MicroRNA-450a-3p Represses Cell Proliferation And Regulates Embryo Development By Regulating Bub1Expression In Mouse

At the early stage of embryo development, normal division of thefertilized egg is very important to it. The fertilized eggs are going onvigorous mitosis, chromosomes separated to the offspring of the twodaughter cells correctly is an important foundation for normal mitosis.Abnormal chromosome segregations may lead to preternatural numbers ofchromosomes, and even provoke cell cycle arrest. To ensure genomestability, eukaryotic cells have developed an inhibitory signaling networkcommonly referred to as the spindle assembly checkpoint (SAC), which candelay anaphase onset until all the sister kinetochores of duplicatedchromosomes are properly aligned and stably attached to microtubulesemanating from opposite spindle poles. Bub1is a critical component of theSAC. As the ‘‘sensor’’ protein of SAC surveillance mechanism, Bub1isknown to regulate cell proliferation and differentiation. We previously foundthat the knockdown of Bub1led to abnormal chromosomes in embryoniccells, and that the expression of Bub1was significantly reduced and thenumbers of spontaneous abortion embryo samples with aberrant numericalchromosome were increased. However, it is unclear how Bub1expression isregulated in this process. It has been shown that microRNAs function asimportant regulators of embryonic development, stem cell differentiation and so on. A vast post-transcriptional regulatory network is mediated bymiRNAs which regulate gene expression through at least two distinctmechanisms: mRNA degradation and mRNA translational repression. In ourprevious study, spontaneous abortion embryos contained low level of Bub1protein but normal mRNA expression, indicating that the Bub1expressionmay be regulated at post-transcriptional level. We first conductbioinformatics analysis and identify eight potential miRNAs that may targetBub1, they including miRNA-30a、30e、494、467a、467e、450a-3p、466a-3pand297b. In this study, we will use miRNAs recombinant adenovirus ormiRNA-mimic to investigate whether the Bub1protein expression isregulated at the post-transcriptional level and the impact of miRNA onmouse fibroblast cell proliferation, apoptosis and cell cycle in vitro; next, weoverexpress miRNA by microinjection technique to observe the affect onearly embryonic development. Study the role of miRNAs in the regulation ofembryonic cell division and the corresponding mechanisms will provide newideas for improving pregnancy quality and prevention embryonicabnormalities abortion.PART ICONSTRUCTION AND IDENTIFICATION OF MICRORNAS ANDBUB1PROTEIN ADENOVIRUS EXPRESSION VECTORObjective:1.Construct miRNA-30a、30e、494、467a、467e、450a-3p、466a-3p、297b and Bub1protein adenovirus expression vector.2. Identification of microRNAs and the Bub1protein adenovirusexpression vector.Methods:1. miRNA genes were amplified from the mouse genome by Nested PCR(contains miRNA precursor sequence), subcloned into the shuttleplasmid pSES-HUS, obtained shuttle plasmid pSES-miRNAs and beingconfirmed by sequencing, digested and electroporated into BJAdEasycompetent cells. Homologous recombination occurs with the backboneplasmid pAdEasy-1, obtained the adenovirus plasmids pAdSES-miRNAs,the plasmid was digested by Pac I then packed in HEK293cells andamplified. Finally identification of mature miRNAs expression was usingFQ-PCR;2. Bub1encoded gene were amplified from the MEF cell cDNA byPCR. subcloned into the shuttle plasmid pAdTrace-TO4, obtained shuttleplasmid pAdTrace-TO4-Bub1and being confirmed by sequencing, digestedand electroporated into BJAdEasy competent cells. Homologousrecombination occurs with the backbone plasmid pAdEasy-5, obtained theadenovirus plasmids pAdEasy-TO4-Bub1, then packed and amplified inHEK293cells. Finally identification of Bub1expression was using FQ-PCRand western-blot.Results:1. The recombinant shuttle plasmid pSES-miRNAs andpAdTrace-TO4-Bub1was constructed successfully confirmed bysequencing.2. Obtained the adenovirus plasmids pAdSES-miRNAs andpAdEasy-TO4-Bub1, then packed and amplified in HEK293cellssuccessfully.3. The adenovirus Ad-miRNAs vector transcribed in cells andexpressed mature miRNAs successfully; adenovirus Ad-Bub1overexpressed Bub1protein in cells successfully.Conclusion: We successfully constructed the adenovirus expression vectorAd-miRNAs and Ad-Bub1by pAdEasy system, they can be applied not only to the subject follow-up studies, but also be applied in other studies related tothe miRNA and Bub1protein. PART ⅡMICRORNA-450A-3P REPRESSES IMEFS PROLIFERATION BYREGULATING BUB1EXPRESSIONObjective:1. Screened the miRNA that regulated Bub1gene from miRNA-30a、30e、494、467a、467e、450a-3p、466a-3p and297b.2. Over-express purpose miRNA in iMEFs, then detect the Bub1protein expression changes, observe its effect on cell proliferation, cycle,apoptosis.3. Take use of "rescue" experimental strategy, we over-express miRNAand Bub1protein to explore the influence of miRNA on cell proliferation,cycle and apoptosis are reversed or weakened.Methods:1. We conduct bioinformatics analysis and identify eight potentialmiRNAs that may target Bub1. Luciferase reporter assay elected the purposemiRNA, then further verify it through protein levels (Western blot) andmRNA levels (FQ-PCR).2.13.5days of embryonic mice were sacrificed, removed the head, taillimbs and internal organs, cut organizations into pieces and digestion,isolated and cultured MEFs. Further established immortalizedMEFs(iMEFs): the retroviral (carrying the the SV40T genes) infected MEFsand get the iMEFs by antibiotic screening methods.3. Cell proliferation were assayed by CCK-8, FCM to detect cell cycle, Hoechst staining and FCM (PE Annexin V staining) to detect the proportionof apoptotic cells. Western blot was taken to analysis of Caspase-3andp-Caspase-3expression.Results:1. Successfully screened miRNA-450a-3p can down-regulate Bub1protein level but no significant changes in the Bub1mRNA level2. MEFs were cultured successfully and immortalized.3. Overexpression of miR-450a-3p in iMEFs repressed cellproliferation, increased apoptosis and enhanced apoptosis-related proteinCaspase-3and p-Caspase-3expression significantly.4. The results of FCM showed that overexpression of miR-450a-3pcould restrict most cells in G1phase of the cell cycle.5."Rescue" experiment found that when Bub1protein levels rebounded,the inhibition on cell proliferation was weakened, the number of apoptoticcells was decreased, and cell cycle arrest disappeared.Conclusion: miR-450a-3p can directly regulate Bub1by binding to the3’-untranslated region of Bub1mRNA. Overexpression of miR-450a-3p inmouse embryonic fibroblast (MEF) cells can down-regulat Bub1proteinlevel, repress cell proliferation, increas apoptosis, restrict most cells in G1phase of the cell cycle. PART IIIEFFECTS OF MICROINJECTION WITH MIRNA-450A-3P ONEARLY EMBRYONIC DEVELOPMENT IN MOUSEObjective:1. To master the mouse fertilized eggs (2-cell stage) acquisition and invitro culture techniques.2. To observe the impact of microinjection with mirna-450a-3p on early embryonic development and the changes of Bub1protein expression.Methods:1. ICR female mice aged6–8weeks were superovulated by consecutiveinjections of PMSG and HCG. Fertilized zygotes at the2-cell stage wereflushed out from the oviducts at42hours after the hCG injection. Embryoswere cultured in M16medium supplemented with10%fetal bovine serumand overlaid with mineral oil at a humidified atmosphere of5%CO2at37oC.2. Fertilized eggs were randomly divided into three groups: blank group,microinjection of control-mimic group and microinjection ofmiR-450a-3p-mimic group. To observe the situation of early embryonicdevelopment after injection, detect the Bub1protein expression duringembryonic development.Results:1. Got a lot of mouse two-cell stage fertilized eggs and cultured in vitrosuccessfully.2. Over-expression of miR-450a-3p significantly reduced cell divisionsof zygotes when compared to those injected with scramble control. About37%of the miR-450a-3p-injected zygotes advanced to develop into the8-cell stage and34%failed to develop into the4-cell, while57%of thescramble control-injected zygotes advanced to develop into the8-cell stageand14%failed to develop into the4-cell stage.3. The results of western blot showed that a significant decrease inBub1protein expression in embryos microinjected with miR-450a-3prelative to the mimic control or the untreated control.Conclusion: Over-expression of miR-450a-3p significantly inhibited mouseearly embryonic development, the one of important reasons maybedown-regulated Bub1protein expression. This part of the experiment provide new research ideas for clinical studies, and the further study ofmicroRNAs is expected to find new therapeutic targets for prevention ofspontaneous abortion.