Study Of Methylation Status On SPINT2 Gene Promoter And Exon 1 And Expression In Cervical Carcinoma

Persistent infection with high risk types of human papillomavirus(HPV) has been widely recognized as a major cause of cervical cancer. Genetic and epigenetic alterations occur after HPV infection, while DNA methylation is an important epigenetic mechanism in cervical carcinogenesis.Objective To explore the promoter and the first exon methylation status of SPINT2 gene and its effect on expression in cervical carcinoma cell lines and cervical cancer tissues, and its correlation with clinicopathologic characteristics.Methods HPV positive cervical cancer cell lines HeLa, CaSki, SiHa and HPV negative cervical cancer cell lines HT-3, C-33 A and 50 cases of cervical carcinoma tissues, 20 cases of normal cervical tissues were used for this study. We used5-aza-2’-deoxycytidine(5-Aza-CdR) to treat HeLa cell lines, and used lentivirus with HPV16 E6/E7 fusion gene to infect HT-3 cell line. Methylation-Specific PCR(MSP) and bisulfite genomic sequencing method(BGS) were used to detect the methylation status of promoter and the first exon of SPINT2 gene, while real-time quantitative PCR was used to detect the mRNA expression.Results ①Methylation status of cervical cancer cell lines: MSP showed that HPV positive cell line HeLa was M type, CaSki and SiHa cell lines were M + U type, while HPV negative cell lines HT-3 and C-33 A appeared only U bands. HeLa cells after5-Aza-CdR treatment showed M + U type, HT-3 cells infected by lentivirus with HPV16E6/E7 fusion gene were still U type. BGS showed that methylation rates of HeLa, CaSki,Si Ha, HT-3 and C-33 A were 95.1%, 46.3%, 32.1%, 0%, 4.9% respectively. After5-Aza-CdR treatment, methylation rate of HeLa cells decreased to 56.0%, while infected HT-3 cells increased to 8.8%. ②Methylation status of cervical tissues: MSP showed that2 of 50 cervical cancer cases were M type, 25 were M + U type, 23 were U type. While 5of 20 normal cervical tissue cases were M + U type, the other 15 were U type. The methylation rate of cervical cancer tissues were higher than normal cervical tissues, the difference was statistically significant(P=0.028). Among cervical cancer cases,methylation status of 46 HPV positive cervical cancer cases was that 2 were M type, 25 were M + U type, 19 were U type, while all the HPV negative cervical cancer cases were U type. The methylation rate of HPV positive cervical cancer tissues were higher than HPV negative cervical cancer tissues, the difference was statistically significant(P=0.038).③The methylation status of SPINT2 gene promoter and the first exon had correlation with pathological grade(P=0.003). The difference of methylation status between G2 and G3 was statistically significant(P=0.001), while the difference between G1 and G2, G1 and G3 were not statistically significant(P>0.05). The methylation status had no significant correlation with age, stage, histological type, tumor size, lymph node metastasis, vaginal invasion(P>0.05). ④The mRNA expression of SPINT2 gene in three HPV positive cell lines was less than in two HPV negative cell lines, the difference was significantly different(P=0.027). The expression of SPINT2 gene in HeLa cell line treated with 5-Aza-CdR raised to 3.945 times then before, while the expression in HT-3 cell line infected by lentivirus with HPV16 E6/E7 fusion gene decreased to 0.688 times. The expression of SPINT2 gene in cervical cancer was significantly higher than in normal cervical tissues(P=0.032). The expression of SPINT2 gene in methylated cervical tissues was significantly higher than in unmethylated tissues(P=0.000).Conclusion ①The methylation rate of the promoter and the first exon of SPINT2 gene in cervical cancer tissues are higher than in normal cervical tissues. ② The methylation rate of SPINT2 gene in HPV positive cell lines are higher than that in HPV negative cell lines, while HPV positive cervical cancer tissues are higher than HPV negative cervical cancer tissues, so the methylation status of SPINT2 gene may have relationship with HPV infection. ③ The downregulated expression of SPINT2 gene is closely related to the methylation of promoter and the first exon. ④ The methylation status of SPINT2 gene promoter and the first exon had correlation with pathological grade, the methylation rate in poorly differentiated tumor is higher than moderately differentiated tumor.