中华肿瘤杂志
中華腫瘤雜誌
중화종류잡지
CHINESE JOURNAL OF ONCOLOGY
2010年
10期
729-733
,共5页
鼻咽肿瘤%细胞系%LTF%突变%甲基化%遗传学%表观遗传学
鼻嚥腫瘤%細胞繫%LTF%突變%甲基化%遺傳學%錶觀遺傳學
비인종류%세포계%LTF%돌변%갑기화%유전학%표관유전학
Nasopharyngeal neoplasms%Cell lines%LTF%Mutation%Methylation%Genetics%Epigenetics
目的 检测鼻咽癌(NPC)细胞系中LTF基因的表达水平,并分析遗传学和表观遗传学改变与其表达的关系.方法 采用逆转录聚合酶链反应(RT-PCR),检测NPC细胞系HNE1、HNE2、HNE3、CNE1、CNE2、5-8F、6-10B和15例慢性鼻咽炎组织中LTF mRNA表达水平,同时通过Western blot法分析6-10B细胞中LTF蛋白水平.运用微卫星分析技术、聚合酶链式反应-单链构象多态(PCR-SSCP)方法和甲基化特异性PCR(MSP)以及亚硫酸盐克隆测序方法,分别检测LTF的杂合性丢失、突变和启动子区甲基化情况.结果 在15例慢性鼻咽炎组织中,LTF基因稳定表达.在NPC细胞系中,除6-10B细胞有LTF基因弱表达外,其余均表达缺失,显著低于慢性鼻咽炎组织中的表达(Z=-3.738,P=0.000),且6-10B细胞中无LTF蛋白的表达.杂合性丢失分析显示,NPC细胞系中不存在LTF微卫星位点的缺失.突变检测显示,LTF在NPC细胞系中的突变率为14.3%(1/7),且突变存在于HNE1细胞系中,即启动子序列-305至-50 bp发生了缺失突变(-218delT).甲基化分析显示,LTF基因在慢性鼻咽炎组织中无甲基化,而在所有NPC细胞系中均有甲基化.用5-杂氮-2-脱氧包苷处理5-8F和6-10B细胞后,LTF mRNA表达上调.结论 LTF基因在NPC细胞系中表达失活,其分子机制与启动子高甲基化和缺失突变有关.
目的 檢測鼻嚥癌(NPC)細胞繫中LTF基因的錶達水平,併分析遺傳學和錶觀遺傳學改變與其錶達的關繫.方法 採用逆轉錄聚閤酶鏈反應(RT-PCR),檢測NPC細胞繫HNE1、HNE2、HNE3、CNE1、CNE2、5-8F、6-10B和15例慢性鼻嚥炎組織中LTF mRNA錶達水平,同時通過Western blot法分析6-10B細胞中LTF蛋白水平.運用微衛星分析技術、聚閤酶鏈式反應-單鏈構象多態(PCR-SSCP)方法和甲基化特異性PCR(MSP)以及亞硫痠鹽剋隆測序方法,分彆檢測LTF的雜閤性丟失、突變和啟動子區甲基化情況.結果 在15例慢性鼻嚥炎組織中,LTF基因穩定錶達.在NPC細胞繫中,除6-10B細胞有LTF基因弱錶達外,其餘均錶達缺失,顯著低于慢性鼻嚥炎組織中的錶達(Z=-3.738,P=0.000),且6-10B細胞中無LTF蛋白的錶達.雜閤性丟失分析顯示,NPC細胞繫中不存在LTF微衛星位點的缺失.突變檢測顯示,LTF在NPC細胞繫中的突變率為14.3%(1/7),且突變存在于HNE1細胞繫中,即啟動子序列-305至-50 bp髮生瞭缺失突變(-218delT).甲基化分析顯示,LTF基因在慢性鼻嚥炎組織中無甲基化,而在所有NPC細胞繫中均有甲基化.用5-雜氮-2-脫氧包苷處理5-8F和6-10B細胞後,LTF mRNA錶達上調.結論 LTF基因在NPC細胞繫中錶達失活,其分子機製與啟動子高甲基化和缺失突變有關.
목적 검측비인암(NPC)세포계중LTF기인적표체수평,병분석유전학화표관유전학개변여기표체적관계.방법 채용역전록취합매련반응(RT-PCR),검측NPC세포계HNE1、HNE2、HNE3、CNE1、CNE2、5-8F、6-10B화15례만성비인염조직중LTF mRNA표체수평,동시통과Western blot법분석6-10B세포중LTF단백수평.운용미위성분석기술、취합매련식반응-단련구상다태(PCR-SSCP)방법화갑기화특이성PCR(MSP)이급아류산염극륭측서방법,분별검측LTF적잡합성주실、돌변화계동자구갑기화정황.결과 재15례만성비인염조직중,LTF기인은정표체.재NPC세포계중,제6-10B세포유LTF기인약표체외,기여균표체결실,현저저우만성비인염조직중적표체(Z=-3.738,P=0.000),차6-10B세포중무LTF단백적표체.잡합성주실분석현시,NPC세포계중불존재LTF미위성위점적결실.돌변검측현시,LTF재NPC세포계중적돌변솔위14.3%(1/7),차돌변존재우HNE1세포계중,즉계동자서렬-305지-50 bp발생료결실돌변(-218delT).갑기화분석현시,LTF기인재만성비인염조직중무갑기화,이재소유NPC세포계중균유갑기화.용5-잡담-2-탈양포감처리5-8F화6-10B세포후,LTF mRNA표체상조.결론 LTF기인재NPC세포계중표체실활,기분자궤제여계동자고갑기화화결실돌변유관.
Objective To investigate the expression of LTF mRNA in several nasopharyngeal cancer (NPC) cell lines, and analyze the relationship between the genetic and epigenetic changes and expression of LTF gene. Methods The expression level of LTF was detected in NPC cell lines HNE1, HNE2, HNF3,CNE1, CNE2, 5-8F, 6-10B cells and tissues of 15 cases of chronic nasopharyngitis by RT-PCR. The LTF protein level was analyzed by Western blotting in 6-10B cells. Then LOH, mutation and methylation status of LTF was examined by microsatellites analysis, PCR-SSCP, MSP and bisulfite genomic sequencing,respectively. Results 15 chronic nasopharyngitis tissues showed stable LTF expression, while there were weak expression in 6-10B cells and absent expression in remaining detected NPC cell lines. There was a significantly lower LTF expression in chronic nasopharyngitis tissues ( Z = - 3. 738, P = 0. 000 ). No LTF protein expression was observed in 6-10B cells. LOH analysis demonstrated that allele loss of LTF wasn't found in NPC cell lines. LTF mutation was noted in 14.3% ( 1/7 ) of NPC cell lines. DNA sequencing confirmed the mutation point in the promoter region ( -305 bp to -50 bp) was at -218 bp (del T) of LTF gene in the HNE1 cell line. Methytation of LTF gene was not found in chronic nasopharyngitis. However,methylation of LTF promoter was detected in all NPC cell lines. LTF mRNA expression was increased in 5-8F and 6-10B cell lines after treatment with 5-aza-2-deoxycytidine. Conclusion There is an inactivation of expression of LTF gene in the NPC cell lines. Its molecular mechanism may be related with methylation of promoter region and deletion mutation.
