CT45-5在DNA损伤应答中作用初探

发布时间：2018-01-01 23:07

  本文关键词：CT45-5在DNA损伤应答中作用初探　出处：《福建农林大学》2012年硕士论文　论文类型：学位论文

  更多相关文章： CT45-5 FHIT DNA损伤应答 siRNA

【摘要】：肿瘤-睾丸抗原45-5(Cancer/testis antigen family45, member A5)是Chen等使用大量平行测序技术和癌/睾丸限制性mRNA表达模型鉴定出的CT抗原基因，这种方法发现了超过20种CT或者CT样基因，其中包括CT45。CT45位于X染色体着丝粒末端Xq26.3，其中包括6个几乎相同的基因拷贝。CT45-5基因全长为567bp，编码189个氨基酸。本课题组先前利用基因芯片技术进行基因差异表达分析显示随着FHIT基因的高表达，有一系列基因的表达也出现上调，其中CT45-5的上调最为明显，对其进行了RNA水平和蛋白水平的验证，并且制备了多克隆抗体。 我们前期的研究发现Fhit-/-细胞对DNA损伤诱导剂具有更强的耐受性，为了进一步研究CT45-5和Fhit之间的关系以及CT45家族在DNA损伤应答中的作用，本课题首先根据CT45-5基因序列，通过生物信息学的方法对靶向CT45-5基因的siRNA进行预测，，从中筛选出2对合理的CT45-5-siRNA，将其克隆到siRNA表达载体pSilencer2.1-U6Hygro中，筛选得到阳性克隆，序列分析得到一条正确的CT45-5-siRNA。用构建成功的CT45-5-siRNA重组载体转染人宫颈癌细胞HeLa及HeLa/Fhit(过表达Fhit的HeLa细胞，3-18)，Western印迹检测siRNA对HeLa及HeLa/Fhit内源性CT45-5以及外源FHIT基因表达的干扰效果，结果显示该siRNA对CT45-5基因具有明显的干扰作用，并且FHIT基因表达也被抑制，提示CT45-5与Fhit可能存在某种功能上的联系。 随后，我们对靶向CT45-5基因的siRNA的功能进行了研究。通过细胞周期实验，证明了在对细胞进行辐射后，转染靶向CT45-5基因的siRNA的细胞出现了明显的G2期延迟现象；通过MTT实验，我们证明了在DNA损伤诱导剂处理的情况下，用靶向CT45-5基因的siRNA表达质粒与空载体分别转染过表达Fhit的HeLa细胞(3-18)，前者对DNA损伤诱导剂具有更强的耐受性，而转染没有Fhit表达的HeLa细胞则不受DNA损伤诱导剂的影响。实验结果提示我们靶向CT45-5基因的siRNA在细胞中可能通过抑制Fhit的表达，从而提高细胞对DNA损伤诱导剂的耐受性。 本实验通过对CT45-5基因的siRNA表达质粒的研究，得到了一条CT45-5-siRNA，为进一步研究Fhit及CT45-5在DNA损伤应答中的作用机制奠定了基础。
[Abstract]:Cancer testis antigen 45-5 (Cancer/testis antigen family45, member A5) CT antigen gene Chen using massively parallel sequencing and expression of cancer / testis restricted mRNA model identified, this method has found more than 20 kinds of CT or CT like genes, including CT45.CT45 X located in the centromeric end of Xq26.3, including 6 almost the same gene copy of.CT45-5 gene was 567bp, encoding 189 amino acids. The expression analysis showed that with the high expression of FHIT gene in our previous use of gene chip technology has a series of genes, gene expression is also up-regulated, the upregulation of CT45-5 was the most obvious, which is verified by RNA and the protein level, and preparation of the polyclonal antibody.
Fhit-/- cell damage tolerance inducing agent is more of DNA found in our previous study, in order to further study the relationship between CT45-5 and Fhit and CT45 in the family in response to DNA damage, firstly, according to the sequence of CT45-5 gene were predicted by Bioinformatics Method of siRNA targeting CT45-5 gene screening. 2 of the reasonable from the CT45-5-siRNA, cloned into siRNA expression vector pSilencer2.1-U6Hygro. The positive clones were screened for sequence analysis, get a correct CT45-5-siRNA. constructed CT45-5-siRNA recombinant vector was transfected into human cervical carcinoma cells (HeLa and HeLa/Fhit Fhit expression in HeLa cells, 3-18, siRNA) Western blot on the interference effect of HeLa and HeLa/Fhit CT45-5 and endogenous exogenous FHIT gene expression, the results show that the siRNA has obvious interference on CT45-5 gene function, and The expression of FHIT gene is also inhibited, suggesting that CT45-5 and Fhit may have some functional relationship.
Subsequently, we targeting siRNA CT45-5 gene function was studied. Through the cell cycle experiments proved that radiation on cells after transfection targeting siRNA gene of CT45-5 cells appeared obvious G2 phase delay phenomenon; through the MTT experiment, we proved that DNA damage inducer treatment case, plasmid and empty vector were transfected into HeLa cells by overexpression of Fhit target expression of siRNA CT45-5 gene (3-18), the former damage tolerance inducing agent has stronger on DNA, but no Fhit expression transfection HeLa cells was not affected by DNA damage inducer. The experimental results suggest that our siRNA targeting CT45-5 the gene may inhibit the expression of Fhit in cells, so as to improve the cell damage tolerance inducing agent of DNA.
In this study, we obtained a CT45-5-siRNA by studying the siRNA expression plasmid of CT45-5 gene, which laid the foundation for further studying the mechanism of Fhit and CT45-5 in DNA damage response.

【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.1

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