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. 2008 Apr 17;27(18):2575-82.
doi: 10.1038/sj.onc.1210919. Epub 2007 Nov 12.

Human papillomavirus type 16 reduces the expression of microRNA-218 in cervical carcinoma cells

I Martinez  1 , A S Gardiner, K F Board, F A Monzon, R P Edwards, S A Khan
Affiliations

Human papillomavirus type 16 reduces the expression of microRNA-218 in cervical carcinoma cells

I Martinez et al. Oncogene. .

Abstract

Human papillomaviruses (HPVs) are involved in the pathogenesis of cancer of the cervix (CaCx). MicroRNA (miRNA) expression analysis using Ambion (Austin, TX, USA) arrays showed that three miRNAs were overexpressed and 24 underexpressed in cervical cell lines containing integrated HPV-16 DNA compared to the normal cervix. Furthermore, nine miRNAs were overexpressed and one underexpressed in integrated HPV-16 cell lines compared to the HPV-negative CaCx cell line C-33A. Based on microarray and/or quantitative real-time PCR and northern blot analyses, microRNA-218 (miR-218) was specifically underexpressed in HPV-positive cell lines, cervical lesions and cancer tissues containing HPV-16 DNA compared to both C-33A and the normal cervix. Expression of the E6 oncogene of high-risk HPV-16, but not that of low-risk HPV-6, reduced miR-218 expression, and conversely, RNA interference of E6/E7 oncogenes in an HPV-16-positive cell line increased miR-218 expression. We also demonstrate that the epithelial cell-specific marker LAMB3 is a target of miR-218. We also show that LAMB3 expression is increased in the presence of the HPV-16 E6 oncogene and this effect is mediated through miR-218. These findings may contribute to a better understanding of the molecular mechanisms involved in cervical carcinogenesis VSports手机版. .

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Figures

Figure 1
Figure 1
Confirmation of miRNA microarray expression data in various cervical cell lines and normal cervical tissue. (A) Northern blot analysis. The housekeeping splicing-related small U6 RNA was used as a loading control. (B) Real-time qRT-PCR analysis. RNU43 served as the endogenous control for miRNAs.
Figure 2
Figure 2
Expression of miRNAs and the SLIT2 gene in cervical tissues. qRT-PCR analysis of three cervical intraepithelial neoplasias type III (CIN III) and five cervical carcinomas (CaCx). The normal cervix sample was obtained from Stratagene. G3PDH served as the endogenous control for SLIT2.
Figure 3
Figure 3
HPV-16 E6 oncogene reduces the expression of miR-218. (A) qRT-PCR analysis of miR-218 and SLIT2 in U2OS-NEO, U2OS-16E6, and U2OS-16E7. (B) Expression of HPV-16 E6 and E7, miR-218 and SLIT2 in the 20861 cell line with or without RNAi against HPV-16 E6/E7. (C) qRT-PCR analysis of miR-218 in NOK-NEO, NOK cell line expressing the E6 gene of either the low-risk HPV-6 (NOK-6E6) or high-risk HPV-16 (NOK-16E6). RNU43 served as the endogenous control for miRNAs, while G3PDH served as the endogenous control for E6, E7 and SLIT2.
Figure 4
Figure 4
Expression of LAMB3 is reduced in the presence of miR-218. (A) Northern blot analysis of miR-218 after transfection of a precursor of miR-218 in SiHa and 20861 cell lines. U6 RNA was used as a loading control. (B) qRT-PCR analysis of LAMB3 in SiHa and 20861 cell lines transfected with a pre-miR-218. (C) Western blot analysis of LAMB3 protein in SiHa cells transfected with pre-miR-218. Location of the 40-kDa LAMB3 protein is indicated. G3PDH was used as a control. (D) Expression of LAMB3 mRNA in the 20861 cell line with or without RNAi against HPV-16 E6/E7, and in the U2OS-NEO and U2OS-16E6 cell lines. G3PDH served as the endogenous control for LAMB3.
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