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Abstract

Cancer of the uterine cervix (cervical cancer) represents 6.6% of all female cancers with 570,000 new cases as of 2018 and is the fourth most frequent cancer worldwide. Human papillomavirus (HPV), especially type 16 has been recognized as a necessary and causal factor in the pathogenesis of this malignancy. Effective screening for HPV-induced precursor lesions plays a huge role in reducing cervical cancer mortality rate globally and HPV DNA testing has recently become one of the recommended screening tests. However, all the HPV DNA tests available while highly sensitive lack high specificity and positive predictive value for advanced lesions and therefore lead to unnecessary anxiety and overtreatment in many screen-positive women. Consequently, HPV RNA tests have been developed for the intelligible discrimination of low (LSIL) and high grade squamous intraepithelial lesions (HSIL). These tests identified a significant upregulation of E1C transcripts in HSIL and CxCa. Therefore, E1C has been proposed as a diagnostically relevant RNA marker, which is capable of distinguishing LSIL from HSIL. In this PhD thesis, HPV16 E1C was characterized to define its role in cervical carcinogenesis. Specifically, E1C was overexpressed in four different HPV16-positive cell lines to analyze its effect on different HPV16 transcripts involved in cell cycle progression and transformation. E1^E4 transcripts showed a significant downregulation upon E1C overexpression, whereas E6*I and E7 transcripts remain unchanged. When investigated for the presence of endogenous E1C protein, the HPV16 E1C transcript positive cell lines tested stained negative in western blot. However, transiently overexpressed E1C protein in HEK-293T cells after proteasome inhibition was detectable in western blot and mass spectrometry, which points towards the instability of the protein. HEK-293T cells co-transfected with E1C expression construct and construct expressing a reporter under control of the HPV16 upstream regulatory region (URR) demonstrated URR activation by E1C transcripts even when E1C translation was abolished by mutation hinting that E1C RNA is the biologically active form of E1C. Additional experiments must be performed to study further about the viral and cellular changes driven by E1C RNA and to understand its upregulation in high-grade lesions.