宫颈癌细胞系中HPV16 E6、E7及E6/E7基因对STK31基因甲基化状态及表达的影响
궁경암세포계중HPV16 E6、E7급E6/E7기인대STK31기인갑기화상태급표체적영향
The roles ofHPV16E6,E7 andE6/E7 genes inSTK31 promoter/exon1 methylation and expression levels in cervical cancer cell lines
  • 万方数据
    中国癌症杂志 중국암증잡지
    China Oncology
    2015年 9期 641-651 ,共11页

    殷复粉%王宁%于啸%毕晓宁%徐小惠%王言奎

    은복분%왕저%우소%필효저%서소혜%왕언규

    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 絲氨痠/囌氨痠蛋白激酶31基因%甲基化%人乳頭病毒16%宮頸癌%生物標誌
    사안산/소안산단백격매31기인%갑기화%인유두병독16%궁경암%생물표지
    Serine/threonine kinases 31 gene%Methylation%HPV16%Cervical cancer%Biomarker
    背景与目的：丝氨酸/苏氨酸蛋白激酶31(serine/threonine kinases 31，STK31)基因在人类多种癌症中扮演重要角色，且STK31基因的表达受其启动子及第一外显子区甲基化状态的影响；病毒感染与肿瘤组织中某些抑癌基因启动子区高甲基化有关。本研究旨在探讨宫颈癌细胞系中HPV16 E6、E7及E6/E7癌基因对STK31基因甲基化状态及表达的影响，以及不同种类甲基转移酶(DNA methyltransferases，DNMTs)基因在STK31基因甲基化中的潜在作用。方法：构建外源性HPV16 E6、E7以及E6/E7基因共表达慢病毒，分别感染人乳头瘤病毒(human papillomavirus，HPV)阴性宫颈癌细胞系HT-3及C33A，获得稳定转染的细胞系；采用亚硫酸氢盐基因组测序法(bisulfite genomic sequencing，BGS)和甲基化特异性PCR (methylation-specific PCR，MSP)检测3种HPV阳性宫颈癌细胞系HeLa、SiHa和CasKi以及HPV阴性宫颈癌细胞系HT-3和C33A转染前后STK31基因的甲基化状态；RT-PCR及蛋白[质]印迹法(Western blot)检测上述宫颈癌细胞系中STK31基因的表达以及DNMT1、DNMT2、DNMT3a、DNMT3b和DNMT3L基因在HPV16转染前后宫颈癌细胞系及HPV阳性、HPV阴性宫颈癌组织中的表达情况。结果：外源性HPV16 E6、E7以及E6/E7基因可在HPV阴性宫颈癌细胞系中稳定表达。3种HPV阳性细胞系HeLa、SiHa和CasKi中STK31基因呈低甲基化状态，STK31 mRNA及蛋白质表达阳性；2种HPV阴性细胞系HT-3、C33A中STK31基因则表现为高甲基化状态，STK31 mRNA及蛋白质表达缺失；与未感染慢病毒HT-3和C33A细胞系比较，外源性HPV16 E7以及E6/E7表达的HT-3和C33A细胞系STK31基因甲基化程度降低，其mRNA及蛋白质重新表达。DNMT1、DNMT3a和DNMT3b基因在HT-3E6/E7和C33AE6/E7细胞系中mRNA水平分别高于HT-3空载细胞系和C33A空载细胞系，差异有统计学意义(P<0.001)。DNMT1、DNMT3a和DN-MT3b基因的mRNA水平在HPV16阳性宫颈癌组织中的表达高于其在HPV阴性宫颈癌组织中的表达，差异有统计学意义(t=5.997，P<0.001；t=6.743，P<0.001；t=7.926，P<0.001)。DNMT2在HT-3E6/E7和C33AE6/E7细胞系中mRNA表达水平分别低于HT-3空载细胞系和C33A空载细胞系，差异有统计学意义(t=7.451，P<0.001；t=2.451， P<0.05)；DNMT2基因转录水平在HPV16阳性宫颈癌组织中低于HPV阴性宫颈癌组织(t=9.134，P<0.001)。DN-MT3L mRNA表达水平在宫颈癌细胞系转染前后及HPV阴阳性宫颈癌组织中的差异无统计学意义(P>0.05)。结论：HPV感染可导致STK31基因启动子及第1外显子区甲基化状态降低，低甲基化状态促进该基因表达。STK31基因的表达受其启动子及第1外显子区甲基化状态的调控。HPV16 E7、E6/E7基因可能通过影响DNMT2的表达参与调控癌基因STK31基因启动子及第1外显子区甲基化状态。
    배경여목적：사안산/소안산단백격매31(serine/threonine kinases 31，STK31)기인재인류다충암증중분연중요각색，차STK31기인적표체수기계동자급제일외현자구갑기화상태적영향；병독감염여종류조직중모사억암기인계동자구고갑기화유관。본연구지재탐토궁경암세포계중HPV16 E6、E7급E6/E7암기인대STK31기인갑기화상태급표체적영향，이급불동충류갑기전이매(DNA methyltransferases，DNMTs)기인재STK31기인갑기화중적잠재작용。방법：구건외원성HPV16 E6、E7이급E6/E7기인공표체만병독，분별감염인유두류병독(human papillomavirus，HPV)음성궁경암세포계HT-3급C33A，획득은정전염적세포계；채용아류산경염기인조측서법(bisulfite genomic sequencing，BGS)화갑기화특이성PCR (methylation-specific PCR，MSP)검측3충HPV양성궁경암세포계HeLa、SiHa화CasKi이급HPV음성궁경암세포계HT-3화C33A전염전후STK31기인적갑기화상태；RT-PCR급단백[질]인적법(Western blot)검측상술궁경암세포계중STK31기인적표체이급DNMT1、DNMT2、DNMT3a、DNMT3b화DNMT3L기인재HPV16전염전후궁경암세포계급HPV양성、HPV음성궁경암조직중적표체정황。결과：외원성HPV16 E6、E7이급E6/E7기인가재HPV음성궁경암세포계중은정표체。3충HPV양성세포계HeLa、SiHa화CasKi중STK31기인정저갑기화상태，STK31 mRNA급단백질표체양성；2충HPV음성세포계HT-3、C33A중STK31기인칙표현위고갑기화상태，STK31 mRNA급단백질표체결실；여미감염만병독HT-3화C33A세포계비교，외원성HPV16 E7이급E6/E7표체적HT-3화C33A세포계STK31기인갑기화정도강저，기mRNA급단백질중신표체。DNMT1、DNMT3a화DNMT3b기인재HT-3E6/E7화C33AE6/E7세포계중mRNA수평분별고우HT-3공재세포계화C33A공재세포계，차이유통계학의의(P<0.001)。DNMT1、DNMT3a화DN-MT3b기인적mRNA수평재HPV16양성궁경암조직중적표체고우기재HPV음성궁경암조직중적표체，차이유통계학의의(t=5.997，P<0.001；t=6.743，P<0.001；t=7.926，P<0.001)。DNMT2재HT-3E6/E7화C33AE6/E7세포계중mRNA표체수평분별저우HT-3공재세포계화C33A공재세포계，차이유통계학의의(t=7.451，P<0.001；t=2.451， P<0.05)；DNMT2기인전록수평재HPV16양성궁경암조직중저우HPV음성궁경암조직(t=9.134，P<0.001)。DN-MT3L mRNA표체수평재궁경암세포계전염전후급HPV음양성궁경암조직중적차이무통계학의의(P>0.05)。결론：HPV감염가도치STK31기인계동자급제1외현자구갑기화상태강저，저갑기화상태촉진해기인표체。STK31기인적표체수기계동자급제1외현자구갑기화상태적조공。HPV16 E7、E6/E7기인가능통과영향DNMT2적표체삼여조공암기인STK31기인계동자급제1외현자구갑기화상태。
    Background and purpose:Studies have proved that the serine/threonine kinases 31 (STK31) gene plays important roles in human cancers. TheSTK31 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 ofHPV16E6,E7, orE6/E7 oncogenes in methylation status and expression of theSTK31 gene, and potential effects of DNA methyltransferases (DNMTs) onSTK31 gene methylation status.Methods:Ectopically-expressed HPV16 E6, E7, or E6/E7 cells were estab-lished by transfectingHPV16E6,E7, orE6/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 theSTK31 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. TheSTK31 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 ofSTK31 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 andDNMT3b 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 ofSTK31 promoter/exon1 that results in the expression of STK31.STK31 gene expression is regulated by methylation status of its promoter/exon1 region. HPV16E7 andE6/E7 oncogenes may influence the methylation status ofSTK31 gene promoter/exon1 region by regulating the expression of DNMT2.
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