ELF3 Overexpression Contributes to the Malignant Transformation of HPV16 E6/E7-Immortalized Keratinocytes by Promoting CCNE2 Expression

Abstract

Current cancer burden caused by persistent infection with human papillomaviruse genotype 16 (HPV16) cannot be ignored. The related mechanisms of oncoproteins E6 and E7 from HPV16 on keratinocyte malignant transformation need to be further elucidated. GSE3292 dataset analysis revealed the upregulation of ETS transcription factor 3 (ELF3) and cyclin E2 (CCNE2). To verify whether there is an interaction between ELF3 and CCNE2, E74 like ELF3 and CCNE2 expression profiles as well as their putative binding sites were analyzed using bioinformatics. Retroviruses encoding HPV16 E6 and E7 genes were used to induce immortalization of human foreskin keratinocytes (HFKs) in vitro. Dual luciferase reporters assay was used to verify the binding of ELF3 and CCNE2. The effect of ELF3 on the immortalized cells was investigated using CCK-8 assay, cell cycle analysis and western blot. ELF3 and CCNE2 presented overexpression patterns in head and neck squamous cell carcinoma. HPV16 E6/E7-expressing HFKs showed enhanced viability, accelerated cell cycle as well as upregulated ELF3 and CCNE2. ELF3 overexpression enhanced the activity of CCNE2 promoter. ELF3 silencing reduced viability, induced cell cycle arrest and suppressed expressions of CCNE2, E6 and E7 in HPV16 E6/E7-expressing HFKs. Downregulation of ELF3 played an inhibiting role in the malignant transformation of HPV16 E6/E7-immortalized HFKs by decreasing CCNE2 expression.

Keywords: E74 like ETS transcription factor 3; Human papillomavirus genotype 16 E6/E7; cyclin E2; keratinocytes; malignant transformation.

Fig. 1

Expression profiles of ELF3 and CCNE2. Volcano plot of differential expression of genes associated with HPV in head and neck squamous cell carcinoma.

Fig. 2. Effects of HPV16 E6/E7 overexpression on viability and cell cycle of HFKs.

(A-B) Analysis of HPV16 E6/ E7 expressions in HFKs by qRT-PCR after transduction with retroviral control (pLXSN) or pLXSN-16E6E7. (C-E) Analysis of HPV16 E6/E7 expressions in HFKs by western blot after transduction with retroviral control or pLXSN-16E6E7. (F) CCK-8 assay was used to assess the viability of HFKs transduced with retroviral control or pLXSN-16E6E7. (G-H) Flow cytometry was performed to detect the cell cycle of HFKs transduced with retroviral control or pLXSN-16E6E7. GAPDH functioned as an endogenous control. Data are shown as mean ± standard deviation. ***P < 0.001. Abbreviation: HPV16, human papillomavirus genotype 16; HFKs, human foreskin keratinocytes; pLXSN-16E6E7, pecombinant pLXSN retroviral vectors encoding HPV16 E6 and E7 genes; qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction; CCK-8, cell counting kit-8.

Fig. 3. ELF3 and CCNE2 expressions in HFKs expressing HPV16 E6/E7 as well as their interaction.

(A-B) Analysis of ELF3 and CCNE2 expressions in HFKs by qRT-PCR after transduction with retroviral control or pLXSN-16E6E7. GAPDH functioned as an endogenous control. (C-D) Bioinformatics analysis of putative binding sites between ELF3 and CCNE2. (E) Dual luciferase reporters assay was performed to verify the combination between ELF3 and CCNE2 in HFKs. Data are shown as mean ± standard deviation. ***P < 0.001. Abbreviation: ELF3, E74 like ETS transcription factor 3; CCNE2, cyclin E2; CCNE2-wt, wild-type reporter plasmids encoding the promoter of CCNE2; CCNE2-mut, consensus sequence mutant of CCNE2 promoter reporter plasmids.

Fig. 4. Effects of the ELF3/CCNE2 axis on malignant transformation of HFKs.

(A) Analysis of ELF3 expression in HFKs by qRT-PCR after transfection with shELF3 or shNC. (B) CCK-8 assay was used to assess the viability of HPV16 E6/E7- expressing HFKs transfected with shELF3 or shNC. (C-D) Flow cytometry was performed to detect the cell cycle of HPV16 E6/ E7-expressing HFKs transfected with shELF3 or shNC. (E-F) Analysis of CCNE2 expression in HPV16 E6/E7-expressing HFKs by western blot after transfection with shELF3 or shNC. (G-H) Analysis of HPV16 E6/E7 expressions in HPV16 E6/E7- expressing HFKs by qRT-PCR after transfection with shELF3 or shNC. GAPDH functioned as an endogenous control. Data are shown as mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviation: shELF3, short hairpin RNA (shRNA) targeting ELF3; shNC, shRNA negative control.

Fig. 5. ELF3 regulated the malignant transformation of HPV16 E6/E7-immortalized keratinocytes through the regulation of CCNE2.