Roles Of RBX1-E3 Ubiquitin Ligase In Flagellar Or Ciliary Disassembly And Carcinogenesis Of The Esophagus

The ubiquitin proteasome system(UPS)is a major cellular protein degradation mechanism in eukaryotic cells and up to 80% of cellular proteins are subjected by UPS for degradation specifically.The process of ubiquitination is carried out by transferring ubiquitin to a target protein,which involves a series of three enzymes: ubiquitin-activating enzyme E1,ubiquitin-conjugating enzyme E2,and ubiquitin ligase E3.The SCF E3 ubiquitin ligases,consisting of Cullins,Skp1,F-box proteins,and Ring box protein-1(RBX1),are the largest family of E3 ligases.And The SCF E3 ubiquitin ligases are crucial to the regulation of critical cellular processes under both physiologic and pathologic conditions.These SCF E3 ubiquitin ligases promote degradation of many cellular proteins,including signal transducers,cell cycle regulators,and transcription factors.And recently it has been identified that proteasome is involved in the biological function of flagella/cilia.So we infer that SCF E3 ubiquitin ligases may be involved in the biological function of flagella/cilia.Flagella and cilia are structurally conserved organelles of many cells during evolution with the same origin of basal body.Cilia were once thought to be vestigial in vertebrates,but are now confirmed as important sensory organelles.Recent studies have revealed that nearly all kinds of human cells have cilia.However,cilia are hard to be observed and are easy to be disappeared during cell culture in vitro.The intraflagellar transport(IFT)machinery was first identified in Chlamydomonas reinhardtii,and then was known in the cilia of higher organelle.Primary cilia play central roles in many different signaling pathways,such as,PDGFαα,Hedgehog and mTOR.In addition,it has been confirmed that cilia are predominantly lost in a number of cancers including pancreatic cancer,glioblastoma cells,breast cancer and renal cell cancer compared with their normal cells.However,the hypothesis that dysfunctional cilia result in tumorigenesis has not been well proved so far.Flagella are longer and could be induced to disassemble by IBMX,which are used as a model organelle to detect the molecular mechanism between ubiquitin ligases and ciliary disassembly.Both Dunaliella salina(D.salina)and Chlamydomonas reinhardtii are unicellular eukaryotic green algae with the difference that the latter has cell wall but the former not.So the flagella of D.salina are more similar with mammalian cilia.In this study,the flagella of D.salina are used as a model organelle to research the function of cilia.The SCF E3 ubiquitin ligases,consisting of Skp1,Cullins,F-box proteins and the RING domain containing Ring box protein-1(RBX1),are the largest family of E3 ubiquitin ligases and critical to regulate lots of cellular processes under pathological and physiological conditions.RBX1,as a crucial subunit of SCF E3 ubiquitin ligases,binds to ubiquitin-loaded E2 at its C terminus and cullins at its N terminus,and catalyzes the transfer of ubiquitin from E2 ubiquitin ligases to specific substrates for protein degradation.RBX1 is essential for embryonic development in Caenorhabditis elegans,Drosophila,mouse and human by regulating cell proliferation and differentiation.Recent researches suggested that RBX1 was overexpressed and played central roles in a large number of human cancers,including liver cancer,gastric cancer and bladder cancer.In this study,in order to provide an evidence for the molecular mechanism of SCF E3 ubiquitin ligases in the ciliary/flagellar disassembly and to find therapy targets for cancer,we investigate whether RBX1 is involved in ciliary/flagellar disassembly and regulates ciliogenesis and tumorigenesis in ESCC cells.Part One Polyclonal antibody preparation of DsRBX1 ObjectiveTo study the effects of cliary disassembly on ESCC,the flagella of D.salina were used as a model organelle to discuss whether DsRBX1 participated in flagellar disassembly,which provided basis for further study on the molecular mechanism of ciliary loss in tumorigenesis.Initially,the DsRBX1 protein was expressed and used to prepare the polyclonal antibody.MethodThe PCR primers were designed to obtain DsRBX1 cDNA fragment corresponding to transcriptome sequencing in flagellar regeneration,and then the 3’ cDNA fragment was amplified.The full length sequence of DsRBX1 gene was obtained from a cDNA contigue.The putative protein sequence was applied to bioinformatics analysis.The DsRBX1 cDNA was subcloned into the prokaryotic expression vector pET28a(+).The recombinant expression vector was identified by sequencing analysis.The recombinant vector was transferred into E.coli BL21.The fusion DsRBX1 protein was induced and then purified by Ni-IDA-Sefinose Kit.The purified DsRBX1 fusion proteins were emulsified with Freund’s adjuvant and immunized rabbits to prepare the polyclonal antibody.The anti-serum was collected and examined by ELISA after 5 times immunization.ResultsA cDNA fragment of 504 bp was cloned from the D.salina corresponding to the result of transcriptome sequencing in flagellar regeneration,and a 455 bp of product was acquired by 3’-RACE PCR.According to the sequencing results,a full-length cDNA sequence of 860 bp containing an ORF of 411 bp was obtained.The ORF encoded a polypeptide of 136 amino acid residues.Homologous analysis revealed that the putative protein shared high identities with the RBX1 s from other organisms.In this study,the fusion DsRBX1 protein was expressed in E.coli BL21 transformed with the pET28a(+)-DsRBX1.The fusion DsRBX1 protein with a molecular weight of 17 kDa was induced by 0.1 mM IPTG and identified by 15 % SDS-PAGE,and was purified by Ni-IDA-Sefinose Kit.The result of indirect ELISA showed that the titer of anti-serum to DsRBX1 was between 1:256 K~1:512 K afte 5 times immunization.ConclusionDsRBX1 which cloned from D.salina was high evolutionarily conserved and was expressed to develop polyclonal antibodies.MethodPart Two Preliminary functional study of DsRBX1 inregulation of flagella ObjectiveTo study the effects of cliary disassembly on ESCC,the flagella of D.salina were used as a model organelle to discuss whether DsRBX1 participated in flagellar disassembly.MethodThe purified DsRBX1 proteins were used to prepare polyclonal antibodies.Initially,the purified DsRBX1 fusion proteins were emulsified with Freund’s adjuvant and immunized rabbits to prepare the polyclonal antibody.The anti-serum was collected and examined by ELISA after 5 times immunization.The expression of DsRBX1 was measured by RT-PCR and Western blotting during the flagellar disassembly of D.salina.ResultsUnder IBMX treatment,the relative abundance of DsRBX1 mRNA was increased by 213 % and then decreased by 25 % rapidly within 30 min,and recovered to normal at 120 min.The Western blot showed a 132% increase in the expression of DsRBX1 at 30 min,followed by a 76% decline,and recovery to normal at 120 min.ConclusionDsRBX1 could be involved in ciliary/flagellar disassembly own to its upregulation significantly during flagellar disassembly of D.salina.Part three Functional study of RBX1 in esophagealsquamous cell carcinoma ObjectiveTo study the expression level of RBX1 in ESCC and its function in ESCC tumorigenesis,we detected whether RBX1 participated in regulation of ESCC tumorigenesis.MethodThe expression levels of RBX1 were examined by Western blotting in non-cancerous esophageal epithelial cell line(Het-1A)and ESCC cell lines(EC9706,Eca109 and EC1).The cell proliferation,cell cycle,cell invasion and cell apoptosis were detected respectively after RBX1 was suppressed by shRNA lentivirus in ESCC cell lines.The cilia expression of ESCC cells were detected by indirect immunofluorescence.Xenograft tumor nude mice models were developed to detect the tumorigenic capacity of EC9706 cells with RBX1 knockdown.The tumor volumes were measured,and the tumor growth curve was drawn and the tumor inhibition rate was calculated.The pathological morphology of Xenograft tumor was observed.Some apoptosis-associated proteins in the tumor cells were surveyed by immunohistochemical.And tumor cell apoptosis in Xenograft tumor was examined by TUNEL method.ResultsThe western blotting showed that the protein levels of RBX1 were,respectively,increased to ~1.78-fold in Eca109 cells(P <0.05),~1.90-fold in EC1 cells(P <0.05)and ~1.81-fold in EC9706 cells(P <0.05)compared with that in Het-1A cells.After the expression of RBX1 was inhibited,the cell proliferation and invasion were inhibited,as well as the cell apoptosis was induced in the three ESCC cell lines.After serum starved for 24 h,cilia of ESCC cells were detected by immunofluorescence with antibody against acetylated α-tubulin.The results showed that cilia were observed at a lower frequency in the three ESCC cell lines than in the RBX1-suppressing ESCC cells.RBX1-suppressing EC9706 cells showed lower growth rate in nude mice.The tumor volume is only the 1/2 of the Control group.Pathological histology examination showed that the treated tumor heterogeneity was reduced.And the treatment groups have more apoptosis cells than the Control by TUNEL method.ConclusionThe high evolutionarily conserved RBX1 is upregulated in ESCC cells.The cell proliferation and invasion are decreased,as well as apoptosis and ciliogenesis are induced in RBX1-suppressing ESCC cells.The results suggested that RBX1 may be involved in tumorigenesis and ciliogenesis.And RBX1 knockdown can inhibit effectively the growth of Xenograft tumor in nude mice by inducing apoptosis of tumor cells.Part four Molecular mechanism of RBX1 in regulation ofESCC tumorigenesis ObjectiveTo study the molecular mechanism of RBX1 in ESCC,we detected some major proteins in regulation of ESCC tumorigenesis.MethodRBX1 downregulation suppressed cell proliferation,invasion and ciliogenesis and induced cell apoptosis in the three ESCC cell lines.We detected the changes of major proteins Akt,p-Akt,mTOR,p-mTOR,p70S6 K,p-p70S6 K,E-cadherin,Bcl-2 and Aurora A proteins by Western blotting.Moreover,we examined the activity of mTOR signaling pathway by the overexpression of RBX1 in non-cancerous esophageal epithelial cell line(Het-1A).ResultsIn shRBX1 ESCC cells,the total protein levels of Akt,p-Akt,mTOR,p-mTOR,p70S6 K and p-p70S6 K were decreased compared with that of shCon cells.The expression of Bcl-2 and Aurora A was decreased compared with shCon ESCC cells as well.Furthermore,the expression of E-cadherein was increased compared with shCon ESCC cells.Moreover,the activity of mTOR signaling pathway was increased by the overexpression of RBX1 in non-cancerous esophageal epithelial cell line(Het-1A).ConclusionRBX1 downregulation inhibits cell proliferation,invasion and ciliogenesis and induces cell apoptosis in ESCC,which may be closely correlated with E-cadherin,Bcl-2,Aurora A and the activity of mTOR signaling pathway.