 12 35 Age 0.041 0.839 ≥ 50 56 14 42 < 50 41 11 30 TNM stage 17.325 0.000 I-II 33 17 16 III-IV 64 8 56 Tissue differentiation 7.698 0.021 Low 65 9 56 Moderate 21 7 14 High 11 5 6 Lymphatic metastasis 10.984 0.001 Yes 74 13 61 No 23 12 11 Tumor diameter 20.248 0.000 ≥ 3 cm 69 9 60 < 3 cm 28 16 12 Infiltration depth 33.847 0.000 Not reaching the serous layer 24 17 7 Breaking through the serous layer 73 8 65. The expression of FOXC1 in non-small cell lung cancer. 12829 Int J Clin Exp Med 2019;12(11):12825-12832. 5-year survival rate of 11.11%, significantly lower than the 28.00% rate of those with nega- tive expressions of FOXC1 (P < 0.05; Table 3).. Expression of FOXC1 in cell lines. Compared with the MRC-5 cell line, FOXC1 was observed to be up-regulated in the A549 cell line (P < 0.05). In the A549-shFOXC1 cell line, FOXC1 was much lower than it was in the A549 cell line or the A549-shNC cell line (P < 0.05). However, no significant difference was ob- served between the A549 cell line and the A549-shNC cell line (P > 0.05; Figure 2).. The effect of FOXC1 on the proliferation ability of the A549 cell line. A plate colony formation assay showed that the knockdown of FOXC1 lead to a decrease in the proliferation ability of A549 cells in comparison with the A549 cells and the A549-shNC cells (P < 0.05). No significant difference was identified. 0.05), the results of the Transwell assay revealed that the knockdown of FOXC1 resulted in a decrease in the invasion ability of the A549 cells, indicating that the acute decrease in the number of cells successfully passed through the Matrigel. However, there was no statistical- ly significant difference between the A549 cells and the A549-shNC cells (P > 0.05; Figure 4).. Discussion. Transcription factors are a group of critical pro- teins involved in biological processes, including the growth, development, differentiation and metabolism of cells, tissues or organs, through modulating the target genes. FOX, existing widely in living creatures, consists of a giant family of transcription factors. Due to the spe- cial wing-like helical structure in the DNA- binding region, namely the fork area [9, 10], it is also called the forkhead box transcription fac- tor. It has been reported that there are more than 100 members of the FOX family in 19 sub- groups (from A to S) [11, 12]. As one member of the FOX family, FOXC1 is involved in many bio- logical events, and the corresponding muta- tion is significantly correlated with anomalous embryonic development [13, 14]. Furthermore, FOXC1 is implicated in the progression of tumors including ovarian cancer, breast cancer, and pancreatic cancer, and its overexpression severely affects patients’ prognosis [15-17]. In this study, the positive expression rate of FOXC1 in NSCLC tissues was significantly high- er than it was in the tumor-adjacent normal tis- sues (P < 0.05), indicating that FOXC1 possibly plays a positive role in cancer, with a high expression in NSCLC. Moreover, the tumor malignancy and positive expression rates of FOXC1 correlated positively with the TNM stage, number, and size of lymphatic metastatic regions, tumor diameter, and tissue infiltration, indicating the underlying positive correlation between the FOXC1 expression and tumor malignancy, as well as the involvement of. Table 3. The correlation of FOXC1 expression with the survival rate of NSCLC patients. 5-year survival rate (%) FOXC1 positive expression group (n = 72) 8 (11.11) FOXC1 negative expression group (n = 25) 7 (28.00) χ2 4.049 P 0.044. in comparing the colony number between the A549 cells and the A549-shNC cells (P > 0.05; Figure 3).. The effect of FOXC1 on the inva- sion ability of A549 cells. Compared with the A549 cells and the A549-shNC cells (P <. Figure 2. The expressions of FOXC1 in various cell lines. (*P < 0.05 vs. MRC-5 cell line; #P < 0.05 vs. A549 cell line).. The expression of FOXC1 in non-small cell lung cancer. 12830 Int J Clin Exp Med 2019;12(11):12825-12832. FOXC1 in the metastasis and invasion of NSCLC. Furthermore, the 5-year survival rate of patients with a positive expression of FOXC1 was significantly higher than it was in those with a negative expression (P < 0.05).. Existing evidence has shown the two-sided role of FOXC1 in tumors. On the one hand, FOXC1 overexpression leads to the abnormal prolifera- tion, invasion, and metastasis of the cells in the basal-like breast carcinoma . On the other hand, FOXC1 overexpression suppresses the proliferation of Hela cells . Meanwhile, FOXO1A, as the upstream tumor suppressor, is involved in the inhibition of tumors. Further- more, FOXC1 is possibly implicated in the inter- action between the notch signal and vascular endothelial growth factor (VEGF) to induce angiogenesis in tumors [20, 21]. Ray et al.  reported that the tumor-free survival duration of the patients with an excessively high mRNA expression of FOXC1 is significantly shortened,. but there is little information regarding the effect of FOXC1 on the biological behaviors of NSCLC. Thus, we constructed the A549 cell lines with a stably interfered expression of FOXC1, aiming to uncover the mechanism of FOXC1 in NSCLC. Compared with the MRC-5 cell line, FOXC1 was observably up-regulated in the A549 cell line (P < 0.05), which coincided with the results of the immunohistochemistry. In the A549-shFOXC1 cells, the knockdown of the FOXC1 gene resulted in a decrease in its level, suggesting that the cell line with the sta- bly interfered expression was constructed suc- cessfully. Previous studies have shown that the development and progression of tumors may be related to the epigenetic changes of the FOXC1 gene that may alter cell proliferation by regulating the expression of Cyclin D1 [22, 23]. In this study, the results of plate colony forma- tion assay showed an acute decrease in the colonies of A549 cells, indicating the positive role of FOXC1 in the proliferation of NSCLC. Figure 3. The effect of FOXC1 on the proliferation ability of the A549 cell line. (*P < 0.05 vs. A549 cell line).. The expression of FOXC1 in non-small cell lung cancer. 12831 Int J Clin Exp Med 2019;12(11):12825-12832. cells. Also, the results of Transwell assay revealed that the knockdown of FOXC1 led to a decline in the number of A549 cells passing through the Matrigel, suggesting that FOXC1 can enhance the invasion ability of NSCLC cells.. FOXC1 is highly expressed in NSCLC and is sig- nificantly correlated with patients’ prognosis. FOXC1 also promotes the effort and might of A549 cells, but the underlying mechanism is still unclear. Further studies will investigate its mechanism of action in NSCLC. Together, FOXC1 is highly expressed in NSCLC, and it closely correlates with patients’ prognosis. Furthermore, FOXC1 promotes the proliferation and invasion of A549 cells.. Disclosure of conflict of interest. None.. Address correspondence to: Rong Chen, De- partment of Respiratory and Critical Care Medicine, Taizhou People’s Hospital of Jiangsu Province, Taizhou 225300, Jiangsu, China. E-mail: jschen- email@example.com. References.  Siegel R, Naishadham D and Jemal A. Cancer statistics. CA Cancer J Clin 2012; 62: 10-29..  Chen W, Zheng R, Zhang S, Zhao P, Li G, Wu L and He J. Report of cancer incidence and mor- tality in China 2009. Chin J Cancer Res 2013; 22: 2-12..  Jemal A, Siegel R, Xu J and Ward E. Cancer sta- tistics, 2010. CA Cancer J Clin 2010; 60: 277- 300..  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