Influence Of Anxa5 On Apoptosis Of Pulmonary Squamous Cells Carcinoma And Its Proteomics Data Analysis

Objective:Lung cancer has been recognized as the leading cause for cancer death in the world.The number of lung cancer patients grows too rapidly,which is closely related to atmospheric pollution and haze weather.Pulmonary squamous cell carcinoma(SCC)accounts for 40%of all lung cancers,so the research on the pathogenesis and treatment of lung squamous cell carcinoma is becoming the priority among all the lung cancer research.Cancer is not only related with the abnormal proliferation and differentiation of cells,but also with the abnormal apoptosis.The development of tumor depends on uncontrolled cells growth and proliferation,and depends on inhibition of the apoptosis mechanism,which can not remove the abnormal cells.At present,the clinical treatment of cancer,such as radiotherapy and chemotherapy are mainly through inducing apoptosis of tumor cells.So the importance of apoptosis in tumor therapy has attracted more and more attention.Protein is the main carrier of all human life activities.Proteomics is the comprehensive analysis of total protein complement in the tumor cells at a given time,and post-translational expression level,the modification status,the interaction between proteins.Comparition of the differential expressions of proteins at the health and disease state can not only provide information to investigate the tumor progress,but also work as a marker to diagnose and treat tumor.In our previous study,we identified the down-regulated protein Anax5 by examining samples of lung SCC and adjacent normal tissues using a combination of 2D-DIGE and MALDI-TOF-MS.Then we constructed Anex5 over-expression plasmid and transfected to lung squamous cell lines NCI-H520 and got positive cloned cell line pcDNA3.1-NCI-H520-ANXA5.The conjunction of the stable isotope dimethyl labeling and mass spectrometry is currently used to investigate the differential expressed proteins and the Western blot is used to validate the results.The proteomics research of pulmonary squamous cell carcinoma is based on the application of large-scale protein separation and identification techniques.Based on the bioinformatics analysis and comparative the expressions of proteins at different statues,we can find valuable differential proteins to delve deeper.Methods:1.The influence of high expressed Anxa5 on squamous cells apoptosis induced by cisplatin.(1)The mock transfection of pcDNA3.1-NCI-H520-NC by empty plasmid,and stable transfected cells pcDNA3.1-NCI-H520-ANXA5-1,pcDNA3.1-NCI-H520ANXA5-2 were achieved by plasmid transfection,(2)The electron microscope was used to detect the morphological characteristics of apoptotic cells;DAPI staining,Hoechst 33342/PI double staining,TUNEL,flow cytometry were used to detect the impact of higher expression of Anxa5 in SCC cells.2.Stable isotope dimethyl labeling and mass spectrometry were performed to detect the protein expression profiles of stable transfected cell line pcDNA3.1NCI-H520-ANXA5 and wild type NCI-H520.3.Bioinformatics analysis(1)Bioinformatics analysis of differential proteome based on DAVID and IP A.(2)Western blot method was used to study the expressions of PDIA6?HSPA5?JUP?KRT6A?FNl?EN01?ALDOA?HSP90 in SCC cells.Result:1.Under the transmission electron microscope,we observed that there were condensed chromation and the crescent-shaped body,cytoplasm was condensed,endoplasmic reticulum became loose and fused with cell membrane,autophagic vesicles are formed,mitochondria brokedown.At the late stage of apoptosis,the nucleus broke into pieces,and apoptotic bodies are formed.2.The results of DAPI staining,Hoechst 33342/PI double staining,TUNEL,flow cytometry showed that the apoptosis rates of NCI-H520,pcDNA3.1-NCI-H520-NC,and pcDNA3.1-NCI-H520-ANXA5-1,pcDNA3.1-NCI-H520-ANXA5-2 were significantly different(P<0.05).The apoptosis rates of pcDNA3.1-NCI-H520-ANXA5 were higher than those of control groups.3.The NanoLC-MS/MS Data analysis was accomplished by using MaxQuant software against IPI human database,with carbamidomethylation+57,0215 selected as the fixed modification and oxidation of methionine(Met)+15,9949,light-marked dimethylation+28,0313(C-and N-terminal)and heavy-marked dimethylation+32,0564(C-and N-terminal)set as the variable modifications.The searching results were combined by MaxQuant automatically.Peptides were processed using fully trypsin cleavage and up to two missed cleavage sites were allowed.The peptide mass tolerance was set at 20 ppm and MS/MS tolerance was set at 0.8 Da.Both the protein and peptides identification false discovery rates(FDR)were<1%.The rest of the parameters follow the default settings of MaxQuant software.We detected 49 differently expressed proteins,among them 27 were up-regulated,and 22 were down-regulated.4.Bioinformatics method showed that the differently expressed proteins involved in metabolic process,biosynthetic process;proteins maily locate in the membrane,organelles,and nucleur,cytoplasm;the differential expressed proteins perform 6 biological functions,structural molecule activity,structural consytituent of cytoskeleton,intramolecular oxidoreductase activity,interconvertng aldoses and ketoses,actin binding,protein complex binding,involving 15 pathways,including carbohydrate metabolism,post-translational modification,actin cytoskeleton signaling,damaged DNA repair.5.Western blot detected pathway related proteins:PDIA6,HSPA5,JUP,KRT6A,FN1,ENO1,ALDOA,HSP90.The results showed that PDIA6,HSPA5,JUP,KRT6A,HSP90 are low-expressed in pcDNA3.1-NCI-H520-ANXA5,whereas FN1,ENO1,ALDOA are high-expressed,which are the same as the mass spectrum.Conclusion:1.High expression of Anxa5 promotes the apoptosis of SCC NCI-H520 cells induced by cisplatin.2.Stable isotope dimethyl labeling and mass spectrometry were performed to detect the protein expression profiles of stable transfected cell line and empty plasmid transfected cell line.49 differently expressed proteins were screened,which involved in carbohydrate metabolism,post-translational modification,actin cytoskeleton signaling,DNA repair..3.The results of Western blot are in good agreement with the mass spectrum experiment,which can be investigated deeper.