Study On The Mechanism Of MicroRNA Ban And Transcription Factor Apontic Co-regulating Endocycle

Drosophila is an important model organism of life science because its various organs are excellent models for studying the development process.The salivary glands are crucial secretory organ of Drosophila,which can secrete the key substances involved in larval metamorphosis and the formation of pupal shell.Endocycle is special cell cycle,which includes interphase and synthesis phase,and lacks division phase.It is an important way for the rapid supplementation of genetic material after cell division,and provides material basis for the rapid synthesis of biological macromolecules.After a few of normal cell cycles,the salivary glands of Drosophila enter into the nuclear cycle and accumulate a large amount of DNA,which is about hundreds of thousands of times that of normal diploid body,providing the basis for the synthesis of a large amount of substances needed for abnormal development.The regulatory process of endocycle is mainly completed by cell cycle and its dependent kinases,which are involved in a variety of factors.However,no clear studies have shown that micro RNAs(miRNA)can participate in the regulation of endocycle.Therefore,we expect to find miRNAs that affect intracellular cycle through screening.miRNAs are non-coding RNAs with a size of about 18-25 nt in organisms.After being transcribed in the nucleus,they will be modified and cut,finally transported to the cytoplasm to form RNA-induced silencing complex(RISC)with Ago proteins to play a role in the regulation of gene post-transcription and translation.Apt is an important transcription factor in Drosophila,which is necessary for organ development and morphogenesis.Apt is known to regulate the expression of a variety of downstream target genes,but its upstream regulatory factors are little known.miRNA mainly plays a role in the regulation of post-transcriptional and translational levels of genes and regulates gene expression.Therefore,we expect to find miRNA acting on the upstream of Apt.Screening of eye discs and salivary glands showed that miR-8,miR-252,miR-263 a and miR-ban could affect the shape of nuclear cells,and some of them could affect apt expression.Studies have found that the salivary gland nuclears change caused by ban overexpression are similar to those of apt mutants,so this study mainly explores the mechanism of ban affecting the endocycle through Apt.In vivo immunostaining results showed that ban overexpression in wing discs and salivary glands resulted in increased Apt protein levels.In vitro western blot showed that Apt protein levels of ban mimics transfected cells were consistent with those in vivo.Bioinformatics predicted that ban conserved binding sites were found on apt 3'UTR.In vitro dual luciferase reporting assay results showed that plasmids containing the binding site did not respond to ban,indicating that ban was indirectly upregulating Apt protein.Our subsequent experiments to explore the indirect regulation mechanism of ban on Apt found that ban did not affect the stability of Apt protein.Meanwhile we demonstrated that ban negatively regulates hid through experiments.Hid negatively regulates E2f1 and Apt and E2f1 can directly activate apt transcription.This suggests that ban affects Apt by inhibiting hid gene and further affecting E2f1 activation of Apt,which is an indirect pathway.In this study,we found that the effect of ban and Apt on the salivary gland organ size is achieved by affecting the endocycle,and the effects of ban and Apt on the endocycle are antagonistic to each other.Then we found that Apt could inhibit ban expression by using reporter gene lac Z,and Ch IP-PCR results showed that this inhibition was direct.In summary,we found miRNAs that affect the nuclear cycle and Apt expression,and preliminarily clarified the mechanism of ban indirectly regulating Apt through Hid and E2f1,then affecting the nuclear cycle.In turn,Apt indirectly activated by ban can directly inhibit the expression of ban,and the two antagonize each other to form a negative feedback regulation on the nuclear cycle and maintain the homeostasis of the nuclear cycle.This study extended the endocycle regulation network,and provided a theoretical basis for the future study of organ size and cell cycle.