Human papillomaviruses (HPVs) are involved in the pathogenesis of different types of human cancers, especially cancer of the cervix (CaCx). MicroRNAs (miRNAs) are post-transcriptional gene regulators that have recently been associated with many types of cancers. We analyzed the expression of cellular miRNAs in HPV-16 positive cervical cell lines and tissues via microarray, Northern blotting, and quantitative RT-PCR. Three miRNAs were overexpressed and 24 underexpressed in cervical cell lines containing integrated HPV-16 DNA compared to the normal cervix. An HPV-negative CaCx cell line, C-33A, showed underexpression of four miRNAs compared to the normal cervix. Also, nine miRNAs were overexpressed and only one underexpressed in cell lines containing integrated HPV-16 DNA compared to C-33A. MicroRNA-218 (miR-218) was specifically underexpressed in cell lines, cervical lesions and cancer tissues containing integrated HPV-16 DNA compared to both C-33A and the normal cervix. Exogenous expression of the HPV-16 E6 oncogene reduced miR-218 expression, and conversely, RNA interference of E6/E7 oncogenes in an HPV-16 positive cell line increased miR-218 expression. Furthermore, exogenous expression of miR-218 in HPV-16 positive cell lines decreased expression of the epithelial-specific gene LAMB3, which is involved in cell migration and tumorigenicity.
We analyzed the expression of cellular miRNAs in additional clinical samples including six HPV-16 positive cervical cancers, three cervical dysplasias, and four normal cervical tissues using TaqMan® MicroRNA Arrays V2.0. Eighteen miRNAs were overexpressed and two underexpressed in cervical cancer tissues compared to normal cervical tissues. Nine miRNAs were consistently overexpressed, and two miRNAs were consistently underexpressed, including miR-218, in cervical dysplasias and cervical cancer tissues compared to normal tissues. We also found that exogenous expression of miR-218 in HPV-16 positive cell lines decreased expression of the extracellular matrix protein MMP3, which is involved in the epithelial-mesenchymal transition.
We also demonstrated that HPV-16 E6 regulates miR-218 via the histone acetyltransferase p300 in cervical cells. The expression of miR-218 was found to be reduced in HPV-negative cells upon p300 knock-down or p300 inhibition with anacardic acid, and expression of miR-218 was reactivated in HPV-16 positive cells upon EZH2 knock-down or inhibition with adenosine dialdehyde. Reactivation of miR-218 was enhanced upon treatment with a combination of 5-azacytidine, trichostatin A, and adenosine dialdehyde. We also demonstrated that miR-218 reduces the migration and invasion of SiHa cervical cancer cells, indicating that miR-218 functions as a tumor suppressor in cervical cancer and that it may have therapeutic value.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-12222010-162351 |
| Date | 23 December 2010 |
| Creators | Gardiner, Amy Sabrina |
| Contributors | Richard Steinman, Saleem Khan, Bino John, James Pipas, Stefan Duensing |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-12222010-162351/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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