| Type 2 diabetes mellitus(T2DM)is a chronic disease that seriously affects health and causes casts great socio-economic burdens.Currently,there is increasing evidence that T2 DM can increase the risk of cancer and worsens the prognosis.However,the biological link between T2 DM and cancer and the underlyingmolecular mechanisms remain unknown.Centrosome amplification has been confirmed as one of the characteristics of tumors,which can initiate tumorigenesis and promote cancer invasion.Clinical data also show that it is associated withpoor cancer prognosis.Recently,we have reported that type 2 diabetes promotes cell centrosome amplification via ROCK1 and 14-3-3?complex,with high glucose,insulin and palmitic acid as triggers,suggesting that centrosome amplification is a candidate biological link between type 2 diabetes and cancer development.In this study,I further investigated the molecular mechanisms underlying the diabetes-associated centrosome amplification using functional proteomic analysis and candidate approach,for which colon cancer HCT116 cells were used as an experimental model.Moreover,I also examined centrosome amplification and its correlation with disease stage in glioma.Theresearchincludes the following four parts:Part One: Functional proteomics analysis for the protein signals for centrosome amplification associated with type 2 diabetes mellitus.I foundthat treatment by high glucose,insulin and palmitic acid increased the centrosome amplification.The treatment-increased centrosome amplification occurred at a level,with 3-5 centrosomes in a cell.Cellular protein were separated using two dimensional gel electrophoresis,which showed 9 differentially expressed proteins,among which 7 were up-regulated and 2 were down-regulated.The 9 proteins were identified by peptide mass fingerprinting.We randomly selectedNPM,PCNA and 14-3-3? as proteins of interests,the increased expressions of which were confirmed by western blots analysis,and individual knockdown of the proteins inhibited the centrosome amplification.Further analyses showed that PCNA promoted the centrosome amplification by targeting at 14-3-3?,while NPM acted alone.In conclusion,my results suggest that high glucose,insulin and palmitic acid trigger the centrosome amplification via two pathways:(1)NPM;and(2)PCNA to 14-3-3?.Part Two: JNK1-Stat3 signaling pathway negatively controls the centrosome amplification.I also found that the activities of JNK1 and Stat3 were increased by high glucose,insulin and palmitic acid.Knockdown of JNK1 and Stat3 using their si RNA species increased the treatment-evoked centrosome amplification,while overexpression of JNK1down-regulated the treatment-elicited centrosome amplification.Meanwhile,the relationship between JNK1 and Stat3 as well as their downstream targets were investigated.JNK1 was found to be the upstream effector of Stat3,and both protein acted by targeting at at14-3-3?.In conclusion,my results suggest that the experimental treatment activates a feedback signaling rout to counteract the centrosome amplification,which is the JNK1-Stat3-14-3-3? signaling pathway.Part Three: Hsp74/14-3-3? complex mediates the centrosome amplification.I examinedthe14-3-3? binding proteins and their functional roles in the centrosome amplification using so-immunoprecipitation in combination with mass spectrometry technologies,since the above results showed that 14-3-3? was important.I identified 134 proteins that interacted with 14-3-3?,which included heat shock 70 k Da protein 4(Hsp74).Gene ontology analyses revealed that many of them wereenriched in binding activity.Bioinformaticsanalysis against Kyoto Encyclopedia of Genes and Genomes database showed that the top 3enriched pathways were ribosome,carbon metabolism and biosynthesis of amino acids.Molecular and functional investigations showed that the high glucose,insulin and palmitic acid increased the expression and binding of 14-3-3? and Hsp74 as well as centrosome amplification,all of which were inhibited by knockdown of 14-3-3? or Hsp74.Moreover,molecular docking analysis showed that the interaction between the14-3-3? and the Hsp74 was mainly through hydrophobic contacts and,to a lesser degree,the ionic interactions as well as hydrogen bond by different amino acids residues.In conclusion,my results suggest that the experimental treatment triggers centrosome amplification via the 14-3-3?and Hsp74 complex.Part Four: Centrosome amplification in glioma and its correlation withdisease stage.Centrosome amplification is one of the characters of cancer.The research investigated the level of centrosome amplification in glioma and its relationship with the clinical stage.Altogether,40 glioma specimens and 10 normal brain tissue specimens were examined.The protein expression levels of Ki67 and ?-tubulin were detected by immunohistochemistry.Centrosome was visialized using immunofluorescent staining.HE staining showed different morphological characteristics in different samples.Compared with normal brain tissue,the glioma samples had significantly higher positivityof Ki67 and?-tubulin(P<0.01).Immunofluorescence analysis results showed that centrosome amplification was occurredin glioma,which was in positive correlation with the disease grade.In conclusion,my results showed that centrosome amplification was present in glioma,in positive correlation with the disease stage,which suggests that centrosome amplification contributes to the development of glioma.In summary,the results showed that the experiment treatment triggered centrosome amplification via multiple signal transduction pathways,which include(1)NPM;(2)PCNA to 14-3-3?;(3)14-3-3? and Hsp74 complex.It also activated a feedback rout to inhibit the centrosome amplification,which was JNK1-Stat3 to 14-3-3?.Centrosome amplification was also found in gliomas,in associated with the disease staging.My results will benefit the assessment of the cause and consequence relationship between type 2 diabetes and cancer development,and the development of protocols for inhibiting centrosome amplification as well as its associated consequences. |
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