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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE46842 | GEO | 2013/05/11

SECONDARY ACCESSION(S): PRJNA202448

REPOSITORIES: GEO

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Project description: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 GeneChipÂ® 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 GeneChipÂ® 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).

Project description:Background. MicroRNAs (miRNAs) are short (~22 nt) non-coding regulatory RNAs that control gene expression at the translational level. Deregulation of miRNA expression has been discovered in a wide variety of tumours and it is now clear that they contribute to cancer development and progression. This prompted the development of miRNA-chips for cancer diagnosis or prognosis, opening a new door to understand carcinogenesis. Cervical cancer is one of the most common cancers in women worldwide. Therefore, there is a strong need for a non-invasive, fast and efficient method to diagnose the disease. We investigated miRNA expression profiles in cervical cancer using a microarray platform developed in house containing probes for mature miRNAs. Results. We have evaluated miRNA expression profiles of a heterogeneous set of cervical tissues from 25 different patients. This set included 19 normal cervical tissues, 4 squamous cell carcinoma, 5 high-grade squamous intraepithelial lesion (HSIL) and 9 low-grade squamous intraepithelial lesion (LSIL) samples. We observed high variability in miRNA expression especially among normal cervical samples, which prevented us from obtaining a unique miRNA expression signature for this tumour type. However, miRNAs deregulation in malignant and pre-malignant cervical tissues was detected after tackling the high variability observed. We were also able to identify putative targets of relevant candidate miRNAs. Conclusions. Our results show that miRNA deregulation may play an important role in the malignant transformation of cervical squamous cells. In addition, deregulated miRNAs highlight new candidate targets allowing a better understanding of the molecular mechanism of this tumour type. In this study we used a common reference design experiment where the common reference used was a commercial RNA from normal cervix (Ambion) and the test samples were 4 pre-treatment squamous cell cervical carcinoma, 7 high-grade Squamous Intraepithelial Lesion (CINII, n=2 and CIN III, n=5) sample, 9 low-grade Squamous Intraepithelial Lesion (CIN I) samples, 19 normal cervix samples and 4 pools of normal cervix samples.

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

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