hOGG1基因特异性锤头状核酶体外切割活性及其在A549细胞内瞬时效应的研究
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摘要
目的采用分子生物学技术与核酶技术抑制人肺腺癌A549细胞中DNA氧化损伤修复基因人8-羟基鸟嘌呤-DNA糖苷酶(Human8-oxoguanineDNAglycosylasel,hOGGl)的表达,以探讨肺癌的发生与发展同DNA氧化损伤修复基因的关系。
方法运用计算机软件人工设计并合成锤头状核酶基因(Ribozyme,RZ),将该核酶基因克隆到真核表达载体pcDNA3.1(+)中,重组子由BamHⅠ和EcoRI酶切,琼脂糖凝胶电泳鉴定及DNA测序证实。多聚酶链反应(PolymeraseChainReaction,PCR)扩增锤头状核酶作用的靶基因hOGGl保守序列,将PCR扩增序列插入体外转录载体pBluescriptSK(+),经SPeⅠ和SacⅡ酶切电泳及测序鉴定。采用地高辛(Digoxigenin,DIG)标记检测系统,体外转录核酶基因及hOGGl基因,获得核酶与带有DIG标记的底物hOGGlmRNA。通过调整核酶体外实验中不同的反应条件,确定核酶体外切割实验方案;然后经RNA变性凝胶电泳分离切割片段,真空转印正电荷尼龙膜固定mRNA,再经DIG的检测系统反映核酶对靶基因的体外切割效率。含有核酶基因的重组载体pcDNA3.1(+)-RZ在类似脂质体的转染试剂FuGENE6的介导下引入人肺腺癌A549细胞,逆转录多聚酶链反应(ReverseTranscriptase-PCR,RT-PCR)扩增耐受新霉素的Neo基因以鉴定阳性转染细胞。以人甘油醛-3-磷酸脱氢酶(Humanglyceraldehydes-3-phosphatedehydrogenase,hGAPDH)为内参照,RT-PCR法半定量检测转染细胞中hOGGlmRNA的表达量。以阿霉素为受试物,MTT法检测转染前后细胞存活率的变化,彗星试验检测转染
Objective To study the correlation between pathogenesis of lung cancer and oxidative DNA damage and repair genes, the expression of human 8-oxoguanine DNA glycosylase gene(hOGG1 gene), which could specially excise 8-oxoguanine induced by reactive oxygen species(ROS), was down-regulated in human lung cancer A549 cells by molecular biological and ribozyme technology.Methods According to design by computer, two specific restriction site BamH I and EcoR I were added to both ends of the hammerhead ribozyme gene, then the modified ribozyme gene was synthesized and cloned into the eukaryotic expression vector pcDNA3.1(+). The positive recombinants were screened by tolerance of ampicillin, and plasmids were extracted from the positive recombinants and digested by BamH I and EcoR I, and then were analyzed by agarose gel electrophoresis and DNA sequencing. By the similar means, the conservative hOGGl gene sequence amplified by polymerase chain reaction (PCR) was inserted into in vitro transcription vector pBluescript SK(+) between restriction site Spe Ⅰ and Sac Ⅱ. Then, the ribozyme gene and hOGGl gene were transcribed in vitro respectively. The transcripts of ribozyme and its target RNA sequence with DIG-11-UTP were mixed, followed by a specific hammerhead ribozyme cleaving reaction to hOGG1 mRNA. In succession, the recombinants containing ribozyme DNA were transiently transfected into A549 cells. The
positive recombinant was identified by reverse transcriptase-polymerase chain reaction (RT-PCR) targeting to Neo gene, which was a neomycin resistance gene for selection of stable cell lines and only appeared in vectors. The changes of hOGGl mRNA in A549 cells were detected by semi-quantitative RT-PCR. Then the cellular sensitivity to adriamycin was tested by comparison between untransfected cells and transfected cells by MTT assay. The adriamycin-induced DNA damage was investigated by comet assay or named single cell gel electrophoresis (SCGE) between untransfected and transfected cells.Results The recombinants containing the ribozyme gene and the recombinants containing hOGGl gene were successfully selected by restriction endonuclease digestion and agarose gel electrophoresis, and were proved by DNA automatic sequencing, respectively. The transcripts of ribozyme and hOGGl were acquired by in vitro transcription, and then cleavage in vitro assay was carried out under different conditions including mole ratio of 1:1, 1:2, 1:4, 1:8 and 1:10, reaction time of 30min, 60min and 90min, reaction temperature of 37°C, 42°C and 50°C, concentration of Mg2+ of lOmmol/L, 15mmol/L and 20 mmol/L. But, under these conditions, the effect of in vitro cleavage of hOGGl mRNA by ribozyme could not be detected. A549 cells containing the recombinants were identified by RT-PCR because Neo genes were amplified only in cells transfected successfully. The expression of hOGGl mRNA in A549 cells transfected ribozyme was 36% significantly less than in control cells(P<0.05). MTT assay showed that on the same concentration, the sensitivity of transfected cells to adriamycin were increased in comparison with untransfected cells. The comet assay showed that the extent of DNA damage induced by 1 |ig/ml adriamycin of transfected cells were worse than unrtransfected cells on the same transfection time (P<0.05), but there was no time-dependent reaction relationship.Conclusion The eukaryotic expression vector with genes of hammerhead ribozyme targeting to hOGGl mRNA and the in vitro transcription vector with its
substrate hOGGl gene were constructed successfully. Though in vitro cleavage assay could not detect ribozyme's cleavage to its substrate mRNA, it effectively inhibited the expression of hOGGl gene in lung cancer A549 cells, and increased the cellular sensitivity to adriamycin, and it helps to study deeply the functions of base excision repair genes hOGGl.
方法运用计算机软件人工设计并合成锤头状核酶基因(Ribozyme,RZ),将该核酶基因克隆到真核表达载体pcDNA3.1(+)中,重组子由BamHⅠ和EcoRI酶切,琼脂糖凝胶电泳鉴定及DNA测序证实。多聚酶链反应(PolymeraseChainReaction,PCR)扩增锤头状核酶作用的靶基因hOGGl保守序列,将PCR扩增序列插入体外转录载体pBluescriptSK(+),经SPeⅠ和SacⅡ酶切电泳及测序鉴定。采用地高辛(Digoxigenin,DIG)标记检测系统,体外转录核酶基因及hOGGl基因,获得核酶与带有DIG标记的底物hOGGlmRNA。通过调整核酶体外实验中不同的反应条件,确定核酶体外切割实验方案;然后经RNA变性凝胶电泳分离切割片段,真空转印正电荷尼龙膜固定mRNA,再经DIG的检测系统反映核酶对靶基因的体外切割效率。含有核酶基因的重组载体pcDNA3.1(+)-RZ在类似脂质体的转染试剂FuGENE6的介导下引入人肺腺癌A549细胞,逆转录多聚酶链反应(ReverseTranscriptase-PCR,RT-PCR)扩增耐受新霉素的Neo基因以鉴定阳性转染细胞。以人甘油醛-3-磷酸脱氢酶(Humanglyceraldehydes-3-phosphatedehydrogenase,hGAPDH)为内参照,RT-PCR法半定量检测转染细胞中hOGGlmRNA的表达量。以阿霉素为受试物,MTT法检测转染前后细胞存活率的变化,彗星试验检测转染
Objective To study the correlation between pathogenesis of lung cancer and oxidative DNA damage and repair genes, the expression of human 8-oxoguanine DNA glycosylase gene(hOGG1 gene), which could specially excise 8-oxoguanine induced by reactive oxygen species(ROS), was down-regulated in human lung cancer A549 cells by molecular biological and ribozyme technology.Methods According to design by computer, two specific restriction site BamH I and EcoR I were added to both ends of the hammerhead ribozyme gene, then the modified ribozyme gene was synthesized and cloned into the eukaryotic expression vector pcDNA3.1(+). The positive recombinants were screened by tolerance of ampicillin, and plasmids were extracted from the positive recombinants and digested by BamH I and EcoR I, and then were analyzed by agarose gel electrophoresis and DNA sequencing. By the similar means, the conservative hOGGl gene sequence amplified by polymerase chain reaction (PCR) was inserted into in vitro transcription vector pBluescript SK(+) between restriction site Spe Ⅰ and Sac Ⅱ. Then, the ribozyme gene and hOGGl gene were transcribed in vitro respectively. The transcripts of ribozyme and its target RNA sequence with DIG-11-UTP were mixed, followed by a specific hammerhead ribozyme cleaving reaction to hOGG1 mRNA. In succession, the recombinants containing ribozyme DNA were transiently transfected into A549 cells. The
positive recombinant was identified by reverse transcriptase-polymerase chain reaction (RT-PCR) targeting to Neo gene, which was a neomycin resistance gene for selection of stable cell lines and only appeared in vectors. The changes of hOGGl mRNA in A549 cells were detected by semi-quantitative RT-PCR. Then the cellular sensitivity to adriamycin was tested by comparison between untransfected cells and transfected cells by MTT assay. The adriamycin-induced DNA damage was investigated by comet assay or named single cell gel electrophoresis (SCGE) between untransfected and transfected cells.Results The recombinants containing the ribozyme gene and the recombinants containing hOGGl gene were successfully selected by restriction endonuclease digestion and agarose gel electrophoresis, and were proved by DNA automatic sequencing, respectively. The transcripts of ribozyme and hOGGl were acquired by in vitro transcription, and then cleavage in vitro assay was carried out under different conditions including mole ratio of 1:1, 1:2, 1:4, 1:8 and 1:10, reaction time of 30min, 60min and 90min, reaction temperature of 37°C, 42°C and 50°C, concentration of Mg2+ of lOmmol/L, 15mmol/L and 20 mmol/L. But, under these conditions, the effect of in vitro cleavage of hOGGl mRNA by ribozyme could not be detected. A549 cells containing the recombinants were identified by RT-PCR because Neo genes were amplified only in cells transfected successfully. The expression of hOGGl mRNA in A549 cells transfected ribozyme was 36% significantly less than in control cells(P<0.05). MTT assay showed that on the same concentration, the sensitivity of transfected cells to adriamycin were increased in comparison with untransfected cells. The comet assay showed that the extent of DNA damage induced by 1 |ig/ml adriamycin of transfected cells were worse than unrtransfected cells on the same transfection time (P<0.05), but there was no time-dependent reaction relationship.Conclusion The eukaryotic expression vector with genes of hammerhead ribozyme targeting to hOGGl mRNA and the in vitro transcription vector with its
substrate hOGGl gene were constructed successfully. Though in vitro cleavage assay could not detect ribozyme's cleavage to its substrate mRNA, it effectively inhibited the expression of hOGGl gene in lung cancer A549 cells, and increased the cellular sensitivity to adriamycin, and it helps to study deeply the functions of base excision repair genes hOGGl.
引文
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[3] Cadet J, Berger M, Douki T, et al. Oxidative damage to DNA: formation, measurement and biological significance[J]. Reviews of Physiology, Biochemistry Pharmacology. 1997; 131: 1-87.
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