中华肿瘤杂志
中華腫瘤雜誌
중화종류잡지
CHINESE JOURNAL OF ONCOLOGY
2013年
6期
405-411
,共7页
王文辉%汪芳裕%魏娟%沈芸竹%刘畅%舒小闯
王文輝%汪芳裕%魏娟%瀋蕓竹%劉暢%舒小闖
왕문휘%왕방유%위연%침예죽%류창%서소틈
结直肠肿瘤%甲基化%HT-29细胞%紧密连接蛋白%细胞凋亡%细胞增殖
結直腸腫瘤%甲基化%HT-29細胞%緊密連接蛋白%細胞凋亡%細胞增殖
결직장종류%갑기화%HT-29세포%긴밀련접단백%세포조망%세포증식
Colorectal neoplasms%Methylation%HT-29 cell line%Claudin%Apoptosis%Cell proliferation
目的 探讨CpG甲基转移酶M.SssI对人结肠癌HT-29细胞中紧密连接蛋白7(claudin-7)和claudin-8表达的调节作用,及其对肿瘤细胞增殖和凋亡的影响.方法 以50 U/ml的M.SssI处理HT-29细胞24h(过甲基化组),采用重亚硫酸盐测序法检测claudin-7和claudin-8基因启动子区域CpG岛的甲基化状态,实时荧光定量PCR法检测claudin-7和claudin-8 mRNA的表达,细胞免疫荧光技术和Western blot法检测claudin-7和claudin-8蛋白的表达,Hoechst 33342荧光染色法和流式细胞术检测HT-29细胞的凋亡,四甲基偶氮唑蓝(MTT)法检测HT-29细胞的增殖.同时设对照组(以2μlPBS处理HT-29细胞)和5-Azacytidine组(以10μmol/L 5-Azacytidine处理HT-29细胞).结果 过甲基化组claudin-7和claudin-8靶序列中发生甲基化的CpG胞嘧啶数目分别为25和10个,对照组分别为9和5个,5-Azacytidine组分别为0和3个.过甲基化组claudin-7和claudin-8mRNA和蛋白的表达均明显低于对照组(均P＜0.05),而5-Azacytidine组claudin-7和claudin-8mRNA和蛋白的表达均明显高于对照组(均P＜0.05).Hoechst 33342荧光染色结果显示,对照组和5-Azacytidine组的HT-29细胞均呈均匀蓝色荧光,细胞呈圆形,大小一致,无明显差异；而过甲基化组HT-29细胞呈高度白色荧光的凋亡细胞明显增多.流式细胞术检测结果显示,过甲基化组HT-29细胞的早期凋亡率比对照组增加84.7％ (P =0.002).MTT法检测结果显示,过甲基化组HT-29细胞的生长速度较对照组缓慢(P =0.002),HT-29细胞的增殖抑制率为32.1％；5-Azacytidine组HT-29细胞的增殖水平与对照组相似(P =0.084).结论 CpG甲基转移酶可以通过上调claudin-7和claudin-8基因启动子区的甲基化程度,下调HT-29细胞中claudin-7和claudin-8 mRNA和蛋白的表达,从而诱导细胞产生凋亡,并抑制其增殖.
目的 探討CpG甲基轉移酶M.SssI對人結腸癌HT-29細胞中緊密連接蛋白7(claudin-7)和claudin-8錶達的調節作用,及其對腫瘤細胞增殖和凋亡的影響.方法 以50 U/ml的M.SssI處理HT-29細胞24h(過甲基化組),採用重亞硫痠鹽測序法檢測claudin-7和claudin-8基因啟動子區域CpG島的甲基化狀態,實時熒光定量PCR法檢測claudin-7和claudin-8 mRNA的錶達,細胞免疫熒光技術和Western blot法檢測claudin-7和claudin-8蛋白的錶達,Hoechst 33342熒光染色法和流式細胞術檢測HT-29細胞的凋亡,四甲基偶氮唑藍(MTT)法檢測HT-29細胞的增殖.同時設對照組(以2μlPBS處理HT-29細胞)和5-Azacytidine組(以10μmol/L 5-Azacytidine處理HT-29細胞).結果 過甲基化組claudin-7和claudin-8靶序列中髮生甲基化的CpG胞嘧啶數目分彆為25和10箇,對照組分彆為9和5箇,5-Azacytidine組分彆為0和3箇.過甲基化組claudin-7和claudin-8mRNA和蛋白的錶達均明顯低于對照組(均P＜0.05),而5-Azacytidine組claudin-7和claudin-8mRNA和蛋白的錶達均明顯高于對照組(均P＜0.05).Hoechst 33342熒光染色結果顯示,對照組和5-Azacytidine組的HT-29細胞均呈均勻藍色熒光,細胞呈圓形,大小一緻,無明顯差異；而過甲基化組HT-29細胞呈高度白色熒光的凋亡細胞明顯增多.流式細胞術檢測結果顯示,過甲基化組HT-29細胞的早期凋亡率比對照組增加84.7％ (P =0.002).MTT法檢測結果顯示,過甲基化組HT-29細胞的生長速度較對照組緩慢(P =0.002),HT-29細胞的增殖抑製率為32.1％；5-Azacytidine組HT-29細胞的增殖水平與對照組相似(P =0.084).結論 CpG甲基轉移酶可以通過上調claudin-7和claudin-8基因啟動子區的甲基化程度,下調HT-29細胞中claudin-7和claudin-8 mRNA和蛋白的錶達,從而誘導細胞產生凋亡,併抑製其增殖.
목적 탐토CpG갑기전이매M.SssI대인결장암HT-29세포중긴밀련접단백7(claudin-7)화claudin-8표체적조절작용,급기대종류세포증식화조망적영향.방법 이50 U/ml적M.SssI처리HT-29세포24h(과갑기화조),채용중아류산염측서법검측claudin-7화claudin-8기인계동자구역CpG도적갑기화상태,실시형광정량PCR법검측claudin-7화claudin-8 mRNA적표체,세포면역형광기술화Western blot법검측claudin-7화claudin-8단백적표체,Hoechst 33342형광염색법화류식세포술검측HT-29세포적조망,사갑기우담서람(MTT)법검측HT-29세포적증식.동시설대조조(이2μlPBS처리HT-29세포)화5-Azacytidine조(이10μmol/L 5-Azacytidine처리HT-29세포).결과 과갑기화조claudin-7화claudin-8파서렬중발생갑기화적CpG포밀정수목분별위25화10개,대조조분별위9화5개,5-Azacytidine조분별위0화3개.과갑기화조claudin-7화claudin-8mRNA화단백적표체균명현저우대조조(균P＜0.05),이5-Azacytidine조claudin-7화claudin-8mRNA화단백적표체균명현고우대조조(균P＜0.05).Hoechst 33342형광염색결과현시,대조조화5-Azacytidine조적HT-29세포균정균균람색형광,세포정원형,대소일치,무명현차이；이과갑기화조HT-29세포정고도백색형광적조망세포명현증다.류식세포술검측결과현시,과갑기화조HT-29세포적조기조망솔비대조조증가84.7％ (P =0.002).MTT법검측결과현시,과갑기화조HT-29세포적생장속도교대조조완만(P =0.002),HT-29세포적증식억제솔위32.1％；5-Azacytidine조HT-29세포적증식수평여대조조상사(P =0.084).결론 CpG갑기전이매가이통과상조claudin-7화claudin-8기인계동자구적갑기화정도,하조HT-29세포중claudin-7화claudin-8 mRNA화단백적표체,종이유도세포산생조망,병억제기증식.
Objective To explore the regulatory effect of CpG methyltransferase (M.SssI) on expression of claudin-7 and claudin-8,promoting apoptosis and inhibiting proliferation of human colorectal cancer HT-29 cells.Methods HT-29 cells were treated with M.SssI (50 U/ml) for 24 hours.The methylation status of claudin-7 and claudin-8 gene promoters was assayed by bisulfite sequencing PCR (BSP).Real-time PCR with SYBR green Ⅰ technique was used to detect the relative expression of claudin-7 and-8 mRNA,and claudin-7 and claudin-8 proteins were tested by cell immunofluorescence and Western blotting,while the effect on cell apoptosis was assessed by Hoechst 33342 fluorescence and flow cytometry.Inhibition of cell proliferation was measured by MTT assay.Results The amounts of methylated claudin-7 and claudin-8 gene CpGs were 25,10 in the M.SssI group,9 and 5 in the PBS group,0 and 3 in the 5-azacytidine group,respectively.Compared with the PBS group,Claudin-7 and-8 were significantly reduced by M.SssI (P ＜ 0.05),but increased by 5-azacytidine (P ＜ 0.05) at both mRNA and protein levels.Hoechst 33342 staining revealed that HT-29 cells treated with PBS and 5-azacytidine were not significantly different,showing even blue fluorescence,round shape and same cell volume.But the M.SssI group presented more apoptotic cells with intensive white fluorescence intensity.Cytometry indicated that early apoptotic index of the M.SssI group was increased by 84.7％,compared with that of the PBS group (P =0.002).Measurement of MTT optical density demonstrated that cell growth of the M.SssI group was significantly lower than that of the PBS group (P =0.002),with an inhibition rate of 32.1％,whereas the proliferation of 5-azacytidine group was similar to that of the PBS group (P =0.084).Conclusions Our findings suggest that M.SssI can down-regulate claudin-7,-8 mRNA and proteins in the human colon cancer HT-29 cells by up-regulating methylation status of claudin-7 and-8 gene promoters,and finally induce apoptosis and inhibit proliferation of the tumor cells.
