Fibronectin 1 promotes migration and invasion of papillary thyroid cancer and predicts papillary thyroid cancer lymph node metastasis

Abstract

Lymph node metastasis (LNM) is common in papillary thyroid cancer (PTC), and is an indicator of recurrence. The detailed molecular mechanism of LNM in PTC has not been well described. This study aimed to investigate the role of fibronectin 1 in PTC LNM and its clinical relevance. The expression of fibronectin 1 was confirmed in PTC tissues and cell lines. A correlation analysis was conducted and a receiver-operating characteristic curve obtained. The effect of fibronectin 1 on the proliferation of PTC cell lines was performed using a colony-formation assay and Cell Counting Kit 8. Cell-cycle analysis was performed with a flow-cytometry assay. Migration and invasion ability were evaluated by transwell and wound-healing assays. Fibronectin 1 was overexpressed in metastasized PTC. Overexpressed fibronectin 1 was positively correlated with PTC LNM. Receiver-operating characteristic analysis showed that the diagnostic accuracy of fibronectin 1 was 81.1%, with sensitivity of 80% and specificity of 82%. Overexpression of fibronectin 1 promoted proliferation, migration, and invasion in PTC. Fibronectin 1 plays a critical role in PTC metastasis by modulating the proliferation, migration, and invasion ability of PTC cells, and it is a valuable diagnostic biomarker for predicting PTC LNM.

Keywords: diagnostic biomarker; fibronectin 1; lymph node metastasis; papillary thyroid cancer.

Figure 1

Fibronectin 1 is overexpressed in metastatic PTC. Notes: **P<0.05. qRT-PCR analysis of the expression levels of fibronectin 1 in PTC tissues (unmetastasized, n=50; metastasized, n=40) (A) and PTC cell lines (TPC1 and K1) (B). The values were normalized to GAPDH mRNA expression. Data expressed as means ± standard deviation of three independent experiments. (C, D) Western blot analysis of fibronectin 1 in TPC1 and K1 cells. GAPDH was used as a loading control. Abbreviations: PTC, papillary thyroid cancer; qRT-PCR, quantitative reverse-transcription polymerase chain reaction; mRNA, messenger RNA.

Figure 2

Potential diagnostic value of fibronectin 1. Notes: The ROC curve of fibronectin 1 predicted the presence of lymph node metastasis in papillary thyroid cancer in terms of sensitivity and specificity: AUC 0.814 (0.723–0.905), P<0.000. Abbreviations: ROC, receiver-operating characteristic; AUC, area under the curve.

Figure 3

Fibronectin 1 knockdown suppresses K1 proliferation. Notes: *P<0.05; **P<0.01. (A) qRT-PCR analysis of mRNA fibronectin 1-expression levels in K1 cells transfected with fibronectin 1-specific shRNA using Lipofectamine on days 3, 5, 7, and 10. (B) Western blot analysis of fibronectin 1 in K1 cells transfected with fibronectin 1-specific shRNA. (C) CCK-8 assays were performed to determine K1 cell proliferation after 0, 12, 24, 36, 48, and 72 hours following transfection with fibronectin 1-specific shRNA. (D) A colony-formation assay was performed to determine the proliferation of K1 cells transfected with fibronectin 1-specific shRNA. (E) Colony-formation assay results. (F) Flow-cytometry images of the cell cycle in K1. (G) Results quantified in the cell cycle shown as a percentage of the total cells. All experiments were independently performed in triplicate. Data presented as means ± standard deviation. Abbreviations: qRT-PCR, quantitative reverse-transcription polymerase chain reaction; mRNA, messenger RNA; shRNA, short hairpin RNA; Scr-shRNA, scrambled shRNA.

Figure 4

Fibronectin 1 knockdown inhibits the migration and invasion of K1. Notes: **P<0.01. (A) Wound-healing assays demonstrated the migration ability of K1 cells. Representative images at 0 and 24 hours are shown. (B) Transwell assays were performed to determine the migration ability of K1 cells with fibronectin 1 knockdown. Representative images of migrating cells at a lower chamber stained with crystal violet. (C) Transwell assays were performed to determine the invasion ability of K1 cells with fibronectin 1 knockdown. Representative images of invasive cells in the lower chamber stained with crystal violet. (D) Quantification of cell migration presented as migrated cell numbers. (E) Quantification of cell invasion presented as invasive cell numbers. All data expressed as means ± standard deviation of three independent experiments. Abbreviations: shRNA, short hairpin RNA; Scr-shRNA, scrambled shRNA.

Figure 4

Fibronectin 1 knockdown inhibits the migration and invasion of K1. Notes: **P<0.01. (A) Wound-healing assays demonstrated the migration ability of K1 cells. Representative images at 0 and 24 hours are shown. (B) Transwell assays were performed to determine the migration ability of K1 cells with fibronectin 1 knockdown. Representative images of migrating cells at a lower chamber stained with crystal violet. (C) Transwell assays were performed to determine the invasion ability of K1 cells with fibronectin 1 knockdown. Representative images of invasive cells in the lower chamber stained with crystal violet. (D) Quantification of cell migration presented as migrated cell numbers. (E) Quantification of cell invasion presented as invasive cell numbers. All data expressed as means ± standard deviation of three independent experiments. Abbreviations: shRNA, short hairpin RNA; Scr-shRNA, scrambled shRNA.

Figure 5

Fibronectin 1 overexpression promotes TPC1 proliferation. Notes: *P<0.05, **P<0.01. (A) qRT-PCR analysis of mRNA fibronectin 1-expression levels in TPC1 cells transfected with lentivirus–fibronectin 1 or lentivirus–vector on days 3, 5, 7, and 10. (B) Western blot analysis of fibronectin 1 in TPC1 cells transfected with fibronectin 1 plasmid. (C) CCK-8 assays were performed to determine TPC1 cell proliferation at 12, 24, 36, 48, and 72 hours following transfection with fibronectin 1 plasmid. (D) Colony formation assays were performed to determine the proliferation of TPC1 cells transfected with fibronectin 1 plasmid. (E) Colony-formation assay results. (F) Flow-cytometry images of the cell cycle in TPC1. (G) Results quantified in the cell cycle shown as a percentage of the total cells. All data expressed as means ± standard deviation of three independent experiments. Abbreviations: qRT-PCR, quantitative reverse-transcription polymerase chain reaction; mRNA, messenger RNA.

Figure 6

Fibronectin 1 overexpression promotes the migration and invasion of TPC1. Notes: **P<0.01. (A) Wound-healing assays were performed to determine the migration ability of TPC1 cells. Representative images at 0 and 24 hours are shown. (B) Transwell assays were performed to determine the migration ability of TPC1 cells with fibronectin 1 overexpression. Representative images of migrating cells in the lower chamber stained with crystal violet. (C) Transwell assays were performed to determine the invasion ability of TPC1 cells with fibronectin 1 overexpression. Representative images of invasive cells in the lower chamber stained with crystal violet. (D) Quantification of cell migration presented as migrated cell numbers. (E) Quantification of cell invasion presented as invasive cell numbers. All data expressed as means ± standard deviation of three independent experiments.