Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 10 (8), 1915-1922
eCollection

Cyclin-Dependent Kinase Inhibitor 3 Promoted Cell Proliferation by Driving Cell Cycle From G1 to S Phase in Esophageal Squamous Cell Carcinoma

Affiliations

Cyclin-Dependent Kinase Inhibitor 3 Promoted Cell Proliferation by Driving Cell Cycle From G1 to S Phase in Esophageal Squamous Cell Carcinoma

Juan Liu et al. J Cancer.

Abstract

Background and aims. Cyclin-dependent kinase inhibitor 3 (CDKN3) has been found playing a varying role in carcinogenesis, but its biological function in esophageal squamous cell carcinoma (ESCC) is unclear. The aim of this study was to demonstrate the role of CDKN3 in ESCC. Materials and Methods: Real-time PCR and Western blot was performed in 15 pairs of ESCC tissues and adjacent normal esophageal tissues. Then cell proliferation ability, cloning ability, cell cycle status and migration and invasion ability were explored in CDKN3 overexpressed TE1 cell line and CDKN3 siRNA transfected TE1 and KYSE70 cell lines. Finally, cell cycle related proteins CyclinD1, CDK4, pAKT, P53, P21, and P27 were tested by Western blot. Results: mRNA level was higher in 11 ESCC tissues compared to adjacent normal tissues, and an increased protein expression was further detected in 8 of those 11 ESCC tissues. Functional assays showed that CDKN3 overexpression promoted ESCC cell proliferation, colony formation, migration and invasion, and facilitated G1/S transition. Opposite results were also got after transfected with CDKN3 siRNA. Cell cycle associated protein pAKT, CyclinD1, CDK4 and P27 were upregulated and P53, P21 and were downregulated under CDKN3 overexpression. All the protein levels were found changed in the opposite direction when CDKN3 expression was disturbed by siRNA. Conclusions: Our study suggested that CDKN3 acted as an oncogene in human ESCC and may accelerate the G1/S transition by affecting CyclinD-CDK4 complex via regulating pAKT-p53-p21 axis and p27 independent of AKT.

Keywords: CDKN3; ESCC; G1/S transition; cell cycle; cyclinD-CDK4 complex.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Expression of CDKN3 was increased in ESCC tissues. (A) mRNA levels of CDKN3 in 15 paired ESCC tissue and adjacent normal tissue. (B) Western blot result of CDKN3 expression level in 11 matched tissues. N is short for normal tissue and T is tumor tissue.
Figure 2
Figure 2
Association between CDKN3 expression and survival rate in patients with esophageal cancer. The data used was from the Cancer Genome Atlas (TCGA). (A) CDKN3 copy number variation (CNV) deletion was associated with higher disease free survival rate. (B) CDKN3 copy number variation (CNV) amplification was associated with poor disease free survival rate. (C) High CDKN3 expression level was related to higher disease free survival rate but (D)lower overall survival rate.
Figure 3
Figure 3
CDKN3 promoted ESCC cell proliferation and colony formation. (A) CDKN3 overexpression promoted cell proliferation in TE1 cell line (left), but TE1 and KYSE70 cells transfected with CDKN3 specific siRNA showed decreased cell proliferation ability (middle and right). (B) CDKN3 overexpression increased colony formation in TE1 cells (up), siRNA expressing decreased colony formation ability in TE1 (middle) and KYSE70 cell line (down). Columns, mean; bars, SE. *P<0.05; **P<0.01; ***P<0.001.
Figure 4
Figure 4
CDKN3 facilitated G1/S transition. Cell cycle was measured by flow cytometer. In TE1 cell line, CDKN3 overexpression decreased the G1 phase cell percentage (up). When transfected with CDKN3 special siRNA, G1 phase cell percentage was increased in both TE1 (middle) and KYSE70 cell lines (down). Columns, mean; bars, SE. *P<0.05; **P<0.01; ***P<0.001
Figure 5
Figure 5
CDKN3 increased ESCC cell migration and invasion. (A) Migration ability was evaluated by Transwell cell assay without Matrigel. Fluorescence microscope result showed that migration was enhanced in CDKN3 overexpressed TE1 cells (up) and was downregulated in TE1 (middle) and KYSE70 (down) cells which were disturbed by CDKN3 siRNA. (B) Invasion ability was evaluated by Transwell cell assay without Matrigel. Fluorescence microscope result showed that invasion was upregulated in CDKN3 overexpressed TE1 cells (up) and was downregulated in TE1 (middle) and KYSE70 (down) cells which were disturbed by CDKN3 siRNA. Columns, mean; bars, SE. *P<0.05; **P<0.01; ***P<0.001
Figure 6
Figure 6
CDKN3 regulate cell cycle associated protein expression which was included in AKT signal pathway. Western blot showed the expression level of AKT, pAKT, P53, P21, P27, CyclinD1 and CDK4. (A) Protein expression level in TE1 cell line, included both CDKN3 overexpressed cells and disturbed cells. (B) Protein expression level in CDKN3 special siRNA transfected KYSE70 cell line.

Similar articles

See all similar articles

Cited by 1 article

References

    1. Torre LA, Bray F, Siegel RL. et al. Global cancer statistics, 2012. CA Cancer J Clin. 2015;65:87–108. - PubMed
    1. Tran GD, Sun XD, Abnet CC. et al. Prospective study of risk factors for esophageal and gastric cancers in the Linxian general population trial cohort in China. Int J Cancer. 2005;113:456–63. - PubMed
    1. Chen W, Zheng R, Baade PD. et al. Cancer statistics in China, 2015. CA Cancer J Clin. 2016;66:115–32. - PubMed
    1. Maak S, Jaesert S, Neumann K. et al. Rapid communication: nucleotide sequence and physical mapping of the porcine cyclin-dependent kinase inhibitor 3 (CDKN3) gene. J Anim Sci. 2002;80:1698–9. - PubMed
    1. Hannon GJ, Casso D, Beach D. KAP: A dual specificity phosphatase that interacts with cyclin-dependent kinases. Proc Natl Acad Sci U S A. 1994;91:1731–5. - PMC - PubMed
Feedback