ZNF292 suppresses proliferation of ESCC cells through ZNF292/SKP2/P27 signaling axis

Abstract

Objective: Increasing evidence has demonstrated that ZNF292 plays a suppressive role in cancer, however, little is known about its function and exact mechanism in esophageal squamous cell carcinoma (ESCC).

Methods: Bioinformatic analysis and immunohistochemistry (IHC) were performed to analyze the role of ZNF292 in affecting the prognosis of ESCC. Cell proliferation and colony formation ability assays were performed to analyze cell growth after inferring the expression of ZNF292. Flow cytometry was used to analyze changes in the cell cycle upon the depletion of ZNF292. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis were used to determine the alteration of cell cycle related RNAs and proteins after knocking down ZNF292. MG-132, cycloheximide (CHX) treatment experiments were performed to analyze the change and half-life time of P27 after knockdown of ZNF292. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays were used to analyze the transcriptional regulation of SKP2 by ZNF292.

Results: We report that low expression of ZNF292 is associated with poor prognosis, and ZNF292 emerges to be highly expressed in adjacent and normal tissues rather than tumor tissues in ESCC. Knockdown of ZNF292 significantly boosts cell growth and S phase entry in ESCC cells. ZNF292 depletion will decrease the expression and half-life time of P27, while knockdown of SKP2 will result in elevated expression of P27. ZNF292 can bind to the promoter region of SKP2, and knockdown of ZNF292 will boost the expression of SKP2.

Conclusions: Knockdown of ZNF292 mediates G1/S cell cycle procession by activating SKP2/P27 signaling in ESCC cells. ZNF292 knockdown promotes SKP2 expression at the transcriptional level, thereby boosting P27 ubiquitin-degradation, and eventually facilitating the S phase entrance.

Keywords: ESCC; SKP2; ZNF292; cell cycle.

Figure 1

Low expression of ZNF292 in ESCC indicates a worse prognosis. (A) Kaplan-Meier survival analysis of ESCC patients’ overall survival based on the ZNF292 mRNA level (P=0.028); (B) mRNA level of ZNF292 in normal tissue and ESCC tissue in TCGA RNA-seq database; (C) IHC assays were performed to analyze the expression profile of ZNF292 in a tissue microarray within 30 primary tumor dots, 30 adjacent dots, and 30 normal dots. ESCC, esophageal squamous cell carcinoma; TCGA, The Cancer Genome Atlas; IHC, immunohistochemistry. ***, P<0.001.

Figure 2

Depletion of ZNF292 promotes ESCC cell proliferation by promoting cell cycle G1/S phase transition. (A) Western blot analysis was used to detect the ZNF292 expression profile in ESCC cell lines; (B) Cell proliferation rates and colony formation ability analyses after knocking down ZNF292 in YES2 cells; (C) Cell proliferation rates and colony formation ability analyses after knocking down ZNF292 in KYSE30 cells; (D,E) Images of representative soft agar plates, revealing accelerated clonogenic growth of ZNF292-knockdown cells compared to control cells in YES2 (D) and KYSE30 (E) cells; (F,G) Cell cycle analysis after knocking down ZNF292 in YES2 (F) and KYSE30 (G) cells; (H,I) EdU dye assays were used to monitor cell proliferation after depletion of ZNF292 in YES2 (H) and KYSE30 (I) cells. ESCC, esophageal squamous cell carcinoma. *, P<0.05; **, P<0.01; ***, P<0.001.

Figure 3

Knockdown of ZNF292 promotes P27 ubiquitin-degradation. (A) qRT-PCR was performed to analyze change in S phase entrance-related genes after knocking down ZNF292 in YES2 cells; (B) qRT-PCR was performed to analyze change in S phase entrance-related genes after knocking down ZNF292 in KYSE30 cells; (C) Western blot analysis was used to detect the change in S phase entrance-related proteins after knocking down ZNF292 in YES2 and KYSE30 cells; (D) IP assays were performed to analyze the expression of ubiquitin-bound P27 after knocking down ZNF292 in YES2 and KYSE30 cells; (E) Culture medium containing 300 µmol/L CHX was added to cells after knocking down ZNF292 for 24 h, and then cells were harvested every 2 h. Western blotting was used to detect change in P27 at different culture times. qRT-PCR, quantitative real-time polymerase chain reaction; IP, immunoprecipitation; CHX, cycloheximide; ns, not significant. *, P<0.05; **, P<0.01; ***, P<0.001.

Figure 4

Knockdown of ZNF292 suppresses the expression of P27 by promoting SKP2 expression. (A) Western blot analysis was used to detect the change in P27 after knocking down SKP2 in YES2 and KYSE30 cells; (B) qRT-PCR was performed to detect the knockdown efficiency of si-SKP2 in YES2 and KYSE30 cells; (C) Western blot analysis was used to detect SKP2 expression upon the depletion of ZNF292 in YES2 and KYSE30 cells; (D) qRT-PCR was performed to detect SKP2 expression upon the depletion of ZNF292 in YES2 and KYSE30 cells; (E) A rescue experiment was performed to analyze the expression of p27. Cells were transfected with si-con (1 unit), si-ZNF292 (1 unit), si-SKP2 (1 unit), si-ZNF292 & si-SKP2 (half and half units) in six well culture plates, 48 h later, cells were harvested, and western blotting was performed to analyze the expression of SKP2 and p27; (F) Genetic information analysis for SKP2 in ESCC from the TCGA database; (G) qRT-PCR was performed to detect the expression profile of SKP2 in ESCC cell lines. qRT-PCR, quantitative real-time polymerase chain reaction; ESCC, esophageal squamous cell carcinoma; TCGA, The Cancer Genome Atlas. *, P<0.05; ***, P<0.001.

Figure 5

ZNF292 functions to suppress SKP2 expression at transcriptional level. (A) Transcription factor binding prediction for ZNF292 on the promoter element of SKP2; (B) ChIP assay for the binding ability of ZNF292 to the promoter element of SKP2 in KYSE30 cells; (C) Construction model of the luciferase reporter plasmids; (D,E) Luciferase reporter results analyses in HEK293 cells (D) and KYSE30 cells (E). These two cell lines were all transfected with pGL+si-con, pGL-SKP2+si-con, pGL-SKP2+si-ZNF292-1, or pGL-SKP2+si-ZNF292-2. After transfection for 48 h, cells were subjected to dual luciferase analysis. The results were presented as the fold change relative to cells transfected with pGL+si-con after normalization to Renilla activity. ChIP, chromatin immunoprecipitation; IgG, immunoglobulin G. ***, P<0.001.

Figure 6

Graphical summary of ZNF292/SKP2/P27 pathway. ZNF292 functions to suppress the expression of SKP2 at the transcription level. The decrease of SKP2 attenuates the ubiquitin-mediated degradation of P27. As a result, the accumulated P27 suppresses cell proliferation of ESCC cells by blocking cell cycle progression. ESCC, esophageal squamous cell carcinoma.