中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2014年
1期
102-104
,共3页
刘泓渊%屈鸣麒%余聚%兰青%步星耀
劉泓淵%屈鳴麒%餘聚%蘭青%步星耀
류홍연%굴명기%여취%란청%보성요
乳腺丝氨酸蛋白酶抑制剂基因%胶质瘤%启动子甲基化%组蛋白乙酰化
乳腺絲氨痠蛋白酶抑製劑基因%膠質瘤%啟動子甲基化%組蛋白乙酰化
유선사안산단백매억제제기인%효질류%계동자갑기화%조단백을선화
Maspin%Glioma%Promoter methylation%Histone acetylation
目的 探讨乳腺丝氨酸蛋白酶抑制剂基因(maspin)在人胶质瘤细胞株U87、U251中沉默与表观遗传学关系.方法 运用逆转录-聚合酶链反应( RT-PCR)、Western blot检测maspin基因在U87、U251的表达.甲基化特异性聚合酶链反应(MSP)测定maspin基因启动子区区域甲基化状态.同时运用亚硫酸氢盐测序法(BSP)检测maspin基因启动子区甲基化CpG位点.RT-PCR检测12 μmol/L 5-氮杂-2’-脱氧胞苷(5-Aza-DC)和/或500 nmol/L曲古霉素(TSA)、15 μmol/L 5-Aza-DC/或500 nmol/L TSA分别处理U87、U251后,maspin基因mRNA表达水平.结果 在胶质瘤细胞株U87、U251中maspin基因表达沉默.maspin基因启动子CpG位点处于甲基化状态.RT-PCR结果显示,未处理组、5-Aza-DC、TSA、5-Aza-DC和TSA分别处理U87后,maspin mRNA相对表达量分别为:0.001 2±0.000 1 、0.0303 ±0.0006、0.019 6±0.001 6、0.052 6 ±0.003 4；未处理组和5-Aza-DC、TSA、5-Aza-DC和TSA分别处理U251后,maspin mRNA相对表达量分别为:0.002 2±0.0030、0.0122±0.0008、0.0249±0.0012、0.038 4±0.003 1.表明5-Aza-DC和/或TSA能恢复U87、U251中maspin基因表达(P＜0.01).结论 maspin在胶质瘤中的表达抑制与其启动子CpG岛甲基化及组蛋白去乙酰化相关,5-Aza-DC和/或TSA能恢复maspin在胶质瘤细株U87、U251中的转录.
目的 探討乳腺絲氨痠蛋白酶抑製劑基因(maspin)在人膠質瘤細胞株U87、U251中沉默與錶觀遺傳學關繫.方法 運用逆轉錄-聚閤酶鏈反應( RT-PCR)、Western blot檢測maspin基因在U87、U251的錶達.甲基化特異性聚閤酶鏈反應(MSP)測定maspin基因啟動子區區域甲基化狀態.同時運用亞硫痠氫鹽測序法(BSP)檢測maspin基因啟動子區甲基化CpG位點.RT-PCR檢測12 μmol/L 5-氮雜-2’-脫氧胞苷(5-Aza-DC)和/或500 nmol/L麯古黴素(TSA)、15 μmol/L 5-Aza-DC/或500 nmol/L TSA分彆處理U87、U251後,maspin基因mRNA錶達水平.結果 在膠質瘤細胞株U87、U251中maspin基因錶達沉默.maspin基因啟動子CpG位點處于甲基化狀態.RT-PCR結果顯示,未處理組、5-Aza-DC、TSA、5-Aza-DC和TSA分彆處理U87後,maspin mRNA相對錶達量分彆為:0.001 2±0.000 1 、0.0303 ±0.0006、0.019 6±0.001 6、0.052 6 ±0.003 4；未處理組和5-Aza-DC、TSA、5-Aza-DC和TSA分彆處理U251後,maspin mRNA相對錶達量分彆為:0.002 2±0.0030、0.0122±0.0008、0.0249±0.0012、0.038 4±0.003 1.錶明5-Aza-DC和/或TSA能恢複U87、U251中maspin基因錶達(P＜0.01).結論 maspin在膠質瘤中的錶達抑製與其啟動子CpG島甲基化及組蛋白去乙酰化相關,5-Aza-DC和/或TSA能恢複maspin在膠質瘤細株U87、U251中的轉錄.
목적 탐토유선사안산단백매억제제기인(maspin)재인효질류세포주U87、U251중침묵여표관유전학관계.방법 운용역전록-취합매련반응( RT-PCR)、Western blot검측maspin기인재U87、U251적표체.갑기화특이성취합매련반응(MSP)측정maspin기인계동자구구역갑기화상태.동시운용아류산경염측서법(BSP)검측maspin기인계동자구갑기화CpG위점.RT-PCR검측12 μmol/L 5-담잡-2’-탈양포감(5-Aza-DC)화/혹500 nmol/L곡고매소(TSA)、15 μmol/L 5-Aza-DC/혹500 nmol/L TSA분별처리U87、U251후,maspin기인mRNA표체수평.결과 재효질류세포주U87、U251중maspin기인표체침묵.maspin기인계동자CpG위점처우갑기화상태.RT-PCR결과현시,미처리조、5-Aza-DC、TSA、5-Aza-DC화TSA분별처리U87후,maspin mRNA상대표체량분별위:0.001 2±0.000 1 、0.0303 ±0.0006、0.019 6±0.001 6、0.052 6 ±0.003 4；미처리조화5-Aza-DC、TSA、5-Aza-DC화TSA분별처리U251후,maspin mRNA상대표체량분별위:0.002 2±0.0030、0.0122±0.0008、0.0249±0.0012、0.038 4±0.003 1.표명5-Aza-DC화/혹TSA능회복U87、U251중maspin기인표체(P＜0.01).결론 maspin재효질류중적표체억제여기계동자CpG도갑기화급조단백거을선화상관,5-Aza-DC화/혹TSA능회복maspin재효질류세주U87、U251중적전록.
Objective To study the relationship between the silenced expression of mammary serine protease inhibitor (maspin) and epigenetics in human glioma cell lines U87 and U251.Methods Reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were used to examine the expression of maspin.Methylation status of the maspin promoter was detected by methylation-specific polymerase chain reaction (MSP) and bisulphite genomic sequencing.RT-PCR was used to examine the expression of maspin after U87 and U251 cells were treated with 12 (or 15) μmol/L 5-Aza-2-deoxyeytidine (5-Aza-dC) and/or 500 nmol/L trichostatin A (TSA).Results A CpG island in the 5' promoter region of maspin was methylated.Maspin was silenced in human glioma cell lines U87 and U251.The relative intensity of maspin was 0.030 3 ±0.000 6,0.019 6 ±0.001 6,and 0.052 6 ±0.003 4 in 5-Aza-DC,TSA,and 5-Aza-DC plus TSA pretreated U87 cells,and 0.001 2 ± 0.000 1 in control group.The relative intensity of maspin was 0.012 2 ± 0.000 8,0.024 9 ± 0.001 2,and 0.038 4 ± 0.003 1 in 5-Aza-DC,TSA,and 5-Aza-DC plus TSA pretreated U251 cells,and 0.002 2 ± 0.00 3 in control goup.U87,and U251could could be restored by treatment with 5-Aza-DC and/or TSA (P ＜ 0.01).Conclusion Maspin is silenced by promoter methylation and histone modification,and 5-Aza-DC and/or TSA can restore the transcription of maspin in U87 and U251 cells.
