Establishment And Functional Study Of LncRNA Knockout Drosophila For E2f1 And P53 Signaling Network

The stability of cellular DNA is constantly challenged by a variety of environmental factors and endogenous genotoxic damage,such as exogenous ultraviolet,ionizing radiation and chemotherapeutic drugs,as well as endogenous reactive oxygen species.These DNA damages affect cell cycle checkpoints,apoptosis,transcriptional activation and cancer formation,so in order to maintain genomic stability,cells have evolved a conservative DNA damage response（DNA damage response,DDR）system.DDR involves a number of processes,including transduction of DNA damage signals,promotion of DNA damage repair,activation of cell cycle checkpoints,and induction of apoptosis when the damage is irreparable.Different DNA damages are repaired by homologous recombination,non-homologous terminal connection,nucleotide excision repair,mismatch repair and so on.Recent studies have identified a key role for longnon-coding RNA（lnc RNA）in the regulation of DNA damage response networks.lnc RNAs are non-coding RNAs greater than 200 nucleotides in length that regulate gene expression at the epigenetic,transcriptional and post-transcriptional levels and are closely linked to the occurrence,development and prevention of human diseases.Current studies have shown that DNA damage changes the expression of a variety of non-coding RNA at many levels,including transcriptional regulation,post-transcriptional regulation and RNA degradation.Lnc RNA participates in DNA damage response by regulating E2f1,p53 or ATM/ATR signal pathways.The E2F family plays an important role in controlling the cell cycle and the function of tumor suppressor genes.Its member E2f1 regulates the transition from the G1 to the S phase of cells,mediates cell proliferation and p53-dependent/independent apoptosis.Wild-type p53 gene is a kind of anticancer gene,and its encoded protein is a transcription factor that controls the beginning of cell cycle.Nutritional deficiency,hypoxia,oxidative stress and DNA damage can activate p53during tumorigenesis or development to promote cell cycle arrest,senescence,apoptosis and DNA recombination.As a target gene of p53,lnc RNA can participate in DDR response by mediating p53-induced cell cycle progression or apoptosis.Therefore,current studies suggest that some lnc RNA,as direct target genes of p53,play an important role in cell cycle progression and apoptosis,thus dealing with DNA damage.Mutant animal phenotype is a research method that can deeply understand the function of genes.At present,good progress has been made in the research of Drosophila melanogaster as a model animal,but most of the functional deletion mutations of lnc RNA have not been described.The purpose of this study is to study the biological functions of lnc RNAs involved in the regulation of E2f1 and p53 signaling networks.lnc RNA CR45742,which is differentially expressed and highly expressed in multiple tissues and developmental stages,and lnc RNA CR46056 in the intron of e2f1gene were screened out through laboratory RNA-seq data.CR45742 knockout Drosophila(CR45742 ^KO)and CR46056 knockout Drosophila(CR46056 ^KO)were constructed using CRISPR Cas9 technology,respectively.Physiological functions of two strains of lnc RNA gene knockout Drosophila were observed,as well as whether drosophila had functional defects after irradiation,so as to study the role of lnc RNA in the body.The results showed that Drosophila CR45742 ^KOwas sensitive to irradiation,and the number of apoptotic cells increased after irradiation,showing G2/M cell cycle test point defect phenotype.CR46056 ^KO showed a homozygous Drosophila reproductive defect,presenting as sterility in male flies.