Identification Of MiRNAsatDNA Damage Response In Drosophila And Their Functions At Apoptosis

Maintaining genome stability is essential for normal functioning of cells and tissues.Everyday,tens of thousands of DNA lesions are occured in about 1013 cells of human body.In response to these DNA damage,the body has evolved a complex system to sense DNA damage,regulate cell metabolism,stop cell cycle progression,repair DNA damage,induce programmed cell death,and so on.And this series of signaling pathways is called the DNA damage response pathway(DDR).If the DNA damage can not be repaired in time,it will lead to mutation of base or chromosome,and eventually lead to the occurrence of malignant diseases such as cancer.At the same time,severe DNA damage can lead to cell death or cell senescence.In the regulation of DNA damage response,many DNA damage respone proteins can be regulated by phosphorylation,sumoylation,ubiquitination,methylation and acetylation.Besides,a series of non-coding RNA,including microRNAs(miRNAs)and long non-coding RNAs(lncRNAs)can regulate target gene expression in a post-transcriptional manner in response to DNA damage.MicroRNAs are small non-coding genes present in all animals and plants,and it with a length of about 22 nucleotides.Previous studies have shown,when the Drice and Ago2 proteins are konckdown,cell survival rate and cell cycle checkpoint blocking level will be significantly reduced after DNA damage.The function of miRNA is to bind the target gene mRNA by the base complementary pairing,which results in the degradation of target gene mRNA or thetranslational inhibition.MiRNA can directly regulate DNA damage response protein,also can adjust the expression of transcription factors,such as p53,E2 F to regulate DNA damage repair process.In this paper,we collected w 1118 with p53 5A-1-4embryos in 2-4 hours,and the total RNA was extracted before or after irradiation with 4Gy,sent to the company for RNA-seq,and analyzed the RNA-seq data to obtain 44 differentially expressed mi RNAs.10 miRNAs were randomly selected from 44 differentially expressed mi RNAs,the qPCR primers were designed for those mi RNAs.qPCR are used to validateexpression level of 10 miRNA,and RNA-seq results are consistent with the results of qPCR,we further confirmed the RNA-seq results are credible.The survival rate of 19 Drosophilas was tested by sensitivity test.The results showed that 10 Drosophilas were sensitive to irradiation,2 Drosophilas are resist to irradiation,and 7 Drosophilas are not sensitive to irradiation.Through the analysis of caspase-3 staining,it was found that level of apoptosis in 7 miRNA mutant Drosophila wing disc are significantly increased after 40 Gy irradiated compared with w 1118 Drosophila.The target genes of miRNA,such as skl,grim,wls and drice are predicted by using the website(http://www.microrna.org ? http://www.targetscan.org).The results of this study have important implications for the study of miRNA in the response to DNA damage and the regulation of apoptosis.