| BackgroundLung cancer is the leading cause of cancer death worldwide.Lung cancer incidence and mortality increase with increasing age.So lung cancer is believed to be the aging-related diseases.Epidemiological evidence suggests that about two-thirds of patients diagnosed with lung cancer more than 65 years,but less than 2%patients younger than 45 years old.In the United States,the median age at diagnosis of lung cancer is 70 years;the age group 75-79 years is the peak incidence of lung cancer.Because of the accelerating aging of population,health threats of human lung cancer will grow.Privious studies found that among with the aging process,genomic defect,the apparent silence genes,oxidative stress,immune surveillance defective telomere abnormalities,chronic inflammation,and changes in tissue microenvironment,will enable significant increase in cancer risk.Among them,the cellular senescence is a common phenomenon in the process of cell aging face,closely related to tumor development.Therefore,to explore the potential link between cellular aging and cancer prevention to anti-aging and cancer are of great significance.Klotho gene is closely associated with aging,found by Japanese scholars Kuro-o in the study of spontaneously hypertensive.The gene product has two isoforms because of different splicing.Both of them are believed to play a role as co-receptors or signaling proteins.Klotho gene deficient mice have lot of aging-related phenotypes,including:atherosclerosis,skin atrophy,emphysema.Studies have shown that there are currently Klotho expression in multiple tissues,and have different functions.Research shows klotho gene has important effect on apoptosis,cardiovascular disease,kidney disease,and metabolic disease.In addition to have a recognized anti-aging effects,klotho gene also play a role as tumor suppressor genes.Klotho play a potential role as tumor suppressor in breast cancer,stomach cancer,prostate cancer,colon cancer,cervical cancer,kidney cancer and so forth.Many studies also show,klotho can affect different signaling pathways and influence the development of lung cancer.A series of our studies on klotho in lung cancer suggest:1.In non-small cell lung cancer cells,overexpressing klotho gene can inhibit the IGF-1 signaling pathway,the expression of apoptosis-related gene Bax/Bcl-2,thereby inhibiting cancer cell growth and promote apoptosis.2.We found that klotho and sCD40L could synergistically to inhibit cancer cell growth and promote apoptosis.3.We have also found klotho by regulating PI3K/AKT signaling pathway increase the sensitivity of cancer cells to chemotherapeutic drug cisplatin,which play an indirect role in the control of tumor development.4.Klotho can also inhibit the Wnt/?-catenin pathway activation,inhibition of cancer cell growth,migration;and play a role as tumor suppressor.Although a lot of effect of klotho was found,but the working model of klotho is still poorly understood.In addition to understanding klotho as a co-receptor of FGF23,how klotho participate in the other signal transmission process,or which molecules is involved in the implementation of klotho function,there are many question still unknowns.To gain further insight into the function of klotho,as well as its role in the complex,in this study,we got klotho binding protein by co-precipitation;identified them by mass spectrometry screening,in order to obtain more klotho relevant protein information,find new ideas for functional studies and the development of new cancer treatments.Methods1.The klotho protein binding protein detection by mass spectrometry in A549 cells1.1 Construction of klotho lentivirus expression vector,the human klotho were cloned into pUltra lentiviral vectors,and the expression of klotho was detected by western.1.2 The pUltra-klotho lentivirus was packaged with a four plasmid system,in 293T cells.Lentivirus Purification Kit was used for purification and enrichment,and efficiency of virus infection was detected by realtime PCR and packaging titer gradient.1.3 Pulldown protein and its binding protein complexes by co-precipitation technique.The klotho lentivirus infected A549 cells,cell lysates were collected after 4 days,klotho proteins and protein complexes were pulldown by FLAG antibody-conjugated agarose beads.The IP products were detected by WB and Coomassie blue staining for detection of specific proteins and protein abundance.1.4 The protein was quantified by BCA,sent to company to perform mass spectrometry assay.The IP protein product was quantified using the BCA,a sufficient amount of sample was underwent acetone precipitation for mass spectrometry.1.5 Analysis of the results of mass spectrometry.Mass spectrometry results obtained were relevant signal pathway analysis.1.6 Several mass spectrometry detected protein were confirmed by immunoprecipitation.Mass spectrometry screening results obtained several potential significance of protein,we construced expression vector of such proteins and co-transfected them into A549 cells,performed co-immunoprecipitation analysis to further verify the mass screening results,eliminate false positives.2.Rab8 and klotho interaction Mechanism2.1 Construction Rab8 shRNA expression vectors and vectorIn the mass spectrometry results,we screened and obtained Rab8 peptides.Then we constructed expression vectors Rab8,and build Rab8 of shRNA vectors,detected the interference efficiency,to prepare for the post-function studies.2.2 Rab8 and klotho interaction detection of CO-IPRab8-HA expression vector and klotho-FLAG plasmids were transfected into A549 cells,co-immunoprecipitation analysis was performed to further verify the mass screening results.2.3 The role of Rab8 on klotho expression.A549 cells were transfected with Rab8-HA or Rab8-shRNA respectively,klotho expression level was detected by Realtime PCR and WB.2.4 Rab8 is involved in surface expression level of klotho.A549 cells were transfected with Rab8QL-HA,Rab8TN-HA or Rab8-shRNA,respectively.Klotho expression on cell surface was detected by surface biotinylation and WB.2.5 Rab8 is involved in klotho regulation of Wnt signal.KL and Rab8 shRNA plasmids were transfected into A549 cells.Wnt3 and ?-catenin expression were detected by WB.2.6 Rab8 is involved in klotho regulation of cell function.KL and Rab8 shRNA plasmids were transfected into A549 cells.Then cells were detected with scratch test,transwell assay for cell migration and invasion in response to the klotho.Changes in cell proliferation ability were detected by CCK8 assay and colony formation.Results1.The klotho protein binding protein detection by mass spectrometry in A549 cells1.1 We successfully constructed and packaged lentivirus expressing human klotho.Klotho expression vector under the ubiquitin promoter,were successfully constructed,the vector carrying 2A separated GFP,can be simultaneously expressed GFP.The lentivirus was successfully packaged in a 108 titer lentiviral particles.And A549 cells with the lentivirus interference obtained klotho overexpression.In fluorescence microscopy,we found the GFP-positive rate of cells was more than 85%;WB detection shown klotho could expressed in A549 cells.1.2 Using FLAG antibody immunoprecipitation,we successfully pull down the protein complexes.The klotho lentivirus was transfected into A549 cells,4 days later,total protein extracts were collected by Flag antibody immunoprecipitation,the obtained precipitate complexes were detected WB,the results indicated the high concentration of klotho.Coomassie brilliant blue staining shows also the high concentration of klotho;and you can see the differences in experimental and control groups(IgG group)pull-down proteins,indicating the specificity of the assay.The BCA protein quantification also indicated that the total protein mass reaches the desired protein requirements.The product was used in subsequent mass spectrometry experiments.1.3 Analysis of the results of mass spectrometry experimentsWe get protein peptide score greater than 20,8265,with the control group compared to the abundance of greater than 2 protein 84,not fully quantified protein 209.1.4 CO-IP confirmed 4 mass spectrometry results with the klotho protein binding ability.We screened klotho protein binding results,and constructed 4 expression vector,then performed the klotho cotransfection experiments and repeated CO-IP.The results show that the presence of three protein interacted with klotho.2.Functional studies of Rab8 and klotho interaction2.1 Rab8 interaction with klothoHA-tagged Rb8(HA-Rab8)and Flag tagged(Flag-klotho)were co-transfected into cultured A549 cells,and followed by co-immunoprecipitation experiments.The results indicatd that HA-Rab8 could be immunoprecipitated by Flag-klotho.The endogenous COIP assay got the same result.2.2 Rab8 does not affect the expression of klotho.A549 cells was transfected with Rab8 inactivation of mutant TN and shRNA,the results indicate Rab8 can not affect the mRNA and protein expression of klotho.2.3 Rab8 influence surface expression level of klotho.With Rab8 sustained inactivation of mutant TN and shRNA transfected into A549 cells,we found Rab8 dysfunction can lead to decreased levels of klotho membrane surface.2.4 Rab8 influences the role of klotho on A549 migration,survival,and signalsRespectively Rab8 shRNA and klotho expression vector was transfected into A549 cells,and cells were performed with transwell,cloning,and scratches CCK8 assay.The results showed that Rab8 influences the role of klotho on A549 migration,survival,and signals.Conclusions1.In the A549,we detected a lot of klotho binding protein may be involved in the implementation of klotho function.2.Rab8 and klotho interaction affects the biological function of klotho and downstream Wnt signaling pathway.Innovations1.By immunoprecipitation and mass spectrometry,we got the first data of a large number of klotho binding protein,which provide clues for future research klotho function.2.We found Rab8 and klotho the interaction and this interaction influence the biological function of klotho and downstream Wnt signaling pathway. |
PDF Full Text Request