中华预防医学杂志
中華預防醫學雜誌
중화예방의학잡지
CHINESE JOURNAL OF
2009年
7期
601-606
,共6页
基因冈报告%启动区(遗传学)%钙黏着糖蛋白类%CpG岛%甲基化
基因岡報告%啟動區(遺傳學)%鈣黏著糖蛋白類%CpG島%甲基化
기인강보고%계동구(유전학)%개점착당단백류%CpG도%갑기화
Genes,reporter%Promoter regions (Genetics)%Cadherins%CpG islands%Methylation
目的 研究细胞内源性上皮型钙黏素基因(E-cadherin,CDH1)启动子CpG岛甲基化状态是否影响报告基因试验的结果 .方法 甲基化特异性PCR法测定8种人肿瘤细胞系CDH1启动子CpG岛甲基化状态,免疫印迹法测定其蛋白表达;根据该基因启动子-73位A/C多态和单倍体型构建2组长短不同的CDH1启动子-荧光素酶报告基因[pGI3-A(-73)/-C(-73)和pGL3-H1/-H4],活性差异采用t检验进行统计分析.结果 (1)CDH1在MCF7、MKN74、PC-3和AGS细胞中COG岛末甲基化,除AGS外均存在CDH1表达;在HeLa、BGC823、A549和RKO细胞中CpG岛甲基化并且无表达;(2)在CDH1未甲基化细胞系MCF7、MKN74、PC-3和AGS中pGL3-C<,(-73)报告基因活性(分别为:0.78±0.10、0.17±0.01、0.11±0.01、1.19±0.18)均显著高于pGL3-A(-73)报告基因活性(分别为:0.30±0.08、0.07±0.01、0.07±0.01、0.39±0.04)(t值分别为:-6.298、-12.349、-8.128、-7.388,P值均<0.01),而在CDH1甲基化细胞中则相反[HeLa、BGC823、A549和RKO细胞中pGL3-C(-73)报告基因活性分别为:0.09±0.02、0.13±0.02、0.05±0.01、0.01±0.00,pGI3-A(-73)活性分别为:0.16±0.01、0.25±0.01、0.11±0.03、0.03±0.00,pGL3-C(-73)活性低于pGL3-A(-73),t值分别为:5.958、11.189、3.661、13.866,P值均<0.05];(3)在MKN74和RKO细胞中,pGL3-H1/-H4的启动子活性分别为1.57±0.23/0.94±0.06和0.38±0.02/0.50±0.04,差异亦存在统计学意义(t值为4.577和-4.915,P值为0.010和0.003),并且在两种细胞巾活性相反.结论 细胞内源性目的 基因CpG岛甲基化状态对报告基凶试验结果 可能有重要影响,高活性基因型的启动子也是容易受抑制的启动子.
目的 研究細胞內源性上皮型鈣黏素基因(E-cadherin,CDH1)啟動子CpG島甲基化狀態是否影響報告基因試驗的結果 .方法 甲基化特異性PCR法測定8種人腫瘤細胞繫CDH1啟動子CpG島甲基化狀態,免疫印跡法測定其蛋白錶達;根據該基因啟動子-73位A/C多態和單倍體型構建2組長短不同的CDH1啟動子-熒光素酶報告基因[pGI3-A(-73)/-C(-73)和pGL3-H1/-H4],活性差異採用t檢驗進行統計分析.結果 (1)CDH1在MCF7、MKN74、PC-3和AGS細胞中COG島末甲基化,除AGS外均存在CDH1錶達;在HeLa、BGC823、A549和RKO細胞中CpG島甲基化併且無錶達;(2)在CDH1未甲基化細胞繫MCF7、MKN74、PC-3和AGS中pGL3-C<,(-73)報告基因活性(分彆為:0.78±0.10、0.17±0.01、0.11±0.01、1.19±0.18)均顯著高于pGL3-A(-73)報告基因活性(分彆為:0.30±0.08、0.07±0.01、0.07±0.01、0.39±0.04)(t值分彆為:-6.298、-12.349、-8.128、-7.388,P值均<0.01),而在CDH1甲基化細胞中則相反[HeLa、BGC823、A549和RKO細胞中pGL3-C(-73)報告基因活性分彆為:0.09±0.02、0.13±0.02、0.05±0.01、0.01±0.00,pGI3-A(-73)活性分彆為:0.16±0.01、0.25±0.01、0.11±0.03、0.03±0.00,pGL3-C(-73)活性低于pGL3-A(-73),t值分彆為:5.958、11.189、3.661、13.866,P值均<0.05];(3)在MKN74和RKO細胞中,pGL3-H1/-H4的啟動子活性分彆為1.57±0.23/0.94±0.06和0.38±0.02/0.50±0.04,差異亦存在統計學意義(t值為4.577和-4.915,P值為0.010和0.003),併且在兩種細胞巾活性相反.結論 細胞內源性目的 基因CpG島甲基化狀態對報告基兇試驗結果 可能有重要影響,高活性基因型的啟動子也是容易受抑製的啟動子.
목적 연구세포내원성상피형개점소기인(E-cadherin,CDH1)계동자CpG도갑기화상태시부영향보고기인시험적결과 .방법 갑기화특이성PCR법측정8충인종류세포계CDH1계동자CpG도갑기화상태,면역인적법측정기단백표체;근거해기인계동자-73위A/C다태화단배체형구건2조장단불동적CDH1계동자-형광소매보고기인[pGI3-A(-73)/-C(-73)화pGL3-H1/-H4],활성차이채용t검험진행통계분석.결과 (1)CDH1재MCF7、MKN74、PC-3화AGS세포중COG도말갑기화,제AGS외균존재CDH1표체;재HeLa、BGC823、A549화RKO세포중CpG도갑기화병차무표체;(2)재CDH1미갑기화세포계MCF7、MKN74、PC-3화AGS중pGL3-C<,(-73)보고기인활성(분별위:0.78±0.10、0.17±0.01、0.11±0.01、1.19±0.18)균현저고우pGL3-A(-73)보고기인활성(분별위:0.30±0.08、0.07±0.01、0.07±0.01、0.39±0.04)(t치분별위:-6.298、-12.349、-8.128、-7.388,P치균<0.01),이재CDH1갑기화세포중칙상반[HeLa、BGC823、A549화RKO세포중pGL3-C(-73)보고기인활성분별위:0.09±0.02、0.13±0.02、0.05±0.01、0.01±0.00,pGI3-A(-73)활성분별위:0.16±0.01、0.25±0.01、0.11±0.03、0.03±0.00,pGL3-C(-73)활성저우pGL3-A(-73),t치분별위:5.958、11.189、3.661、13.866,P치균<0.05];(3)재MKN74화RKO세포중,pGL3-H1/-H4적계동자활성분별위1.57±0.23/0.94±0.06화0.38±0.02/0.50±0.04,차이역존재통계학의의(t치위4.577화-4.915,P치위0.010화0.003),병차재량충세포건활성상반.결론 세포내원성목적 기인CpG도갑기화상태대보고기흉시험결과 가능유중요영향,고활성기인형적계동자야시용역수억제적계동자.
Objective To investigate the effects of methylation status of CpG islands of endogenous E-cadherin (CDH1) gene on the promoter activity of corresponding genes in reporter assays. Methods The methylation statuses of CpG island of CDHI in 8 different cell lines were detected by methylation-specific PCR. CDH1 protein was analyzed by Western blotting. Two sets of pGL3 reporter vectors with different genotypes/haplotypes of the CDH1 promoter were constructed [pGI3-A(-73)/-C(-73)pGL3-H1/-H4]and used to transfect these cell lines. The differences between these promoter reporter vectors were analyzed by t-test. Results (1) CDH1 CpG island was unmethylated in AGS, MCF7, MKN74, and PC-3 cell lines,expressed in MCF7, MKN74, and PC-3 ,but not in AGS. Expression of CDH1 was silenced by methylation in HeLa, BGC823, A549, and RKO cell lines. (2) In the four CDH1 -unmethylated MCFT, M KN74, PC-3, and AGS cell lines ,the promoter activities of pGI3-C(-73)(as 0. 78±0. 10,0. 17±0.01,0. 11±0. 01,1.19±0. 18)were significantly higher than those of pGL3-A(-73)(as 0. 30±0. 08,0. 07±0. 01,0. 07±0. 01,0. 39±0. 04) (t values are -6. 298, -12. 349, -8. 128, -7.388, and P<0. 01). However, in the four C DH1 -methylated HeLa, BGC823, A549, and RKO cell lines, the promoter activity of pGL3-C(-73)(as 0. 09±0. 02,0. 13±0. 02,0. 05±0. 01,0. 01±0. 00) was significantly lower than that of pGL3-A(-73)(as 0. 16±0. 01,0.25±0.01,0. 11±0.03,0.03±0.00) (t valued at 5.958,11. 189,3. 661,13. 866,and P<0.05). (3) In the unmethylated MKN74 and methylated RKO cell lines, the promoter activities of pGI3-H1/-H4 were obviously and contrarily different(as 1.57±0. 23/0. 94±0. 06 and 0. 38±0. 02/0. 50±0. 04 ,t values were 4. 577 and -4. 915 ,P values were 0. 010 and 0. 003). Conclusion The methylation status of CpG island of the target gene in the tested cell lines affects the promoter activity in Reporter Assay significantly. The most active one may be the most suppressive one.
