MicroRNA-34a, Which Regulates DNA Damage Repair And Apoptosis, Forms A Negative Regulatory Loop With Cloud Acetylase SIRT1

To maintain the faithful genome transmission through dividing, cells evolved coordinated pathways such as cell cycle checkpoint activation, DNA repair, senescence and apoptosis to counteract inevitable DNA damages. Among different types of DNA lesions, double-strand breaks (DSBs) induced by either ionizing radiation or some cancer therapeutic drugs lead to chromosome rearrangement and translocation, which are extremely dangerous to the genome. There are two main pathways to repair DSBs: non-homologous end joining (NHEJ) or homologous recombination (HR). The DSB damage response and repair are tightly regulated, as dysregulation of DSB repair underpins many human diseases and even predisposes individuals to tumorigenesis.The regulation of DSB repair mainly lies at the crossroads of various protein modifications including phosphorylation, ubiquitylation, acetylation, methylation and even the combination of modification marks. In addition, DSB repair genes can be regulated at the transcriptional level and beyond. The fact that HuR regulates the expression of the DSB repair related deacetylase SIRT1 mRNA through binding to the 3'-UTR suggests other DSB repair genes may also be regulated at the posttranscriptional level.MicroRNAs (miRNAs) are posttranscriptional regulatory molecules that have been proved to be implicated in a wide variety of normal physiological processes and many human diseases. We hypothesize certain miRNAs may target DSB repair genes. Using target prediction tools we found miR-34 family members as candidates targeting Rad51.The bioinformatics analysis also predicted that miR-34a potentially targets SIRT1, a NAD+ dependent class-?histone deacetylase. SIRT1 is known to deacetylate DSB repair related proteins p53, Ku70, NBS1, WRN and FoxO3a and regulate their activity or stability. Another study using SIRT1 overexpression and knockdown cells showed that SIRT1 promotes DSBR. Furthermore, recent studies demonstrated that miR-34s can be activated by p53 in response to DNA damage and promote apoptosis.We thus propose that this DNA damage responding miRNA probably forms regulation circuit with SIRT1, p53 and other transcription factors, which may cooperate to control DNA damage repair and cell survival. We constructed the expression vectors of miR-34s and confirmed their correct expression using Northern blot. Overexpression of miR-34s in HeLa cells proved our prediction that Rad51 and SIRT1 are direct targets of miR-34s. Then we tested if miR-34s affect the DSBR activity. The results showed that miR-34s repress the HRR activity in the conditions of DR-GFP reporter assay. In the luciferase assay based single strand break repair and plasmid re-ligation experiments, miR-34s have no apparent effect on repair activity. In addition, it was proved by comet assay that miR-34a suppresses DSBR activity in the cells treated with etoposide.Apoptosis is induced due to severe DNA damages or dysfunction of repair proteins. In our study, it was shown that miR-34a can cause apoptosis in Saos-2 cells, but have no apparent effects in HeLa and 293 cells. It indicates that the proapoptotic effect of miR-34a may differ in different cell lines and inhibition of SIRT1 by miR-34a may partially contribute to cell cycle arrest and apoptosis in some conditions.To test the hypothesis that miR-34a forms regulatory feedback loop with SIRT1, we used SIRT1 inhibitors and SIRT1 knockdown cell clones and found that SIRT1 negatively control the expression of miR-34a. miR-34a was previously reported to be transcriptionally activated by p53, and this was confirmed in our study. Another transcription factor FoxO3a that promotes DSBR through interaction with ATM was predicted to regulate miR-34a. The results then proved that FoxO3a can suppress miR-34a expression at the transcriptional level. ChIP and CoIP data showed that SIRT1, along with p53 and FoxO3a can be recruited to the promoter region of miR-34a gene and SirTl can interact with p53. Because SIRT1 and FoxO3a were reported to be associated in response to oxidative stress, we did not detect the interaction under normal culture conditions.In summary, we provide evidence to support that miR-34a suppresses DSBR activity and forms regulatory feedback loop with SIRT1.