LHS cDNA的部分克隆及其表达对肝癌细胞行为的影响

目的:克隆在肝癌组织中低表达的差异显示片段L2的基因cDNA序列,并初步探讨其在肝癌发生、发展过程中的作用.方法:将L2在GenBank中拼接后,分析其序列结构,并构建正义真核表达质粒,转染BEL-7402肝癌细胞后,对转染细胞增殖、粘附、迁移和侵袭等指标进行测定.结果:克隆得到一条1 005 bp的cDNA,其5′端712 bp与人Liprinβ1 cDNA 5′端调控序列和外显子1,2,3序列同源(100%),而3′端293 bp与Liprinβ1基因内含子4同源(100%).其间有一个510 bp开放阅读框架,起始密码位置与Liprinβ1基因相同,推断的蛋白产物除C端13个氨基酸残基外,其余均与Liprinβ1同源.此cDNA暂命名为LHS (Liprinβ1-homologous sequence).LHS cDNA正义转染BEL-7402肝癌细胞系,对于细胞的增殖活性和侵袭能力无明显影响,但可降低肝癌细胞在层粘连蛋白上的粘附和迁移能力.结论:LHS表达下调可能在肝癌的转移过程中起重要作用.     

用荧光差异显示法分离新的肝细胞癌相关基因

目的 :寻找新的肝细胞癌 (hepatocellularcarcinoma ,HCC)相关基因。方法 :应用荧光 差异显示 (fluorescentdifferentialdisplay)技术分析人HCC、配对非癌肝、胎肝、正常肝等组织之间的基因差异表达 ,差异表达cDNA片段 ,经测序后 ,再用RNA狭缝杂交对部分差异片段在上述 4种组织中的表达进行验证。结果 :共得到差异表达cDNA片段 110条。经狭缝杂交筛选 ,获得了 4条阳性片段 ,未知cDNA片段L2和与人醛缩酶B 99.5 %同源的cDNA片段L7在部分HCC中特异性低表达 ,未知cDNA片段LC2 7和与人Nip3基因 99.4%同源的cDNA片段LC2 0在部分HCC中特异性高表达。结论 :确认了 4条HCC相关基因。其中L2和人醛缩酶B基因可能为阻抑HCC发生、发展的基因 ,而人Nip3基因和LC2 7可能为促进HCC发生、发展的基因。L2和LC2 7为目前未知的新基因。     

Biological function of a novel gene overexpressed in human hepatocellular carcinoma

Objective To clone the full-length of a differentially expressed cDNA fragment, LC27, and study its biological function tentatively. Methods Northern blot was used to analyze the expression pattern of LC27 in hepatocellular carcinoma, matched nontumor liver tissues, fetal liver and normal adult liver tissues, as well as BEL-7402 hepatocellular carcinoma cell line ESTs splicing and 5’ rapid amplification of cDNA ends (5’ RACE) were used to clone the full-length of LC27 cDNA.An antisense oligodeoxynucleotide approach was used to investigate the biological role of the gene in the proliferation of BEL-7402 cells. Results A 2186 bp novel cDNA with an open reading frame encoding a 283 amino acid protein was cloned.Analysis of the deduced amino acid sequence indicated that it is 38% (88/229) identical to human Golgi 4-transmembrane spanning transporter MTP.The gene and the encoded protein was termed hepatocellular carcinoma overexpressed transmembrane protein (hotp) and HOTP, respectively.Hotp mRNA was almost undetectable in normal adult liver and fetal liver tissues.However, it was significantly up-regulated in hepatocellular carcinoma and some matched nontumor liver tissues, as well as BEL-7402 cells.The proliferation of BEL-7402 cells was suppressed by an antisense oligodeoxynucleotide against hotp mRNA at a concentration of 50 μg/ml. Conclusion HOTP may be an integral membrane transporter protein.The overexpression of the gene in hepatocellular carcinoma may play an important role in hepatocarcinogenesis and disease progression.     

用荧光差异显示法分离新的肝细胞癌相关基因

目的：寻找新的肝细胞癌（ｈｅｐａｔｏｃｅｌｌｕｌａｒ ｃａｒｃｉｎｏｍａ,ＨＣＣ）相关基因。方法：应用荧光－差异显示（ｆｌｕｏｒｅｓｃｅｎｔ ｄｉｆｆｅｒｅｎｔｉａｌ ｄｉｓｐｌａｙ）技术分析人ＨＣＣ、配对非癌肝、胎肝、正常肝等组织之间的基因差异表达,差异表达ｃＤＮＡ片段,经测序后,再用ＲＮＡ狭缝杂交对部分差异片段在上述４种组织中的表达进行验证。结果：共得到差异表达ｃＤＮＡ片段１１０条。经狭缝杂交     

LHScDNA的部分克隆及其表达对肝癌细胞行为的影响

目的：克隆在肝癌组织中低表达的差异显示片段Ｌ２的基因ｃＤＮＡ序列,并初步探讨其在肝癌发生、发展过程中的作用。方法：将Ｌ２在ＧｅｎＢａｎｋ中拼接后,分析其序列结构,并构建正义真核表达质粒,转染ＢＥＬ－７４０２肝癌细胞后,对转染细胞增殖、粘附、迁移和侵袭等指标进行测定。结果：克隆得到一条１００５ｂｐ的ｃＤＮＡ,其５’端７１２ｂｐ与人Ｌｉｐｒｉｎβ１ｃＤＮＡ５’端调控序列和外显子１,２,３序列同源（１００％）,而３’端２９３ｂｐ与Ｌｉｐｒｉｎβ１基因内含子４同源（１００％）。其间有一个５１０ｂｐ开放阅读框架,起始密码位置与Ｌｉｐｒｉｎβ１基因相同,推断的蛋白产物除Ｃ端１３个氨基酸残基外,其余均与Ｌｉｐｒｉｎβ１同源。此ｃＤＮＡ暂命名为ＬＨＳ（Ｌｉｐｒｉｎβ１－ｈｏｍｏｌｏｇｏｕｓ ｓｅｑｕｅｎｃｅ）。ＬＨＳ ｃＤＮＡ     

人肝细胞癌相关新基因的筛选

目的:寻找肝细胞癌(hepatocellular carcinoma,HCC)相关的新基因,并从分子水平进一步探讨HCC发生发展的机制.方法:对通过荧光-差异显示(fluorescent-differential display,FluoroDD)技术从人HCC组织、配对非癌肝(paired non-cancerous liver,PNL)组织、胎肝(fetal liver,FL)组织和正常肝(normal liver,NL)组织获得的110条差异表达cDNA片段(DD片段)中的25条进行测序,将测序结果在GenBank NR数据库中进行同源性分析,并对其中12条DD片段用RNA狭缝杂交法验证其在HCC、PNL及NL组织中的表达规律.结果:25条DD片段中18条与已知基因具有高同源性(＞96%);5条与已知序列(基因结构与功能未知)具有高同源性(＞85%);2条与GenBank NR数据库中的序列无明显同源性.RNA狭缝杂交筛选到5条在HCC中特异性高或低表达并与已知哺乳类动物基因高度同源(97%～99%)的cDNA片段:在HCC中特异性高表达的3条片段是LC13(人热休克蛋白90β)、LC21(智人异源性核内核糖核蛋白C)和LC22(干扰素诱导的智人鸟嘌呤结合蛋白2);在HCC中特异性低表达的2条片段是LC5(智人甲状腺激素受体相关蛋白240)和FL25(分化相关基因1).结论:新发现5条与HCC相关、与已知哺乳动物基因高度同源的cDNA片段.其中3条在HCC中高表达、2条低表达,它们可能分别作为促进与阻抑HCC发生、发展相关的基因,其确切的生物学作用有待进一步研究.