人肝癌父系表达基因10的遗传印记状态

The imprinting status of genetic imprinted gene PEGIO in human hepatocellular carcinomas

目的研究人肝癌组织及肝癌细胞株中父系表达基因10（PEG10）的遗传印记状态。方法从40例肝癌及其癌旁组织、15例正常肝组织、5株肝癌细胞（PLC／PRF／5、SMMC7721、HepG2、Hep3B、SK—HEP—1）、2株正常肝细胞（changliver、HL7702）中提取基因组DNA，针对PEG10基因单核苷酸多态性位点设计引物进行PCR，扩增片段经测序分析基因型，从杂合样本中提取总RNA进行RT—PCR，对扩增产物测序以检测等位基因表达状态，同时进行实时荧光定量RT—PCR检测PEGl0表达水平。计量资料以均数±标准差（x-±s）表示，组间比较用t检验与方差分析；两组率的比较用x2检验。结果40例肝癌及其癌旁组织中16例呈杂合状态，15例正常肝组织中3例呈杂合状态，肝癌细胞HepG2扩增片段测序检测到一杂合突变位点，其余组织及细胞株均为纯合状态。杂合样本中，82．4％（14／17）肝癌样本（包括组织及肝癌细胞株）中PEG10基因呈双等位基因表达，发生印记丢失；17．6％（3／17）肝癌样本呈单等位基因表达，提示印记存在。PEG10在癌旁及正常肝组织中几乎不表达。发生印记丢失的肝癌组织与印记存在的肝癌组织相比，PEG10表达水平的差异无统计学意义（t=1．311，P〉0．05）。结论大多数肝癌组织中存在PEG10印记丢失现象，PEG10印记状态与其在肝癌组织中的表达水平无明确关系。

Objective To study the imprinting status of genetic imprinted gene PEG10 （pertemally expressed gene 10） in human hepatocellular carcinoma（HCC） tissues and liver cancer cell lines, Methods Genomic DNA was extracted from 20 HCC tissues and its adjacent tissues, 15 normal liver tissues, 5 liver cancer cell lines （PLC/PRF/5, smmc-7721, HepG2, Hep3B, SK-HEP-1） and 2 normal human liver cell lines （changliver, HL7702）. The DNA fragments containing single nucleotide polymorphism （SNP） site of PEG10 were amplified by polymerase chain reaction （PCR） and the genotype of samples was detected by DNA sequencing. Total RNA was extracted from heterozygous samples, the imprinting status and expression level of PEG10 were evaluated by quantitative real time reverse transcription-polymerase chain reaction （qRT- PCR） and DNA sequencing. Results It was found that 16/40 of HCC and its adjacent tissues were heterozygous, 3/15 of normal liver tissue were heterozygous. A site of heterozygous mutation was found in HepG2 cells by DNA sequencing. The other liver tissues and cell lines were all homozygous. PEG10 was biallelically ex- pressed and showed loss of imprinting （LOI） in 82.4% （14/17） liver cancer samples （16 HCC tissues and HepG2）, however it was a monoallelic expression and showed genomic imprinting in17.6% （3/17） liver cancer samples. In HCC, the expression levels of PEG10 were increased apparently, but it was negative expressed in cancer adjacent tissues and normal liver tissues. Expression levels of PEG10 were not signifi