肝癌相关新基因HC6的全长cDNA克隆及分析

IDENTIFICATION OF THE FULL-LENGTH cDNA OF ANOVEL GENE (HC6) RELATED TO HEPATOCELLULARCARCINOMA IN PAC579

目的 染色体17p13.3区域内多态性位点D17S926是肝细胞肝癌(HCC)杂合性缺失热点,PAC579克隆含有D17S926位点,本实验目的在于从PAC579克隆中寻找与肝癌相关的表达序列(ESTs),对有意义的EST片段克隆其全长cDNA。方法 根据PAC579克隆中现有的9个代表新基因的EST序列设计引物,对正常人肝cDNA文库作多聚酶链式反应(PCR)扩增检测,确定在肝组织中表达的EST,然后采用Southem杂交检测肝组织中表达的EST在27对肝癌和癌旁标本中杂合性生缺失(LOH)频率,选择缺失频率最高的EST克隆其全长cDNA,Northern杂交验证后测序并作进一步分析。结果PAC579现有9个代表新基因的DST中有3个在肝组织表达,其中EST6在肝癌中LOH频率高达54.6%(6/11),通过RACE-PCR方法,得到约l.8kb全长cDNA克隆,与Northern杂交结果一致,结构及同源性分析显示该基因是一新基因,编码一个包含134个氨基酸,分子量约为14.7kD的蛋白质,另外发现碱基序列313-592属于ALu同源序列,重新命名为肝癌相关基因6(HC6)。结论 通过定位克隆方法从肝癌杂合性缺失热点得到一个新基因HC6,该基因与肝癌的关系有待进一步研究。

ve To identify the expression sequence tags(ESTs)related to hepatocellular carcinoma(HCC)in PAC579 and toclone the full-length cDNA.Methods LOH was tested in 27 casesof HCC and adjacent normal liver tissues by Southern blot analysis.Using Marathon RACE-PCR method,the full-length cDNA exhibiting high rate of LOH was cloned.Results LOH rate of EST6 in HCC is about 54.6%(6/11).The full-length cDNA OF THIS NOVEL GENE(designated as HC6)is about 1.8kb and consistent with the size oftranscript shown by Northern blot.Conclusion A novel gene inPAC579 from liver cDNA library has been cloned,which could be related to HCC.This new gene is about 1.8kb in length andcontains an open reading frame(ORF)encoding an 134-amino acid polypeptide.