The Cell Cycle-Associated Protein CDKN2A May Promotes Colorectal Cancer Cell Metastasis by Inducing Epithelial-Mesenchymal Transition

Abstract

Colorectal cancer (CRC) is a common gastrointestinal malignancy, and recurrence and metastasis contribute considerably to its high mortality. It is well known that the epithelial-mesenchymal transition (EMT) accelerates the rate of cancer cell dissemination and migration, thus promoting cancer metastasis. Targeted therapy is a common modality for cancer treatment, and it can play a role in inhibiting cancer progression. In this study, bioinformatics was used to search for genes associated with the prognosis of CRC. First, differential analysis was performed on colon and rectal cancer samples to obtain 2,840 and 3,177 differentially expressed genes (DEGs), respectively. A Venn diagram was then used to identify 262 overlapping genes from the two groups of DEGs and EMT-related genes. The overlapping genes were subjected to batch survival analysis and batch expression analysis successively, and nine genes were obtained whose high expression in CRC led to a poor prognosis. The least absolute shrinkage and selection operator (LASSO) prognostic model was then constructed to obtain the risk score formula. A nomogram was constructed to seek prognostic independent factors to obtain CDKN2A. Finally, CCK-8 assay, flow cytometry and western blotting assays were performed to analyze the cellular biological function of CDKN2A. The results showed that knockdown of CDKN2A expression inhibited HT-29 cell proliferation, promoted apoptosis and cell cycle progression, and affected the EMT process in CRC.

Keywords: CDKN2A; cancer metastasis; cell proliferation and migration; colorectal cancer; epithelial-mesenchymal transition.

Figure 1

Differential analysis results from screening of EMT-related genes associated with prognosis of colorectal cancer (CRC). Volcano plot. Red indicates upregulated genes, and blue indicates downregulated genes. (A) Differential analysis of colon cancer and para cancer; (B) differential analysis of rectal cancer and para cancer; and (C) intersection results of differentially expressed genes (DEGs) in colon cancer, DEGs in rectal cancer, and EMT-related genes.

Figure 2

Batch survival and expression analysis. (A) Results of batch survival analysis of overlapping genes, HR > 1 indicates that the gene is a protective factor for cancer; and (B) expression analysis of genes with survival significance. **P < 0.01, ***P < 0.001.

Figure 3

Establishment of the least absolute shrinkage and selection operator (LASSO)-Cox prognostic model. (A, B) Selected characteristic coefficients are shown by lambda parameters, and the number of prognostic factors was determined using LASSO regression analysis; (C) distribution of high and low risk samples;(D) KM survival analysis of the high- and low-risk samples for CRC in the TCGA dataset.

Figure 4

Identification of independent prognostic factors for CRC and single-gene prognostic analysis. (A) Single-factor Cox regression analysis; (B) multi-factor Cox regression analysis; and (C, D) the nomogram model used to predict the OS of patients with CRC at one, three, and five years. p < 0.05 was considered statistically significant.

Figure 5

Comparison of different survival types. (A–C) The OS, PFS and DSS of patients with CRC who expressed high levels of CDKN2A. (D–F) ROC curves of OS, PFS and DSS. p < 0.05 was considered statistically significant.

Figure 6

In vitro experiments validating the biological role of CDKN2A. (A) CCK-8 assay; (B) Protein expression of CDKN2A, E-cadherin, N-cadherin, and vimentin. (C) Flow cytometric analysis of the cell cycle and statistical results of the cell cycle; (D) Flow cytometric analysis of apoptosis and statistical results of apoptosis; The results are shown as the mean ± SD of the data from three sets of replicate experiments, *P < 0.05, ns, non significant.