| ObjectiveCancer is deadly disease all over the world.Understanding the mechanisms of tumorigenesis helps to design novel strategies to treat cancer patients,whereas development of novel methods for early detection is the key to reducing mortality of cancer patients.To this end,the purpose of this thesis is as follows.(1)Study the characteristics of IRS1 and p110? E545 K and the related downstream signaling pathway in CRC cell lines.Validate the important role of both proteins in the development of CRC.(2)Discover new biological function and the molecular mechanism of p85?,a regulatory subunit of PI3 K,in CRC harboringp110? E545 K mutation.(3)Find out the key gene information of p85? and study the relationship between the bio-function of p85? and the key information.(4)Set up simple,rapid and visible analysis methods for 8-hydroxydeoxyguanosine(8-OHdG)and folate receptor1(FR1),two biomarkers related to cancers,providing methods for early screening of cancer.Methods1.Knock out IRS1 gene,in the DLD1 and DLD1 Mut-only(DLD1cells with the WT allele of p110? deleted)colorectal cancer(CRC)cell lines,which harbor PIK3CA/p110?.Compare the related protein expression level,cell apoptosis,colony formation and soft agar foci formation,in the parental and IRS1 knockout(KO)cell lines.2.Construct p110? D933 A mutation that inactivates its lipid kinase activity and p110? KO cell lines,in DLD1 Mut-only cells.Compare the related protein expression level,cell apoptosis,colony formation and soft agar foci formation,in the parental and the p110? KO cell lines.3.Study the interaction of p110?,IRS1,p85a and p85? in the cell lines of DLD1,DLD1 WT-only(DLD1 cells with the E545 K mutant allele of p110? deleted),DLD1 Mut-only,HCT116,RKO and SW480,with co-immunoprecipitation.4.Knock out p85? based on the DLD1 Mut-only cell line.Analyze phenotype,including cell apoptosis,plate colony formation,soft agar formation and xenograft tumor growth model,before and after p85? KO,in order to discover the bio-function of p85?.Furthermore,study the molecular mechanism of p85? associated with the bio-function,using western blot technique.5.Find out the cellular localization of p85? in the cell lines of DLD1 WT-only,DLD1 Mut-only,HCT116 WT-only(HCT116 cells with the H1047 R mutant allele of p110? deleted)and HCT116 Mut-only(HCT116 cells with the WT allele of p110? deleted),with immunofluorescence staining and confocal techniques.6.Predict the key nuclear localization signal of p85? with “cNLS Mapper”.Overexpress HA-tagged WT p85? and HA-tagged Mut p85?(mutated at the key nuclear localization signal)on the basis of p85? KO cell line.Validate the predicted nuclear localization signal of p85? with immunofluorescence staining and confocal techniques.7.Construct p85? with mutation at the nuclear localization signal knock-in cell line based on DLD1,using CRISPR-Cas9 technique.Validate the key gene information related to the oncogenic function of p85? in DLD1 parental and p85? KI cell lines with the xenograft tumor growth model.8.Set up an absorption method to determine 8-hydroxydeoxyguanosine(8-OHdG),a biomarker related to cancer,with specific interaction between antibody and antigen,and immune colloidal gold techniques.9.Set up a resonance-Rayleigh-scattering method for the determination of folate receptor 1(FR1),a tumor biomarker,using specific interaction between antibody and antigen,andimmune colloidal gold techniques.ResultsCompared to the parental cells,p110? E545 K protein levels,but not p85?,are decreased in the IRS1 knockout cell lines.For the downstream signal,pAKT expression level is attenuated,but MAPK signaling pathway is not affected by IRS1 KO.Moreover,IRS1 KO cells exibit increased apoptosis,reduced colony formation and soft agar foci formation.Compared to the parental cells,IRS1 and p85? protein levels are not affected by p110? D933 A mutation or p110? knockout.For the downstream signal,pAKT expression level is attenuated dramatically,but MAPK signaling pathway is not affected.Moreover,p110? KO cells exibit increased apoptosis,reduced colony formation and soft agar foci formation.p85a binds to p110? in both DLD1 WT-only and DLD1 Mut-only cell lines.p85? binds to p110? and IRS1 in DLD1 WT-only cell line,exerting the function of regulatory subunit,but dissociates from p110? E545 K in DLD1 Mut-only cell line.And the dissociated p85? does not bind to IRS1,either.The fact that p85? dissociates from p110? is specific to the cell line with p110? E545 K mutation,which does not exist in other CRC cell lines.To interrogate if the freed p85? plays a role in tumorigenesis in tumors with ap110? E545 K mutation,we genetically engineered DLD1 p85? knockout cell lines.Knockout of p85? impairs colony and soft agar foci formation,slows in vivo xenograft tumor growth and increases apoptosis.Togther,these data suggest that the freed p85? promotes tumorigenesis in cancer cells harboring a p110? E545 K mutation.And what is shown in the study of molecular mechanism is that the oncogenic function of p85? may be associated with JNK/c-jun signaling pathway.What we found in the cellular localization of p85? with immunofluorescence staining is that the dissociated p85? goes into nucleus only in the cells with p110? E545 K mutation.While,it still stays in cytoplasm in other CRC cell lines without p110? E545 K mutation.What is shown in the analysis of nucleus localization signal of p85? is that the lysine and arginine“KR”,which is located at 477 and 478 in the whole amino acid sequence of p85?,is really crucial for the p85? action of going into nucleus in the cells with p110? E545 K mutation.Overexpress HA-tagged WT p85? and HA-tagged Mut p85?(“KR” mutated to “AA”,double alanine)on the basis of p85? KO cell line.p85? goes into nucleus in WT-p85?-overexpressed stable clone cells,while it stays in cytoplasm in Mut-p85?-overexpressed cells,which is shown in the immunofluorescence staining of HA.The ability of xenograft tumor growth is decreased apparently after knock in KR-to-AA mutation based on DLD1 cells.What we confirmed in this experiment is that “KR” is indeed the key nucleus localization signal of p85? and it is really important for the oncogenic function of p85?.On the other hand,what we get from the determination of 8-OHdG,a biomarker related to cancer,is that there is an excellent linear relationship between the red-shift absorption wavelength of antibody-capped Au NPs and 8-OHdG concentration,with a detection range of 0.25–5.00 mg/mL and LOD of 25 ng/mL.And the solution color changes from red to purple or even to blue,with the increasing concentration of 8-OHdG.So the semi-quantitative determination of 8-OHdG comes true with the visible color change.The red-shift and color change are associate with the aggregation status of gold nanoparticles,which is validated by TEM images.The simple and rapid determination of folate receptor 1(FR1)is realized by the linear relationship between enhancement of resonance Rayleigh scattering(RRS)intensity and FR1 concentration,with a detection range of 0.50-37.50 ng/mL and LOD of 0.05 ng/mL.The determination sensitivity is improved dramatically with RRS as the response signal.ConclusionWhat we found in the study of molecular mechanism for the colorectal cancer is that IRS1 and p110? E545 K are crucial for the progression of cancer with DLD1 cells.The oncogenic function is realized through AKT signaling pathway.p85?,but not p85a,specifically dissociates from DLD1 cells with p110? E545 K mutation.And it does not bind to IRS1,either.The freed p85? transloactes into the nucleus in p110? E545 K cells and promotes tumorigenesis.And the function is highly associated with the lysine and arginine “KR”,which is located at 477 and 478 in the whole amino acid sequence of p85?.Moreover,we establish two determination methods for biomarkers(8-OHdG and folate receptor 1)related to cancer.The methods are simple,rapid,easy to operate and with good selectivity.Innovations(1)Discover the new bio-function of p85? dissociated from p110?,and figure out the key gene information for the function,providing a new promising target for the drug screening and clinical therapy.(2)Set up two simple and rapid determination methods for biomarkers related to tumor,providing theoretical foundation for early screening of cancer.Limitations and future planThere are still limitations in this thesis.(1)The phenotype analysis is not enough for p85? KR mutation KI cell line based on DLD1 cells.(2)The detailed mechanisms by which nuclear p85? drives tumorigenesis remained to be determined.(3)The sensitivity should be improved for the determination of 8-OHdG.According to the limitations above,we will improve and optimize the expetiments as follows.(1)Do more phenotype analysis for p85? KR KI cell line,such as cell apoptosis,plate and soft agar colony formation.(2)Read more related references,analyze the downstream signaling pathway for the p85? bio-function and try to find the reasonable molecular mechanism to explain the oncogenic function of p85?.(3)Try other modifications for Au NPs to improve the sensitivity for 8-OHdG determination. |
PDF Full Text Request