Mitotic Arrest-Deficient Protein 2B Overexpressed in Lung Cancer Promotes Proliferation, EMT, and Metastasis

Abstract

As the noncatalytic subunit of mammalian DNA polymerase, mitotic arrest-deficient protein 2B (MAD2B) has been reported to play a role in cell cycle regulation, DNA damage tolerance, gene expression, and carcinogenesis. Although its expression is known to be associated with poor prognosis in several types of human cancers, the significance of MAD2B expression in lung malignancies is still unclear. Our study showed that MAD2B expression significantly increased in lung cancer, especially in the metastatic tissues. We also found that knockdown of MAD2B inhibited the migration, invasion, and epithelial-mesenchymal transition of lung cancer cells in vitro and the metastasis in vivo, while overexpression of MAD2B had the opposite effect. Microarray and Western blotting data indicated that slug might be its downstream target since knockdown of MAD2B inhibited, while overexpression increased, the expression of slug. Moreover, the expression of MAD2B was found to be positively correlated with slug in lung cancer tissues as well. Collectively, these findings indicate an oncogenic role of MAD2B in lung cancer, and slug might be involved in the process.

Figure 1

Mitotic arrest-deficient protein 2B (MAD2B) is highly expressed in human metastasis lung cancer tissues. (A) MAD2B mRNA expression was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in 87 lung cancer tissues. (B) qRT-PCR results between nonmetastatic and metastatic lung cancer tissues were summarized by a statistical software. (C) Survival analysis of patients with lung cancers was based on the Kaplan–Meier survival analysis in The Cancer Genome Atlas (TCGA). A value of p < 0.001 is based on the Student’s t-test. Error bars, standard deviation (SD).

Figure 2

MAD2B is highly expressed in metastasis lung cancer cell lines. (A) MAD2B expression in lung cancer cell lines was analyzed by Western blotting. (B) Quantitative chart of MAD2B protein levels in liver cancer cell lines. (C) MAD2B expression in lung cancer cell lines was analyzed by qRT-PCR. A value of p < 0.001 is based on the Student’s t-test. Error bars, SD.

Figure 3

Knockdown of MAD2B inhibits capacities of migration and invasion of lung cancer cells in vitro. (A) MAD2B protein’s low expression in the A549-shMAD2B cell lines was verified by Western blot assay compared with the control cells. (B) MAD2B mRNA’s low expression in the A549-shMAD2B cell lines was verified by qRT-PCR assay compared with the control cells. (C) A549-shMAD2B and its control cells were subjected to Transwell migration and Matrigel invasion assays. (D) Quantification of migrated and invasion cells through the membrane is shown as proportions of their vector controls in a column diagram from (C). A value of p < 0.001 is based on the Student’s t-test. Error bars, SD.

Figure 4

Knockdown of MAD2B inhibits metastasis capacity of lung cancer cells in vivo. (A, B) Fewer metastasis foci in the liver were counted in each mouse injected with lung cancer cells silencing MAD2B. A value of p < 0.01 is based on the Student’s t-test. Error bars, SD.

Figure 5

Overexpression of MAD2B promotes capacities of migration and invasion in lung cancer cells in vitro. (A). Western Blot analysis of MAD2B levels in the established cell lines. (B) qRT-PCR analysis of MAD2B levels in the established cell lines. (C) Lung cancer cells with high expression of MAD2B possessed stronger invasion abilities in Transwell and Matrigel assays. (D) Quantification of migrated and invasion cells through the membrane is shown as proportions of their vector controls in a column diagram from (C). A value of p < 0.01 is based on the Student’s t-test. Error bars, SD.

Figure 6

Overexpression of MAD2B promotes metastasis capacity of lung cancer cells in vivo. (A, B) More metastasis foci in the liver were counted in each mouse injected with lung cancer cell overexpressing MAD2B. A value of p < 0.01 is based on the Student’s t-test. Error bars, SD.

Figure 7

Knockdown of MAD2B inhibits epithelial–mesenchymal transition in lung cancer cells. (A) Western blot analysis showed that silencing MAD2B upregulated the epithelial cell markers (E-cadherin and α-catenin) and downregulated the mesenchymal cell markers (N-cadherin and vimentin) in A549 cells. (B) qRT-PCR analysis showed that silencing MAD2B upregulated the epithelial cell markers (E-cadherin and α-catenin) and downregulated the mesenchymal cell markers (N-cadherin and vimentin) in A549 cells. A value of p < 0.01 is based on the Student’s t-test. Error bars, SD.

Figure 8

Overexpression of MAD2B promotes epithelial–mesenchymal transition. (A) Western blot analysis showed that overexpression of MAD2B downregulated the epithelial cell markers (E-cadherin and α-catenin) and upregulated the mesenchymal cell markers (N-cadherin and vimentin) in DSM53 cells. (B) qRT-PCR analysis showed that overexpression of MAD2B downregulated the epithelial cell markers (E-cadherin and α-catenin) and upregulated the mesenchymal cell markers (N-cadherin and vimentin) in DSM53 cells. A value of p < 0.01 is based on the Student’s t-test. Error bars, SD.

Figure 9

MAD2B regulates the expression level of slug. (A) Clustering of the genes differentially expressed after silencing of MAD2B in A549 cells. (B) The enrichment scores of differentially expressing genes in the MAD2B silencing cell line.

Figure 10

Slug expression was affected by MAD2B. (A) The slug expression levels in the MAD2B silencing cell line were assayed by Western blot. (B) The slug expression levels in the MAD2B silencing cell line were assayed by qRT-PCR. (C) The slug expression levels in the MAD2B overexpression cell line were assayed by Western blot. (D) The slug expression levels in an MAD2B overexpression cell line were assayed by qRT-PCR. (E) The expression of slug was positively correlated with MAD2B in the lung cancer tissues. A value of p < 0.01 is based on the Student’s t-test. Error bars, SD.