Aberrant Hypermethylation of SALL3 with HPV Involvement Contributes to the Carcinogenesis of Cervical Cancer

Abstract

Objective: This study aimed to investigate the methylation status of the promoter region of spalt-like transcription factor 3 (SALL3) and the expression of SALL3 in cervical cancer to explore the function of this gene in cervical cancer carcinogenesis.

Methods: The methylation status of SALL3 was detected by methylation-specific PCR, and SALL3 gene expression was assessed by real-time quantitative PCR in the cervical cancer cell lines, SiHa, HeLa and C33A, as well as in cervical cancer tissue samples (n = 23), matched pericarcinomatous tissue samples (n = 23) and normal cervix tissue samples (n = 17). MTT was used to measure the cell viability and proliferation capacity of SiHa and HeLa cells.

Results: The SALL3 promoter was completely methylated in SiHa cells, unmethylated in C33A cells and partially methylated in HeLa cells. After treatment of SiHa and HeLa cells with 5 μM and 10 μM of 5-Azacytidine (5-Aza), respectively, the methylation level of the SALL3 promoter decreased and observed increase in the degree of unmethylation in a dose-dependent manner. Moreover, the relative expression of SALL3 mRNA increased as the concentration of 5-Aza increased in SiHa (p<0.05) and HeLa (p<0.05) cells. This above-mentioned increase in SALL3 mRNA in SiHa cells was more remarkable than that observed in HeLa cells. Cell proliferation capacity also decreased after administration of 5-Aza to SiHa and HeLa cells (p<0.05). Methylation of the SALL3 promoter was observed in 15 of 23 (65.21%) cervical cancer tissue samples, 15 of 23 (65.21%) matched pericarcinomatous tissue samples and 5 of 17 (29.41%) normal cervical tissue samples (p<0.05). SALL3 mRNA expression was significantly lower in cervical cancer and pericarcinomatous tissues compared with normal cervical tissues (p<0.05). In all cervix tissue samples, HPV infection was positively associated with hypermethylation of the promoter region of SALL3 (p<0.05, r = 0.408), and the expression of SALL3 mRNA in HPV-positive tissues was lower than that in HPV-negative tissues (p<0.05).

Conclusion: The aberrant hypermethylation of SALL3 together with HPV involvement inactivated its function as a tumor suppressor and contributed to carcinogenesis in cervical cancer.

Fig 1. Detection of the methylation status in the promoter regions of SALL3 in cervical cancer cell lines.

(A) Predicted CpG islands in the promoter region of SALL3. Numbers indicate the positions in bp relative to the transcription start site. The blue region represents the CpG islands and the red vertical bars are the CpG loci in these input sequences. (B) Detection of SALL3 methylation status by MSP in SiHa, C33A and HeLa cell lines. (M: methylated; U: unmethylated).

Fig 2. Detection of the methylation status in the promoter regions of SALL3 in SiHa(A) and HeLa(B) cells after treated with 5-Aza.

(M: methylated; U: unmethylated).

Fig 3. Relative expression of SALL3 mRNA in SiHa and HeLa cells after treatment with different concentrations of 5-Aza.

(*p<0.05).

Fig 4. Cell viability and proliferation capacity of the cervical cancer cell lines SiHa(A) and HeLa(B) after treatment with 5-Aza(*p<0.05).

Fig 5. Detection of SALL3 methylation status by MSP in cervical cancer tissues (A), matched-pericarcinomatous tissues (B) and normal cervix tissues(C) (M: methylated; U: unmethylated).

Fig 6. (A) Relative expression of SALL3 mRNA in cervical cancer, pericarcinomatous and normal cervical tissues. These data are represented by the median with an interquartile range (*p<0.05) (B) Relative expression of SALL3 mRNA in methylated and unmethylated cervical tissues.

These data are represented by the median with an interquartile range (*p<0.05).

Fig 7. Relative expression of SALL3 mRNA in HPV-positive and HPV-negative tissues.

These data are represented by the median with an interquartile range (*p<0.05).