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The deacetylase SIRT1 regulating DNA damage repair and apoptosis microRNA-34a cloud form a negative regulation loop

Author: LiLei
Tutor: LiangZhiQuan;LiuDePei;LvXiang
School: Beijing Union Medical College
Course: Biochemistry and Molecular Biology
Keywords: SIRT microRNA-34a DNA Negative regulation Expression vector Beijing Union Medical College homologous DSB Reporter gene Bioinformatic analysis Homologous recombination Damage repair Promoter region Protein interactions Sequence Base excision repair Retroviral Gene silencing Protein factors Mammalian cells
CLC: Q255
Type: PhD thesis
Year: 2008
Downloads: 110
Quote: 0
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Abstract


In a variety of DNA damage, double-strand breaks (DSB) is the most serious, it can cause chromosomal rearrangements or deletions to bring great harm to the genome. Through the two conserved pathway for DSB, cell abundance repair, a non-homologous not end connection (NHEJ), another is homologous recombinant (HR). Can not be repaired the DSB or abnormal repair product often related to the occurrence of many diseases, even more serious is the DSB repair abnormal also greatly increase the probability of individuals suffering from cancer, the fine regulation of DSB repair process to complete the normal physiological function in cell the process has important significance. DSB repair and repair of the interaction between the protein factor and modified: including the most studied protein phosphorylation and other protein modifications such as acetylation, ubiquitination, methylation, or even two or adjusting the core part of the network a combination of two or more species of the modified state jointly involved in regulating the activity of DSB repair. Transcriptional regulation constitutes another level of regulation of DSB repair, for example, the core factor of the HR pathway Rad51 may be, including p53, including several transcription factors regulate, thus affecting the activity of HR repair. Recent studies have found that a DSB repair a closely related gene SIRT1 mRNA by the RNA-binding protein HuR in regulating post-transcriptional level, which suggests there may be more DSB repair related genes regulated by the post-transcriptional level. microRNA (miRNA) are a class of small RNA molecules achieve transcription by binding to the target gene mRNA 3'-UTR District regulation, recent studies suggest that miRNA molecules play a role in a variety of physiological processes, and to the occurrence of many diseases. DSBR projects related genes may also be subject to miRNA molecules mediated transcription regulation. We use a target site prediction software to analyze the possible role of Rad51, XRCC3 DSB repair gene miRNA miR-34s eventually selected as candidate molecules. Bioinformatics analysis, we found that miR-34s, another possible target genes of SIRT1. SIRT1 NAD-dependent class III histone deacetylase, promote cell survival under stress and play a regulatory role in maintaining genome stability. Past research has found that, of SIRT1 can go acetylation multiple DSB repair protein factor affecting their activity or stability of these repair proteins including p53 of FoxO3a of Ku70, WRN and NBS1 and SIRT1 can promote DSB repair. In addition, the latest research findings, p53 can trans-activate the expression of miR-34s and the latter will promote apoptosis. The analysis of the above information, we hypothesized that miR-34s and of SIRT1, p53, and other unknown transcription factors may constitute regulation loop, play a role in the regulation of DSB repair and cell survival. We first constructed: niR-34s of the expression vector and verify that they can correct expression by Northern blot method. 3'-UTR reporter gene experiments, DSB repair the related protein Rad51 and SIRT1 is the direct target genes of miR-34s. Further, we found DSB repair experiments miR-34s can be suppressed HR repair activity, whereas the activity of the injury and plasmids of the single chain reconnection no effect. In etoposide processing caused by endogenous genome DSB case, found that miR-34a inhibits overall DSB repair activity by comet assay. Serious DSB repair proteins dysfunction may lead to apoptosis, and therefore we also detected in different cell lines, miR-34s apoptosis. The results show that, miR-34a can significantly promote apoptosis in Saos-2 cells but has no effect in HeLa and 293 cells, and other cells having a similar phenomenon has also been reported in the literature. This suggests that miR-34 to promote apoptosis may be completed by a different target gene, and we found that the target gene SIRT1 may in some cases have a partial contribution. Through the use of an inhibitor of SIRT1 and knock low SIRT1 expression by RNA interference, we found that SIRT1 is involved in the negative regulation of miR-34a, and detected by a combination of SIRT1 in the promoter region of miR-34a. It has been confirmed p53 transcriptional activation of the expression of miR-34s, and repeat the same results in our study. In addition, the study found, FoxO3a can inhibit miR-34a expression at the transcriptional level. Further by ChIP, we detected p53 and FoxO3a these two transcription factor binding to the promoter region of the gene of miR-34a. In summary, we found that miR-34a can inhibit the activity of DSB repair by suppressing Rad51 and SIRT1 expression, its expression is also subject to the regulation of SIRT1, p53, and FoxO3a. miR-34a SIRTl, p53, and is FoxO3a common to create a control loop, play a role in the regulation of DSB repair and cell survival.

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CLC: > Biological Sciences > Cell Biology > Cell physiology > Cell aging and death
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