Roles Of Peroxiredoxin1in Flagellar Or Ciliary Disassembly And Carcinogenesis Of The Esophagus

Esophageal cancer (EC) is one of the most common lethal malignancies and thesixth most frequent cause of death in the world. Esophageal cancer is composed oftwo histological types, including esophageal esophageal adenocarcinoma cellcarcinoma and squamous cell carcinoma (ESCC). The former is mainly occurring inin western country, and the latter is common in China and other Asian countries.Despite a large number of advances in basic research and clinic therapeuticapproaches, the prognosis of the patients with ESCC remains quite poor and the5-year survival rate of this disease is only10-20%after surgery and radiation therapy.The poor prognosis of patients with ESCC is due to the lack of efficient diagnosticmarker and therapeutic target, and the dismal outcome of ESCC is attributed tolargely unknown molecular mechanism in its carcinogenesis and progression.microtubule-basedFlagella and cilia are evolutionarily conserved organelles, which transmitssignaling to cell nucleus and triggers cell reactions by accepting external environment,or other physical and chemical signalings. Recent studies have revealed that primarycilia, for example, play important roles in several crucial signaling pathways ofcarcinogenesis, such as PDGFαα, Hedgehog and mTOR. In addition, it has beendemonstrated that cilia are predominantly lost in several cancers including renal cellcancer, breast cancer, glioblastoma cells and pancreatic cancer in comparison with their normal cellular counterparts. Althouth it is well known that cilia may play anessential role in carcinogenesis, the mechanisms regarding ciliary disassembly andassembly remain unclear. At present, studies have revealed that cilia are universallydisplayed in all type cells in human, but it is very difficult to study the human ciliadirectly. The main causes are as follows: short cilia, disassembly or lost duringcarcinogenesis. Therefore, the knowledge of flagella/cilia is manily from the modelorganelles. Flgella exist in lower eukaryotes whose genome structures are simple andeasy to investigate. For example, the intraflagellar transport mechanism was firstidentified in Chlamydomonas reinhardtii (C. reinhardtii), and then was confirmed inthe cilia of higher organisms. So far, some flagella/cilia-related genes wereidentified and their related functions were investigated in model organisms, such as C.reinhardtii, Caenorhabditis elegans, Drosophila melanogaster and zebra fish.Peroxiredoxin1(Prdx1) is preliminarily identified as an enhanced natural killingcell factor and “scavenger” of oxygen freeradicals in cells. Subsequent investigationdemonstrated that Prdx1plays an important role in H2O2mediated signaling pathway.Recently, the functions of Prdx1in carcinogenesis remain elusive. Prdx1was firstidentified as a tumor suppressor in several malignancies, and Prdx1could inhibit theexpressions of oncogenes c-Abl and c-Myc, and was closely associated with clinicstaging of ESCC. Additionally, Prdx1mediated carcinogenesis is related to theinactivity of PTEN. However, more and more evidence has demonstrated that Prdx1is associated with carcinogenesis based on its up-regulation or over-expression inseveral human cancers including lung, colorectal, ovarian and ESCC, which wasconsidered as an underlying diagnostic marker and therapeutic target of tumors.Importantly, Prdx1protein was identified in D. salina, C. reinhardtii flagella andHomo sapiens (H. sapiens) cilia by proteomics, however, its localization andregulative functions remains to be elucidated in ESCC.In this study, Prdx1isolated from the D. salina flagellar disassembly SSH cDNAlibrary was investigated. Our results showed that highly evolutionarily conservedPrdx1was localized to flagella and basal bodies of D. salina and involved in flagellardisassembly. The gene of Prdx1was overexpressed and cilia lost in ESCC cells.When Prdx1was inhibited, the cell proliferation, invasion were decreased, ciliogenesis was restored, and the activity of LKB1-AMPK-mTOR signaling pathwaywas suppressed. These results suggest that the abnormal expression of thecilia-related gene including Prdx1may affect both ciliogenesis and cancernogenesisand Prdx1is an underlying therapetic target of ESCC.Part1The role of Prdx1in flagellar disassembly of D. salinaObjectiveTo study the roles of ciliary disassembly in the occurrence and development ofESCC, the D. salina flagella were used as model organelles. Firstly, the D. salinaflagella disassembly related genes were screened from the flagella disassemblyrelated genes cDNA library, and then its function was preliminary investigated duringthe flagella disassembly, which will provide the basis for further study on themolecular mechanism of ciliary loss or disassembly in carcinogenesis.MethodChapter1Construction of flagellar disassembly SSH cDNAlibrary in D. salina1. The flagellar disassembly was induced by IBMX and the flagellar length wasdetected during the flagellar disassembly.2. The total RNA was extracted from wild type and IBMX induced D. salina.The D. salina flagellar disassembly SSH cDNA library was constructed byPCR-SelectTMcDNA Subtraction kit, and the differentially expressed genes wereisolated from the library. Chapter2Cloning and prokaryotic expression of the Prdx1gene in D. salina1. The PCR primers were designed according to the sequences obtained in theSSH cDNA library, and then the5’ and3’ cDNA fragements were amplified. The fulllength of DsPrdx1gene was obtained from a cDNA contigue that the putativeseqences cloned and was applied to bioinformatics analysis.2. The Prdx1cDNA was subcloned into the prokaryotic protein expressionvector pET28a (+), and the recombinant plasmid was identified by sequencing. Therecombinant plasmid was expressed in E. coli BL21and the recombinant funsionDsPrdx1protein was induced by IPTG and then purified using Ni-IDA-Sefinose Kit.Chapter3Polyclonal antibody and functional analysis of thePrdx1in D. salina1. The purified DsPrdx1recombinant funsion proteins were emulsified withFreund’s adjuvant, and then immunized rabbits for the preparation of polyclonalantibody. After immunization for5times, the anti-serum was collected and detectedby indirect ELISA.2. The location of Prdx1in D. salina cell was examined by indirectimmunofluorescence and Western blotting.3. The protein expression levels of Prdx1were examined by Western blottingduring the flagellar disassembly in D. salina.ResultsChapter1Construction of flagellar disassembly SSH cDNAlibrary in D. salina1. After flagellar disassembly was induced by IBMX for30min, the flagellarlength of D. salina was shorted from~11.5μm to~7.5μm;2. Total RNA was extracted from D. salina cells untreated and treated withIBMX for30min, respectively, and then the D. salina flagellar disassembly SSHcDNA library was constructed by PCR-SelectTMcDNA Subtraction kit. By BLAST homology search,6flagellum-associated genes and6unkonwn EST sequences wereobtained from the library.Chapter2Cloning and prokaryotic expression of the Prdx1gene in D. salina1. A cDNA fragment (372bp) of Prdx1was isolated from the D. salina SSHcDNA library, and541bp and843bp of products were obtained by5’ and3’-RACEPCR, respectively. Based on these results, a full-length cDNA sequence of956bpcontaining an ORF of606bp was obtained. The ORF encodes201amino acidresidues, and homologous analysis revealed that the putative protein Prdx1shareshigh identities with the known Prdx1from other organisms;2. The Prdx1was expressed in E. coli strain BL21(DE3) transformed with thepET28a(+)-Prdx1. The fusion proteins with a molecular weight of22kDa wereinduced by IPTG and identified by SDS-PAGE, and were purified usingNi-IDA-Sefinose Kit.Chapter3Polyclonal antibody and functional analysis of thePrdx1in D. salina1. The result of Indirect ELISA showed that the titers of anti-serum againstDsPrdx1were between1:256K~1:512K after immunization;2. The results of Western blotting and indirect immunofluorescence showed thatPrdx1was mainly localized to the flagella and basal bodies of D. salina;3. The results of Western blotting revealed that the expression levels of Prdx1were increased significantly during flagellar disassembly of D. salina.ConclusionPrdx1which isolated from the D. salina SSH cDNA library was highevolutionarily conserved and may be involved in flagellar/ciliary disassembly due toits marked up-regulation during the flagellar disassembly of D. salina. Part2Functional study of Prdx1in the occurrence anddevelopment of ESCCObjectiveTo study the expression levels of Prdx1in ESCC cells and its function in ESCCcarcinogenesis. Based on the results of Prdx1being involved in flagellar disassembly,we explore whether Prdx1was participated in ciliary disassembly or loss in ESCCcells, and its molecular mechanism in regulation of ESCC carcinogenesis and cilia.MethodChapter1The detection of Prdx1expression and frequencyof cilia in ESCC cells1. The Prdx1expression levels were detected by Western blotting innon-cancerous esophageal epithelial cell Het-1A as well as ESCC cell lines EC9706,Eca109and EC1and HeLa229cells;2. The frequency of cilia in ESCC cells were examined by indirectimmunofluorescence.Chapter2The preliminary funcional study of Prdx1onregulation of cilia and ESCC carcinogenesis1. The cell proliferation, cell cycle, invasin and apoptosis were examinedrespectively after Prdx1was inhibited by shRNA lentivirus in ESCC cells;2. The cilia and p-Aurora A expression levels were detected in Prdx1-depletionESCC cells;3. The activity of LKB1-AMPK signaling pathway was examined in Prdx1-suppressing ESCC cells;4. The activity of mTOR-p70S6K signaling pathway was examined in Prdx1-suppressing ESCC cells. ResultsChapter1The detection of Prdx1expression and frequencyof cilia in ESCC cells1. The results of Western blotting showed that the expression of Prdx1inEC9706, Eca109and EC1cells were increased by~5.9-fold (P<0.001),~3.8-fold(P<0.01) and~3.3-fold (P<0.01), respectively, compared to that of Het-1A cells;2. After serum starved for24h, cilia of esophageal cells were examined usingimmunofluorescence with antibodies against acetylated α-tubulin (a cilia/flagellarmarker), and the results demonstrated that cilia were observed at a lower frequency inEca109(14.27%, n=496) and EC9706(12.52%, n=576) cells compared with that inHet-1A cells (39.95%, n=431)(P<0.05).Chapter2The preliminary funcional study of Prdx1onregulation of cilia and ESCC carcinogenesis1. The protein level of Prdx1was reduced by~2.2-fold (P<0.05) inPrdx1-suppressing EC9706cells compared with those transfected with the controlshRNA lentivirus. The proliferation of the EC9706cells and control cells was41.37%(n=985) and37.87%(n=1005), respectively, whereas that of the Prdx1-suppressingEC9706cells was significantly decreased to25.83%(n=1108)(P<0.05). The resultsshowed that the invaded cell numbers of Prdx1-suppressing EC9706cells was24.5±3.7compared with9.4±4.7of control cells (P<0.05). The apoptosis of thePrdx1-suppressing EC9706cells was increased by~3.1-fold (P<0.05) compared withthe control cells. Furthermore, the expression of caspase-3was increased by~0.62-fold compared to control cells (P<0.05) as well. However, the results of flowcytometry analysis showed that shRNA lentivirus against Prdx1had no effect on cellcycle distribution of EC9706cells (P>0.05);2.24.73%(n=454) of the EC9706cells were ciliated after transfecting withPrdx1-shRNA lentivirus, but only11.11%(n=580) of the control cells were ciliated(P<0.05), similarly,25.67%(n=447) of the Eca109cells were ciliated after Prdx1inhibition, but only11.32%(n=578) of the control cells were ciliated (P<0.05), and the the protein levels of p-AMPK were increased to~1.13-fold and~4.22-fold inEC9706and Eca109Prdx1-shRNA cells compared with their control cells (P<0.05),respectively;3. The protein levels of LKB1were increased to~1.12-fold and2.01-fold inEC9706Prdx1-suppressing cells and Eca109Prdx1-shRNA cells compared with theircontrol cells (P<0.05), respectively. Meanwhile, the protein levels of p-AMPK wereincreased to~1.13-fold and~4.22-fold in EC9706and Eca109Prdx1-shRNA cellscompared with their control cells (P<0.05), respectively;4. In Prdx1-suppressing EC9706cells, the total protein levels of mTOR andp70S6K were respectively decreased by~1.5-fold (P<0.001) and~3.48-fold(P<0.001) compared with that of control cells. Furthermore, the expressions ofproteins of p-mTORSer2448and p-p70S6KThr421/Ser424were also respectively decreasedby~1.2-fold (P<0.001) and~3.3-fold (P<0.05).ConclusionThe high evolutionarily conserved Prdx1is upregulated and cilia are lost inESCC cells. The cell proliferation and invasion are decreased, apoptosis andciliogenesis are induced, but cell cycle distribution is not changed in Prdx1-suppressing cells. Our results suggest that Prdx1may be involved in carcinogenesisand ciliogenesis through the regulation of LKB1-AMPK-mTOR/p70S6K pathway inESCC.