Inhibitors of Differentiation-1 Promotes Transformation of Human Papillomavirus Type 16-immortalized Cervical Epithelial Cells

Our previous study implied a correlation of inhibitors of differentiation-1 (Id-1) to cervical cancer development. However, how Id-1 contributes to cervical carcinogenesis is unknown. In the present study, we investigated the role of Id-1 in transforming cervical cells with an in vitro transformation model. The human papillomavirus (HPV) immortalized cervical epithelial cells (H8) were successfully transformed by exposure to the carcinogen N-nitrosopyrrolidine (NPYR). The results showed that both Id-1 RNA and protein expression were significantly increased in transformed H8 cells, suggesting a possible role of Id-1 in cervical cell transformation. Ectopic expression of Id-1 in H8 cells potentiated NPYR-induced cell transformation. In contrast, silencing of Id-1 suppressed NPYR-induced H8 cell transformation. A cDNA microarray assay was performed, which identified suggested potential cell signaling pathways for NPYR-induced H8 cell transformation. The results suggest that Id-1 plays an oncogenic role in the cervix, which sheds light on cervical cancer development and implies potential target for cervical cancer prevention and therapy. Id-1-shRNA H8 and the control pGIPZ-H8 cells, with or without NPYR treatment, were used for cDNA microarray assay. Total RNA was extracted by using TRIZOL Reagent (Life Technologies, Carlsbad, CA, US) following the manufacturer's instructions. Total RNA was further purified by RNeasy micro kit (QIAGEN, GmBH, Germany) and RNase-free DNase Set (QIAGEN, GmBH, Germany). The RNAs were amplified, labeled and purified by using GeneChip 3' IVT Express Kit (Affymetrix, Santa Clara, CA, US) following the manufacturer's instructions. Array hybridization and wash were performed using GeneChip® Hybridization, Wash and Stain Kit (Affymetrix, Santa Clara, CA, US) in a Hybridization Oven 645 (Affymetrix) and Fluidics Station 450 (Affymetrix) following the manufacturer's instructions. The slides were scanned with GeneChip® Scanner 3000 (Affymetrix) using the Command Console Software 3.1 (Affymetrix) with default settings. Raw data were normalized by using the MAS 5.0 algorithm of GeneSpring Software 11.0 (Agilent Technologies, Santa Clara, CA, US).