中华肝胆外科杂志
中華肝膽外科雜誌
중화간담외과잡지
CHINESE JOURNAL OF HEPATOBILIARY SURGERY
2011年
2期
132-137
,共6页
王少平%亢黎莉%陈孝平%周鹤俊%隋玉军%司文章
王少平%亢黎莉%陳孝平%週鶴俊%隋玉軍%司文章
왕소평%항려리%진효평%주학준%수옥군%사문장
癌,肝细胞%抑癌基因%KLF6%基因表达%细胞增殖
癌,肝細胞%抑癌基因%KLF6%基因錶達%細胞增殖
암,간세포%억암기인%KLF6%기인표체%세포증식
Carcinoma,hepatocellular%Tumor suppressor gene%Kruppel-like factor 6%Gene expression%Cell proliferation
目的 研究潜在抑癌基因KLF6在肝细胞癌的表达、变异及对肝癌细胞增殖的影响,探讨KLF6基因在肝细胞癌中的作用机制.方法 分别用荧光定量PCR、Western-Blot和DNA直接测序、荧光标记多态性微卫星分析,检测肝细胞癌中KLF6基因的表达及变异情况;构建野生型KLF6真核表达质粒pcDNA3.0/KLF6,转染肝癌细胞HepG2,观察转染后细胞生长情况.结果 KLF6基因在肝细胞癌的表达明显下调(P＜0.01).在21例肝癌组织中的突变率为29%.3个错义突变--Y97C、T145A和T147A均位于KLF6蛋白活性域的非保守氨基酸序列,而配对癌旁组织均未发现突变.其中,错义突变--T147A(base 423 A-G)破坏了脯氨酸蛋白激酶的磷酸化作用位点(XTPX*).微卫星分析KLF6在肝细胞癌表现出较高频率的杂合子缺失,缺失率为52%.在出现杂合子缺失的11例样本,KLF6基因均有明显的表达下调.其中,9例样本同时检测出突变.导人外源KLF6基因可明显抑制转染细胞HepG2生长(42.7%,P＜0.05).实验还发现,pcDNA3.0/KLF6转染HepG2细胞后,可诱导转染细胞凋亡.结论 KLF6基因可能为新的肝癌相关抑癌基因,在肝细胞癌病理演变过程中可能扮演重要角色.
目的 研究潛在抑癌基因KLF6在肝細胞癌的錶達、變異及對肝癌細胞增殖的影響,探討KLF6基因在肝細胞癌中的作用機製.方法 分彆用熒光定量PCR、Western-Blot和DNA直接測序、熒光標記多態性微衛星分析,檢測肝細胞癌中KLF6基因的錶達及變異情況;構建野生型KLF6真覈錶達質粒pcDNA3.0/KLF6,轉染肝癌細胞HepG2,觀察轉染後細胞生長情況.結果 KLF6基因在肝細胞癌的錶達明顯下調(P＜0.01).在21例肝癌組織中的突變率為29%.3箇錯義突變--Y97C、T145A和T147A均位于KLF6蛋白活性域的非保守氨基痠序列,而配對癌徬組織均未髮現突變.其中,錯義突變--T147A(base 423 A-G)破壞瞭脯氨痠蛋白激酶的燐痠化作用位點(XTPX*).微衛星分析KLF6在肝細胞癌錶現齣較高頻率的雜閤子缺失,缺失率為52%.在齣現雜閤子缺失的11例樣本,KLF6基因均有明顯的錶達下調.其中,9例樣本同時檢測齣突變.導人外源KLF6基因可明顯抑製轉染細胞HepG2生長(42.7%,P＜0.05).實驗還髮現,pcDNA3.0/KLF6轉染HepG2細胞後,可誘導轉染細胞凋亡.結論 KLF6基因可能為新的肝癌相關抑癌基因,在肝細胞癌病理縯變過程中可能扮縯重要角色.
목적 연구잠재억암기인KLF6재간세포암적표체、변이급대간암세포증식적영향,탐토KLF6기인재간세포암중적작용궤제.방법 분별용형광정량PCR、Western-Blot화DNA직접측서、형광표기다태성미위성분석,검측간세포암중KLF6기인적표체급변이정황;구건야생형KLF6진핵표체질립pcDNA3.0/KLF6,전염간암세포HepG2,관찰전염후세포생장정황.결과 KLF6기인재간세포암적표체명현하조(P＜0.01).재21례간암조직중적돌변솔위29%.3개착의돌변--Y97C、T145A화T147A균위우KLF6단백활성역적비보수안기산서렬,이배대암방조직균미발현돌변.기중,착의돌변--T147A(base 423 A-G)파배료포안산단백격매적린산화작용위점(XTPX*).미위성분석KLF6재간세포암표현출교고빈솔적잡합자결실,결실솔위52%.재출현잡합자결실적11례양본,KLF6기인균유명현적표체하조.기중,9례양본동시검측출돌변.도인외원KLF6기인가명현억제전염세포HepG2생장(42.7%,P＜0.05).실험환발현,pcDNA3.0/KLF6전염HepG2세포후,가유도전염세포조망.결론 KLF6기인가능위신적간암상관억암기인,재간세포암병리연변과정중가능분연중요각색.
Objective To investigate the expression and genetic alterations of KLF6 in hepatocellular carcinoma (HCC) and explore their functional mechanisms in the oncogenesis and development of HCC. Methods Real-time quantitative-PCR, direct sequencing and LOH approaches were used to detect KLF6 genetic abnormalities in HCC. The experiment had 2 groups, an experimental group and a control group. In the experimental group, the transfected plasmid pcDNA3.0 was recombined with KLF6 and tranfected into HCC HepG2 cells. MTT, flow cytometry and Western blotting were used to observe the effect of anti-oncogene wild type KLF6 on HepG2 cells by transgenic method for 48 h.Results Expression levels of KLF6 were significantly downregulated in HCCs(P＜0. 01), as detected by qRT-PCR. LOH occurred in 11 (52%) of the 21 tumors, and all the samples with LOH showed KLF6 down-regulation. The mutational frequency was 29%, and sequence changes located in activation domain of KLF6. Meanwhile, MTT assay showed a significant antiproliferative effect of the transfected wtKLF6 on HepG2 cells(42.7%, P＜0.05). Fluorescence-activated cell sorting analysis revealed that KLF6 induced apoptosis. Conclusion The deregulation of KLF6 together with genetic abnormalities of allelic imbalance and mutations may play an important role in HCC pathogenesis.
