p16 promotes proliferation in cervical carcinoma cells through CDK6-HuR-IL1A axis

Abstract

The Cyclin-Dependent Kinase Inhibitor p16 (p16) acts as a tumor suppressor in most cells, but for HPV transformed cervical cancer, in which oncoprotein E7 expressed by human papillomavirus (HPV) mediates the degradation of retinoblastoma protein (Rb), p16 exhibits oncogenic activity. Our study was conducted to study the mechanism underling p16 mediated promoting effect of cell proliferation in cervical cancer cell lines. CCK8 assay and EdU incorporation were conducted to evaluate cell proliferation. Loss-of-function assay was used to silence p16 in Ca Ski and SiHa cells. Next, western blot, qPCR, RNA silencing, luciferase activity assay, run-on assay, mRNA stability assay, RNA immunoprecipitation, co-immunoprecipitation Immunofluorescence were performed to examine the interaction between CDK6, HuR, and IL1A mRNA in p16 mediated proliferation promoting effect. Our results showed that: (1) Silencing p16 inhibited the proliferation of cervical cancer cells by decreasing the half-life of IL1A mRNA in CDK6 dependent manner; (2) The stabilization of IL1A mRNA was regulated by HuR which could be inactivated by p16/CDK6 mediated phosphorylation at Ser202; (3) IL1A mediated the oncogenic activity of p16 in cervical carcinoma cell lines. In conclusion, p16 promotes proliferation in cervical carcinoma cells through CDK6-HuR-IL1A axis.

Keywords: Cervical carcinoma; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p16; ELAV-Like Protein 1; Interleukin-1alpha..

Figure 1

p16 regulated IL1A expression in cervical cancer cell lines: (A) p16 transcript levels of Ca Ski sh-ctrl, Ca Ski sh-p16#1 and Ca Ski sh-p16#2 were determined by SYBR Green qRT-PCR analyses. (B) p16 expression levels of Ca Ski sh-ctrl, Ca Ski sh-p16#1 and Ca Ski sh-p16#2 were determined by Western blotting. GAPDH served as the loading control. (C) Cells proliferation assay of Ca Ski sh-ctrl, Ca Ski sh-p16#1 and Ca Ski sh-p16#2 for 60 hours. (D) A microarray identified 95 genes comparing Ca Ski sh-ctrl and Ca Ski sh-p16#1. The threshold was set as 1.5-fold changes and p < 0.05. (E) Heat maps of 8 genes involved in the regulation of cell proliferation. (F) IL1A transcript levels of Ca Ski sh-ctrl, Ca Ski sh-p16#1 and Ca Ski sh-p16#2 were determined by Taqman qRT-PCR analyses. (G) IL8 transcript levels of Ca Ski sh-ctrl, Ca Ski sh-p16#1 and Ca Ski sh-p16#2 were determined by SYBR Green qRT-PCR analyses.

Figure 2

p16 regulated IL1A expression in CDK6 dependent manner: CDK4 (A) and CDK6 (B) transcript levels of Ca Ski sh-ctrl+ctrl, Ca Ski sh-p16#1+ctrl, Ca Ski sh-p16#1+CDK4#1, Ca Ski sh-p16#1+CDK4#2, Ca Ski sh-p16#1+CDK6#1 and Ca Ski sh-p16#1+CDK6#2 were determined by SYBR Green qRT-PCR analyses and IL1A transcript levels (C) were determined by Taqman qRT-PCR analyses. CDK6 (D) and p16 (E) transcript levels of Ca Ski ctrl and Ca Ski CDK6R31C were determined by SYBR Green qRT-PCR analyses and IL1A transcript levels (F) were determined by Taqman qRT-PCR analyses. (G) Cell cycle (H) of synchronized Ca Ski sh-ctrl and Ca Ski sh-p16#1 was measured. (I) CDK6 and Cyclin D expression levels of synchronized Ca Ski sh-ctrl and Ca Ski sh-p16#1 were determined by Western blotting. GAPDH served as the loading control. (J) IL1A transcript levels of synchronized Ca Ski sh-ctrl and Ca Ski sh-p16#1 were determined by Taqman qRT-PCR analyses.

Figure 3

p16/CDK6 regulated IL1A mRNA stability: (A) A schematic diagram of IL1A promoter luciferase was constructed. (B) The activity of IL1A promoter in Ca Ski sh-ctrl, Ca Ski sh-p16#1 and Ca Ski sh-p16#2 was analyzed by luciferase-based reporter assay. (C) Nuclear run-on assay of Ca Ski sh-ctrl, Ca Ski sh-p16#1 and Ca Ski sh-p16#2. (D) The stability of endogenous IL1A mRNA was influenced by p16 knockdown. (E) The half-life of IL1A mRNA in Ca Ski sh-ctrl, Ca Ski sh-p16#1 and Ca Ski sh-p16#2 were calculated.

Figure 4

HuR was involved in IL1A mRNA stability regulation: (A) A schematic diagram of IL1A 3'-UTR luciferase was constructed. (B) The activity of luciferase-based reporter of the IL1A 3'-UTR in Ca Ski cells was analyzed. (C) The interaction between RNA binding proteins and IL1A mRNAs was examined by RNA immunoprecipitation. Mouse IgG was included as a negative control. (D) HuR transcript levels of Ca Ski sh-ctrl, Ca Ski sh-HuR#1 and Ca Ski sh-HuR#2 were determined by SYBR Green qRT-PCR analyses. (E) The activity of luciferase-based reporter of the IL1A 3'-UTR in Ca Ski sh-ctrl, Ca Ski sh-HuR#1 and Ca Ski sh-HuR#2 was analyzed. (F) The stability of endogenous IL1A mRNA was affected by HuR knockdown. (G) The half-life of IL1A mRNA in Ca Ski sh-ctrl, Ca Ski sh-HuR#1 and Ca Ski sh-HuR#2 were calculated.

Figure 5

CDK6 phosphorylated HuR at Ser202: (A) HuR expression level in synchronized Ca Ski sh-ctrl and Ca Ski sh-p16#1 were determined by Western blotting. (B) Confocal microscopy images of Ca Ski sh-ctrl, Ca Ski sh-p16#1 and Ca Ski sh-p16#1+CDK6#2. HuR (red) and DAPI staining (blue) was visualized by immunfluorescence. (C) The phosphorylation level of HuR in synchronized Ca Ski sh-ctrl and Ca Ski sh-p16#1 were examed by Co-Immunoprecipitation. (D) Immunoprecipitation and western blot analysis were performed by using the indicated antibodies in the panel. (E) In vitro kinase assay was performed to examine the phosphorylation of HuR by CDK6+CCND1. The phosphorylation level of wild-type HuR (WT) or mutant HuR (S202A, S221A, and S242A) were examined by Co-Immunoprecipitation. (F) The localization of wild-type (top panel) or mutant EGFP-HuR (S202A, middle panel, or S202S, bottom panel) was showed by confocal assay.

Figure 6

IL1A mediated the oncogenic activity of p16 in cervical carcinoma cell lines: (A) Ca Ski Cells were transfected with independent siRNA against Non-Target control or IL1A. IL1A transcript levels were determined by Taqman qRT-PCR analyses. IL1A knockdown affected cell viability (B) and proliferation (C)(D) of Ca Ski. (E) IL1A transcript levels of Ca Ski ctrl-sh-ctrl, Ca Ski ctrl-sh-p16#1, Ca Ski ctrl-sh-p16#2, Ca Ski IL1A-sh-p16#1 and Ca Ski IL1A-sh-p16#2 were determined by Taqman qRT-PCR analysis. IL1A overexpressing affected the cell viability (F) and proliferation (D)(H) of p16 silencing Ca Ski.