关键词: 丝氨酸/苏氨酸蛋白激酶31基因, 甲基化, 人乳头病毒16, 宫颈癌, 生物标志

Abstract: Background and purpose: Studies have proved that the serine/threonine kinases 31 (STK31) gene plays important roles in human cancers. The STK31 gene expression was demonstrated to be regulated by the methylation status of its promoter/exon1 region. Viral infection was revealed to be associated with the hypermethylation of some tumor suppressor genes in some tumor samples. The purposes of this paper were to study the roles of HPV16 E6, E7, or E6/ E7 oncogenes in methylation status and expression of the STK31 gene, and potential effects of DNA methyltransferases (DNMTs) on STK31 gene methylation status. Methods: Ectopically-expressed HPV16 E6, E7, or E6/E7 cells were established by transfecting HPV16 E6, E7, or E6/E7 oncogenes with lentivirus vectors into HPV-negative cervical cancer cell lines HT-3 and C33A. Bisulfite genomic sequencing PCR (BGS) combined with TA clone and MSP (methylation-specific PCR) were used to analyze methylation status of the STK31 gene promoter/exon1 region in HPV-positive cervical cancer cell lines (HeLa, SiHa, CaSki), HPV-negative cervical carcinoma cell lines (C33A, HT-3) and the transfected cells. The mRNA and protein expression of STK31, DNMT1, DNMT2, DNMT3a, DNMT3b and DNMT3L were detected by RT-PCR and Western blot. Results: Transfection efficiency was tested by Western blot, which showed that the transfected cells successfully expressed E6, E7, or E6/E7 proteins, respectively. The STK31 gene promoter/exon1 was hypomethylated in HPV-positive cell lines HeLa, SiHa and CasKi resulting in detection of mRNA and protein expression. STK31 gene promoter/exon1 showed hypermethylation leading to silenced expression in the two HPV-negative cervical cancer cells HT-3 and C33A. Compared with primary HT-3 and C33A cells, the methylation status of STK31 promoter/exon1 was down-regulated that led to expression of STK31 in the ectopically-expressed HPV16 E7 and E6/E7 cells. Expressions of DNMT1, DNMT3a and DNMT3b genes at the level of transcription were higher in C33AE6/E7 and HT-3E6/E7 cells than those in C33A-vector and HT-3 vector cells, respectively (P<0.001). mRNA levels of DNMT1, DNMT3a and DNMT3b were higher in HPV16-positive cervical cancer tissues than those in HPV-negative cervical cancer tissues, respectively (t=5.997, P<0.001; t=6.743, P<0.001; t=7.926, P<0.001). DNMT2 mRNA level was lower in C33AE6/E7 and HT-3E6/E7 cells than those in C33A-vector and HT-3 vector cells, respectively (t=7.451, P<0.001; t=2.451, P<0.05). mRNA level of DNMT2 gene was lower in HPV16-positive cervical cancer tissues than in HPV-negative cervical cancer tissues (t=9.134, P<0.001). There was no statistically significant difference in expression levels of DNMT3L mRNA between cervical cancer cell lines before and after transfection, or HPV16-positive and HPV-negative cervical cancer tissues, respectively (P>0.05, data not shown). Conclusion: HPV infection leads to the down-regulated methylation status of STK31 promoter/exon1 that results in the expression of STK31. STK31 gene expression is regulated by methylation status of its promoter/exon1 region. HPV16 E7 and E6/E7 oncogenes may influence the methylation status of STK31 gene promoter/exon1 region by regulating the expression of DNMT2.

Key words: Serine/threonine kinases 31 gene, Methylation, HPV16, Cervical cancer, Biomarker