The Role of HPV16 E1 in Cervical Carcinogenesis

Abstract

Objectives: This study aimed to determine the expression profile of HPV-16 E1 in cervical samples and the role of E1 in cervical carcinogenesis. In addition, this work also aimed to determine whether the HPV-16 E1 63bp duplication is present in the Thai population.
 
 Methods: One-hundred and twenty-four HPV16 positive cervical samples ranging from normal, CIN1, CIN2/3, and SCC lesions were studied. E1 mRNA expression was determined by ddPCR. Methylation of promoters p97 and p670 was quantified by pyrosequencing, while PCR and sequencing were used to determine the physical state and variations of HPV16 E1 genome. HEK 293T cells were transfected with pEGFP-C1 containing HPV16 E1. Cell proliferation of transfected cells was measured using cell viability count and cell metabolism assay. Apoptosis and necrosis was determined in transfected cells treated with or without QVD-OPH (pan-caspase inhibitor) by Annexin V and propidium iodide staining and quantitated using flow cytometry. RNA expression Microarray analysis was performed on FACS sorted HPV16 transfected cells. RNA expression was confirmed by Real-time reverse transcription (RT) PCR. Plasmid containing pEGFP-C1 was used as a vector control throughout the experiments.
 
 Results: Increased E1mRNA expression related to disease progression (normal 0.18, CIN 1 0.41, CIN 2/3 0.65 and SCC 0.79) was demonstrated with significant positive correlation (r = 0.661, p =0.019). No association between physical state and E1 expression was found.� Methylation of p97and p670 promoters showed significant elevation in SCC compared to normal samples. Only 4.2% showed genomic variations of HPV16 E1 63 bp duplication. HPV16 E1 transfected HEK293T cells showed a significant decrease in number of viable cells and cell proliferation 24 h post-transfection (p < 0.0001). HPV16 E1 significantly induced both apoptotic and necrotic cell death 48 h post-transfection. Treatment of HPV16 E1 transfected cells with QVD-OPH showed a decreasing trend without statistical significance. Microarray and real-time RT PCR results revealed that E1 dysregulated genes involved in protein synthesis (RPL36A), metabolism (ALDOC), cell proliferation (CREB5, HIF 1A, JMJD1C, FOXO3, NFKB1, PIK3CA, TSC22D3), DNA damage (ATR, BRCA1 and CHEK1), and immune response (ISG20) pathways. Kinetic host gene expression in HPV16 E1 transfected cells indicated that most genes were downregulated after 48 h post-transfection.
 
 Conclusion: Detection of E1 mRNA and p97 methylation were beneficial as cancer prognostic markers. The presence of 63 bp duplication in HPV16 E1 was observed in the Thai population and related to low grade lesions. The presence of HPV16 E1 protein alone was able to dysregulate many cellular pathways in a time dependent manner. Notable genes disrupted by HPV16 E1 included genes involved in cell proliferation and host DNA damage response. This study suggests that E1 may play a role in cancer development however HPV16 E1 transfection experiments did not provide a definite carcinogenic pathway which warrants further studies.