MiRNA-968 Targets Expanded In HIPPO Signaling Pathway To Regulate DNA Damage

DNA damage is a change in cellular DNA structure caused by external environmental factors or intrinsic factors.There are many types of DNA damage,including mutations and deletions of DNA bases,DNA single-strand breaks,and DNA double-strand breaks.In order to protect the stability of the genome,there is a complex network in the body that responds to and repairs DNA damage,called DNA damage response.After DNA damage,the DNA damage response is activated,and the transcription of a series of genes is regulated,causing cell cycle arrest and DNA damage repair.When DNA damage cannot be completely repaired,the cells with DNA damage undergo apoptosis.Abnormal DNA damage response can lead to a series of diseases in the body.Studies have shown that DNA damage responses require not only the involvement of protein-coding genes,but also the involvement of non-coding RNA genes.Non-coding RNA was once considered to be"junk RNA".With the deepening of research,the function of non-coding RNA is gradually revealed,and it plays a wide-ranging regulatory role in life activities.miRNA is a non-coding RNA,first discovered in 1993,the first miRNA was identified in the Caenorhabditis Elegans.The miRNA binds to the 3'non-coding region of the target gene mRNA mainly through the"seed sequence",and inhibits the expression of the target gene or degrades the target gene at the post-transcriptional level.miRNAs are involved in DNA damage response,mainly induced by DNA damage,or involved in DNA damage signal transduction,cell cycle checkpoints,DNA damage repair and apoptosis.There have been many studies on the function of miRNAs in mammals,and the understanding is clear.However,little is known about how the miRNAs in the classical model organism Drosophila participate in DNA damage responses.Drosophila has a short growth cycle,simple genome,convenient genetic manipulation,and a variety of powerful genetic tools,so it plays an important role in genetics,development,cell biology and other fields.In this paper,Drosophila was used as the research object.In the previous research,109 miRNAs that responded to DNA damage were discovered by RNA sequencing.In the study,63 miRNA mutants obtained from the research group were tested for DNA damage sensitivity,and 30 of them were found to be sensitive to DNA damage.Further studies revealed that miR-968-1002 ^KOO and miR-986 ^KO fruit flies had S-phase checkpoint defects after DNA damage,while miR-284 ^KO and miR-995 ^KOO fruit flies showed S-phase checkpoint enhancement,other miRNA mutants Drosophila exhibits a normal S-phase checkpoint after DNA damage.To investigate the mechanism of action of miRNAs that respond to DNA damage,the study analyze target genes of miRNAs with miRanda and the targetscan tool software,focusing on genes involved in cell cycle or DNA damage responses.Using the dual luciferase reporter system,this study demonstrated in vitro the predicted target gene in Drosophila S2 cells and found that miR-968 inhibits the reporter gene constructed by the expanded 3?UTR in the HIPPO signaling pathway;when the miR-968 seed sequence is recognized After the site was mutated,the inhibition was significantly reduced,and it was initially proved that miR-968 was targeted.Since miR-986 ^KO and miR-968-1002 ^KO fruit flies all showed S phase checkpoint defects after DNA damage,this study established a miR-986 miR-968miR-1002 mutant Drosophila by genetic manipulation.After the S phase test point detection after DNA damage,there was no significant change in the defect of the S-phase checkpoint of the double mutant fruit fly.This study identified miR-968targeting expanded which participate in the HIPPO signaling pathway,and regulating the S phase checkpoint caused by DNA damage.Providing a new basis for elucidating miRNA regulation of HIPPO signaling pathway and participating in DNA damage response.