Estrogen-Related Receptor (ERR) Participates In Regulating Glycolysis And Influences Embryonic Development In Silkworm Bombyx Mori

As a member of the transcription factor family,Estrogen-related receptors?ERRs?are involved in the regulation of many important biological processes.ERRs are associated with energy metabolism and cancer in mammals,so ERRs are used as a target for detection of breast and cervical cancer currently.The reported showed that ERR could be used as a switch of energy metabolism to regulate the development in Drosophila,but the mechanism of regulating metabolism is unknown.At present,ERR had identified and cloned in the silkworm,which could participate in the regulation of the expression of vitellogenin.Whether ERR has other functions in the silkworm?In this study,we selected the silkworm embryo as the material,whose development process is a period of vigorous metabolism,and the results are as follows:?1?Expression analysis of BmERR and glycolytic-related genes during silkworm mbryo developmentWe detected the mRNA and protein expression profiles of BmERR in the embryonic stage by RT-PCR and western bloting,and confirmed that BmERR had produced during embryonic stage,suggesting BmERR had certain biological functions.To investigated the effect of BmERR on energy metabolism,we also examined the expression profiles of B.mori phosphofructokinase?BmPFK?,B.mori hexokinase?BmHK?and B.mori pyruvate kinase?BmPK?three rate-limiting enzymes during glycolysis.We also found that BmERR expression trend was very similar with that of glycolytic rate-limiting enzymes were,suggesting that there may be a correlation between them.?2?Effect of BmERR on the expression of glycolytic-related genesTo determine whether BmERR plays a role in regulating expression of glycolytic rate-limiting enzymes including BmHK,BmPK,and BmPFK,we performed overexpression,RNAi and inhibition assays for BmERR in BmE cells.The BmERR was successfully overexpressed in BmE cell line,and the expression of BmHK,BmPK and BmPFK were also increased respectively.Meanwhile,the BmERR was decreased when the RNAi was performed to interference the expression of BmERR in the BmE cells,and the expression of BmHK,BmPK,and BmPFK were also reduced compared to the control respectively.Finally,BmERR was inhibited by mammalian ERR-specific inhibitor XCT-790.The results of the inhibition assays are consistent with those of the BmERR interfered assays.These results indicated that BmERR would participate in glycolysis through regulating the glycolytic rate-limiting enzymes such as BmHK,BmPK,and BmPFK.In addition,interference experiments were also performed on silkworm eggs.The double-stranded BmERR and the control dsEGFP were separately injected into the newly-born silkworm eggs at a concentration of 1000 ng/?L,and the final concentration of the dsRNA was about 3 ng/egg.We counted the time when the eggs started to hatch.67.5%of eggs were hatch from the 10^th day to 13^th day in the control groups;however,64%of the eggs were hatched at the 10^th-14^th day after knocking down BmERR.All data showed there was about one-day delayed in the development of embryo when BmERR expression was decreased by dsRNA.Furthermore,the glucose level of the eggs was detected at day 4 after injection.The results showed that the glucose concentration in the experiment group was higher than that of the control.Simultaneously,when embryos develop to stage of their heads becoming pigment,total RNAs were extracted for analysis.qRT-PCR analysis revealed the expression level of BmERR,BmHK,BmPK,and BmPFK were decreased by compared to the control respectively.These results indicated that BmERR could affect the development of embryonic by participating in glycolysis in silkworm.?3?Molecular mechanism of BmERR affecting embryo developmentIn order to investigated whether BmERR colud affect the expression of rate-limiting enzymes by regulated the activity of the rate-limiting enzyme genes promoter?In this study,JASPAR software was used to analyzed the promoter sequences of BmHK,BmPK and BmPFK and to predicted ERRE elements.The results showed that ERRE elements were predicted on their promoters.Based on the previous interference assays and expression profile,BmPFK was selected for further analysis.First,a series of pGL₃-BmPFKP cell transfection vectors with different mutations were constructed.Then,the effect of the regulatory elements on the promoter activity of the BmPFK gene was analyzed using the dual luciferase reporter system.The results showed that promoter activity was unchanged after the mutation of the ERRE-like1 element;however,the mutant of ERRE-like2 element could significantly reduced the activity of the promoter.In order to verify the previous experiment,we overexpressed BmERR in the silkworm embryo cell line BmE-SWU1,and examined the effect of ERRE-like2 on promoter activity.The results showed that ERRE-like2 significantly upregulated the activity of the promoter.To determine whether BmERR protein interacts with the ERRE-like2 motif of the BmPFK promoter in vitro,we incubated biotin-labelled ERRE-like2 probes with recombinant BmERR DNA-binding domain?BmERR DBD?proteins.Electrophoretic mobility shift assay?EMSA?experiments revealed a lag band.which increased with an increase in BmERR DBD proteins and decreased with an increase in competitive probes.When ERRE-like2 probes were incubated with BmERR DBD proteins,the concentration of ERRE-like2 probes increased,accompanied by a increase in lag bands.However,no band shift was observed when the probe sequence was mutated.At the same time chromatin immunoprecipitation?ChIP?experiments to further confirm whether BmERR specifically interacted with the ERRE-like2 motif in vivo.In conclusion,the present results indicated that BmERR binds to ERREs in promoter of glycolytic genes,thereby regulates glycolytic gene expression and influences silkworm embryonic development.Silkworm as a model insect,the present data will promote to study the regulatory association among ERRs,energy metabolism,and development.