The Expression And Purification Of ULK1 And The Regulation Research Of MiRNAs To G4R1 Expression In Human Cervical Cancer Cell Hela

Autophagy is one of the main important ways of macromolecular material degradation in eukaryotic cells, which is highly conservative and regulated by complex mechanisms. Ulk1(unc-51 like autophagy activating kinase 1) is one of the two kinds of homologues of yeastautophagy startgene Atg1 in mammals, whichis a downstream molecule of the AMP activated protein kinase(AMPK) to participates in mammalian autophagy. At the same time, ULK1 is a negative regulatory factor of mammalian target of rapamycinprotein(mTOR) signaling pathway of. The expression level of ULK1 has a close relationship with the normal start or not of autophagy in human cells, andULK1 is the target site of tumor suppressor p53.In order to obtain the purified recombinant protein ULK1, we induced and expressed ULK1 protein in E coli. We obtained ULK1 gene by PCR(Polymerase chain reaction, PCR); and then combine it with the expression vector pET-28a-SUMO to construct a recombinant plasmid and transferinto aE.coli Transetta(DE3) Chemically Competent Cell; use isopropyl glucosinolates galactose glucoside(IPTG) to induce the expression of purposeprotein;and identify whether it expressed and cofirm the ULK1 expression form in E.Coliby SDS-PAGE; Using BeaverBeads?His-tag Protein Purification magnetic beads to purify the recombinantprotein.Through the research of looking for the best inducing condition to expressULK1 in E.coli Transetta(DE3)Cells, we have found that the best induction time is 3 h, and the best induction density is OD600 1.0, and when ULK1 is induced by 0.6 mmol/L IPTG, ULK1 expression is the most of all. The results of this research are as follows:1、pET-28a-SUMO cannot efficiently induce the soluble expression of ULK1 protein;2、The expressionform ofULK1 protein in E.coliis inclusion body;2、Failure to obtain purified ULK1 protein;G-quadruplex(G4) are four-stranded tetrad structures mediated by Hoogsteen hydrogen bonds formed between four guanines in G-rich nucleic acid sequences. G4 presents widely in the genome and telemore, and has important role in the maintenance of genome stability and regulation of gene expression. Some helicases specifically bind and resolve G4-DNA, which is important for genome metabolism. G4 resolvase 1(G4R1),which is responsible for the major G4-DNA resolving activity in HeLa cells, shows binding G4-DNA and G4-RNA with high affinity and unwinding activity in vitro.Some studies demonstrated G4R1 regulates gene expression through G4 resolvasing activity. However, the mechanism of G4R1 binding and resolving G4-DNA in vivo and regulation of gene expression and cell proliferation are less studied. Micro RNA(miRNA) is a kind of18 to 24 ntnoncoding RNA, which widely exists in animals, plants and viruses, and can identify and bind mRNA 3’-untranslational region(3’UTR) of the target gene. Itcan inhibit or degradate mRNA by specific binding the target sequence, and then realize the negative regulation of the expression of target genes, involving in regulating the process of cell growth, differentiation and apoptosis, physiological.We use the commonly used biological information software Target scan(www.target.org), Pictar Pictar(mdc-berlin.de) and miRanda(www.microrna.org) to predicted possible mi RNAs. The results show that many mi RNAs(miR-30a、miR-150、miR-340 and miR-200b/c) can matchwithconservative sequences of G4R1 3’UTR. In order to study the expression regulation of mi RNAs to G4R1 in Hela cells, we cloned G4R1 3’-UTR and successfully construted pGK-Gluc-G4R1-3 ’UTR vector, and then transfected it into Helacell, alonging with mi RNAs(mi R-30a、miR-150、miR-340 and miR-200b/c). Results showed that miRNAs can significantly inhibit the Gulc luciferase activity of Hela cells, which were transfected along with pGK-Gluc-G4R1-3’-UTR. Then mutate G4R1 3’-UTR and transfecte it into Hela cell, alonging with miR-30 a, the Gulc luciferase activity of Hela cells does not decrease,, which were transfected along with pGK-Gluc-G4R1- 3’-UTR, showing that miR-30aregulatesG4R1 by its 3’-UTR. Western blot also shows the same result, miR-30 a can decrease G4R1 expression level. And G4R1 expression suppression gradually enhanced while the increasing of miR-30 a,which further confirms that miR-30 a suppresses G4R1 expression through binding the 3’UTR.In addition, this study also confirmed that mi R-340 and miR-200/c can also regulate G4R1.But the effect of miR-150 and mi R-200/b to G4R1 protein expression is not obvious.