Methylation-mediated transcriptional repression of microRNAs during cervical carcinogenesis

Abstract

Deregulated expression of microRNAs (miRNAs) is common and biologically relevant in cervical carcinogenesis and appears only partly related to chromosomal changes. We recently identified 32 miRNAs showing decreased expression in high-grade cervical intraepithelial neoplasia (CIN) and carcinomas not associated with a chromosomal loss, 6 of which were located within a CpG island. This study aimed to investigate to what extent these miRNAs are subject to DNA methylation-mediated transcriptional repression in cervical carcinogenesis. Methylation-specific PCR (MSP) analysis on a cell line panel representing different stages of human papillomavirus (HPV) induced transformation revealed an increase in methylation of hsa-miR-149, -203 and -375 with progression to malignancy, whereas expression of these miRNAs was restored upon treatment with a demethylating agent. All three miRNAs showed significantly increased levels of methylation in cervical carcinomas, whereas methylation levels of hsa-miR-203 and -375 were also significantly increased in high-grade CIN. A pilot analysis showed that increased hsa-miR-203 methylation was also detectable in HPV-positive cervical scrapes of women with high-grade CIN compared with controls. Similar to recent findings on hsa-miR-375, ectopic expression of hsa-miR-203 in cervical cancer cells decreased both the proliferation rate and anchorage independent growth. We found evidence for methylation-mediated transcriptional repression of hsa-miR-149, -203 and -375 in cervical cancer. Methylation of the latter two was already apparent in precancerous lesions and represent functionally relevant events in HPV-mediated transformation. Increased hsa-miR-203 methylation was detectable in scrapes of women with high-grade CIN, indicating that methylated miRNAs may provide putative markers to assess the presence of (pre)cancerous lesions.

Keywords: CIN lesion; DNA methylation; HPV; MSP; microRNA; squamous cell carcinoma.

Figure 1. Methylation patterns of selected miRNAs in the cell line panel. Methylation as determined in human foreskin keratinocytes (HFKs), HPV-transformed keratinocyte cell lines FK16A, FK18A and FK18B (reminiscent of high-grade CIN), and cervical cancer cell lines SiHa, HeLa and CaSki is shown for (A) hsa-miR-149, (B) hsa-miR-203, (C) hsa-miR-375, (D) hsa-miR-572 and (E) hsa-miR-638. In (F) ACTB results are shown, indicating successful modification and comparable input for all samples. In vitro methylated DNA (IVD) and unmodified DNA (UD) were included as a positive and negative control, respectively.

Figure 2. Re-expression of methylated miRNAs after demethylating treatment. Expression levels of (A) hsa-miR-149, (B) hsa-miR-203, (C) hsa-miR-375 and (D) hsa-miR-638 were determined in SiHa cells treated with PBS (mock), 200 nM and 5,000 nM DAC. (E) MSP results for hsa-miR-149, -203, -375 and -638 in SiHa cells treated with PBS (mock), 200 nM and 5,000 nM DAC are shown. The ACTB PCR results indicate successful modification and comparable input for all samples.

Figure 3. Methylation levels in cervical specimens. Methylation levels were determined in tissue specimens of normal cervix (n = 16), CIN1 (n = 4), CIN3 (n = 13) and SCCs (n = 20) for (A) hsa-miR-149, (B) hsa-miR-203 and (C) hsa-miR-375. In (D) methylation levels of hsa-miR-203 are shown in cervical scrapes of hrHPV-positive women with normal cytology and without any CIN disease during 5-y follow-up (n = 13) and in scrapes of hrHPV-positive women with abnormal cytology who presented with high-grade CIN disease within 18 mo (n = 17). hsa-miR-149, -203 and -375 methylation was undetectable in 0, 9 and 11 normals; 0, 2 and 2 CIN1; 1, 1 and 3 CIN3; and 0, 2, and 7 SCCs. Methylation of hsa-miR-203 was undetectable in 6 scrapes with normal cytology and 5 with abnormal cytology. Average methylation levels per sample group are indicated by the horizontal lines. * p < 0.05; ** p < 0.01.

Figure 4. The effect of ectopic expression of hsa-miR-203 in SiHa cells on cellular proliferation and anchorage independent growth. (A) Expression of hsa-miR-203 in SiHa cells transduced with either the control vector (SiHa + ctrl) or hsa-miR-203 (SiHa + miR-203). In (B) cell viability is shown in SiHa + ctrl cells and SiHa + miR-203 cells. In (C) representative pictures of colony formation in soft agar of SiHa + ctrl cells and SiHa + miR-203 cells and in (D) quantification of the number of colonies in soft agar formed by SiHa + ctrl cells and SiHa + miR-203 cells.