P16INK4a and p14ARF mRNA expression in Pap smears is age-related

Abstract

Expression of high-risk HPV oncogenes results in a strong overexpression of cellular protein p16INK4a. Immunohistochemical staining for p16INK4a is widely used as diagnostic marker. However, p16INK4a upregulation was also described as a biomarker of age. Here we analyzed p16INK4a expression in cervical smears to investigate if patient age may influence p16INK4a-based cervical cancer diagnosis. p14ARF was analyzed as a related supportive biomarker. Cervical scrapes were taken and stored in RNAlater. Total RNA was extracted, and cDNA was analyzed for expression of p16INK4a and p14ARF relative to β-actin, by real-time reverse transcriptase PCR SYBR-Green I assays. Patient-derived smears referred as HSIL (n=45) had 6.27-fold higher p16INK4a mRNA expression than smears of cytologically normal and HPV-negative persons (n=48). Expression of p14ARF was 4.87-fold higher. When women with normal diagnoses were stratified for age, a significantly enhanced p16INK4a (2.88-fold) and p14ARF (1.9-fold) expression was observed as a consequence of ageing. A significant age-dependent upregulation was also observed in older HSIL patients (2.54-fold). Our study revealed significantly enhanced expression of p16INK4a/p14ARF mRNA in cervical scrapes referred to as HSIL compared with normal women. An age-dependent bias has to be considered when quantifying these tumor suppressor genes, with respect to cervical cancer development.