NUCKS1 Promotes Proliferation, Invasion and Migration of Non-Small Cell Lung Cancer by Upregulating CDK1 Expression

Abstract

Background: Non-small cell lung cancer (NSCLC) is a predominant type of lung cancer with a high mortality rate.

Objective: The aim of this study is to investigate the roles of nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1) in NSCLC and to identify the potential mechanisms.

Materials and methods: The expression of NUCKS1 in several NSCLC cells was detected firstly. Then, NUCKS1 was overexpressed or silenced in both A549 and NCI-H460 cells, where cell proliferation, invasion and migration were, respectively, determined, using CCK-8, colony formation assay, transwell and wound healing assays. Cell cycle analysis was performed, and the expression-associated proteins were detected by Western blotting. Subsequently, NCI-H460 cells with NUCKS1 overexpression for the subsequent tumor-bearing experiment. And the NUCKS1 expression in tumor tissues was measured by means of immunohistochemistry and Western blotting. Additionally, the STRING database predicted that Cyclin-Dependent Kinase 1 (CDK1) would bind to NUSK1, which was verified by the co-immunoprecipitation assay. Then, CDK1 was silenced by transfection with short hairpin RNA (shRNA)-CDK-1 or by exposure to CDK1 inhibitor p2767-00. And the biological characteristics of proliferation, invasion and migration were examined.

Results: Results indicated that NUCKS1 was overly expressed in NSCLC cells, and its overexpression promoted proliferation, invasion and migration of both A549 and NCI-H460 cells while NUCKS1 knockdown displayed the opposite effects. Moreover, the results of the xenograft experiments revealed that NUCKS1-upregulation promoted the tumor growth. Furthermore, the immunoprecipitation assay verified CDK1's interaction with NUCKS1, and CDK1 knockdown alleviates the impact of NUCKS1 overexpression on NSCLC cell proliferation, invasion and migration.

Conclusion: Taken together, these findings demonstrated that NUCKS1 promotes proliferation, invasion and migration of NSCLC by upregulating CDK1, providing a novel putative target for the clinical treatment of NSCLC.

Keywords: CDK1; NUCKS1; migration; non-small cell lung cancer; proliferation.

Figure 1

NUCKS1 expression was significantly upregulated in NSCLC cells. The expression of NUCKS1 in several NSCLC cells (A549, NCI-H292, NCI-H358 and NCI-H460) and one normal human bronchial epithelioid cell line (BEAS-2B) was detected using (A, B) RT-qPCR and Western blot analysis. **P<0.01 and ***P<0.001. The levels of NUCKS1 in A549 cells were determined using (C, D) Western blot analysis and RT-qPCR after transfection with OE-NUCKS1. ***P<0.001. The expression of NUCKS1 in NCI-H460 cells was evaluated by (E, F) Western blot analysis and RT-qPCR after transfection with OE-NUCKS1. *P<0.05 and ***P<0.001.

Figure 2

Overexpression of NUCKS1 promotes the proliferation and inhibits cell cycle arrest of A549 and NCI-H460 cells. (A) Cell viability was tested using a CCK-8 kit. ***P<0.001 vs A549-Oe-NC; ^###P<0.001 vs NCI-H460-Oe-NC. (B) Cell proliferation was assessed using colony formation assay. (C and D) Cell cycle distribution was detected using flow cytometer. (E) The expression of cell cycle-related proteins was measured using Western blot analysis. *P<0.05, **P<0.01 and ***P<0.001.

Figure 3

Overexpression of NUCKS1 accelerates the invasion and migration of A549 and NCI-H460 cells. (A) The invasive ability of A549 and NCI-H460 cells was determined using a Transwell assay. (B) Relative number of invaded cells. (C) Migratory activity of cells was detected using a scratch wound-healing assay. (D) Relative migratory rate was calculated. (E) Western blot analysis was used to evaluate the expressions of migration-related proteins. *P<0.05, **P<0.01 and ***P<0.001.

Figure 4

NUCKS1 silencing attenuates the proliferation and promotes cell cycle arrest of A549 and NCI-H460 cells. The expression of NUCKS1 in A549 cells was tested using (A, B) Western blot analysis and RT-qPCR after transfection. NUCKS1 level in NCI-H460 cells was detected using (C, D) Western blot analysis and RT-qPCR. **P<0.01 and ***P<0.001. (E) Cell viability was measured using a CCK-8 kit. ***P<0.001 vs A549-shRNA-NC; ^###P<0.001 vs NCI-H460-shRNA-NC. (F) Colony formation assay was employed to determine the proliferation of A549 and NCI-H460 cells. (G and H) Cell cycle distribution was detected using flow cytometer. (I) Expression of cell cycle-related proteins was measured using Western blot analysis. *P<0.05, **P<0.01 and ***P<0.001.

Figure 5

NUCKS1 silencing suppresses the invasion and migration of A549 and NCI-H460 cells. (A and B) Cell invasion of A549 and NCI-H460 cells was evaluated using a Transwell assay. (C and D) Migratory activity of cells was detected using a scratch wound-healing assay. (E) The expressions of migration-related proteins was assessed by Western blot analysis. *P<0.05, **P<0.01 and ***P<0.001.

Figure 6

Overexpression of NUCKS1 promotes the growth of lung cancer cells in vivo. (A and B) A total of 2x10⁶ NCI-H460 cells were subcutaneously implanted into the right armpit of every male nude mice. The images of tumors were obtained at day 15 of inoculation. (C) The weight and (D) tumor volume were calculated every two days. **P<0.01 and ***P<0.001 vs OE-NC. The expression of NUCKS1 in tumor tissues was detected using (E) Western blot analysis and (F) Immunohistochemistry analysis. **P<0.01. (G) The expression level of Ki67 was measured using Immunofluorescence assay.

Figure 7

CDK1 silencing reverses the impact of NUCKS1-upregulation on proliferation and cell cycle arrest in NCI-H460 cells. (A) The STRING website predicted CDK1’s interaction with NUCKS1. (B) The combination between CDK1 and NUCKS1 was verified by co-immunoprecipitation assay. The expression of CDK1 was measured using (C) Western blot analysis and (D) RT-qPCR after transfection with shRNA-CDK1. **P<0.01 and ***P<0.001. (E) Cell viability was tested using a CCK-8 assay. ***P<0.001 vs OE-NUCKS1; ^###P<0.001 vs OE-NUCKS1+shRNA-CDK1. (F) Cell proliferation was determined using colony formation assay. (G and H) Cell cycle distribution was detected using flow cytometer. (I) Expression of cell cycle-related proteins was measured using Western blot analysis. *P<0.05 and ***P<0.001.

Figure 8

CDK1 silencing restores the inhibitory effects of NUCKS1 overexpression on invasion and migration of NCI-H460 cells. (A and B) Cell invasion of NCI-H460 cells was evaluated using a Transwell assay. (C and D) Migratory activity of cells was measured using a scratch wound-healing assay. (E) The expression of migration-related proteins was evaluated using Western blot analysis. *P<0.05, **P<0.01 and ***P<0.001.