Effects Of Trichinella Recombinant Protein Tropomyosin On H446 Cells Of Small Cell Lung Cancer And Its Molecular Mechanism

Lung cancer is a malignant tumor with high incidence all over the world,and its mortality is at a high level among all malignant tumors and gradually rising.Among them,Small cell lung cancer(SCLC)accounts for 12%-15% of the total number of lung cancers.It is highly malignant and invasive,with a short natural course of disease,very fast disease progression,often early metastasis,limited effect of traditional chemotherapy,and easy to lead to drug resistance and relapse,poor prognosis and high mortality.Trichinella spiralis(T.spiralis)parasitism in humans can cause Trichinella spiralis,which is a zoonotic parasitic disease.In recent years,a large number of studies have shown that T.Spiralis has anti-tumor effects,whether the tumorbearing mice are directly infected with Trichinella spiralis,Or its active ingredients or related antigens have antitumor effects.Our previous studies have shown that the excretion and secretion of trichinella spiralis protein can induce apoptosis of H446 cells through mitochondrial pathway.Further Liquid chromatography-tandemmass spectrometry(LC-MS/MS)analyzed the excretion-secreted proteins of Trichinella spiralis,and it was found that six known proteins had anti-tumor effects.One of them is Tropomyosin(TM).It has been shown that TM is a tumor suppressor gene that is downregulated in a variety of human cancers,such as colon,breast,renal cell carcinoma,myeloma,stomach cancer and bladder cancer.In this study,TM eukaryotic expression plasmid was constructed to observe the influence of TM overexpression plasmid on malignant phenotype,cycle and apoptosis of SCLC H446 cells and its mechanism of action,and further LC-MS/MS technology was used to analyze the changes of protein components and pathways in SCLC H446 cells overexpressed by TM.Differentially expressed proteins(DEPs)functional enrichment analysis of GO(Genetic ontology),KEGG(Kyoto encyclopedia of genes and genomes)pathway analysis and Protein-protein interactions(PPI)were performed Bioinformatics analysis,and Western blotting(WB)experiments verified the expression level of NDRG1 in proteomics,and explored the effect of TM on SCLC H446 cells and the potential molecular mechanism.Part I: Effects of Trichinella spiralis recombinant protein TM on malignant phenotype,cycle and apoptosis of small cell lung cancer H446cellsPurpose:By constructing TM eukaryotic expression plasmid,the effects of TM recombinant plasmid on malignant phenotype,cycle and apoptosis of SCLC H446 cells were observed,and the possible mechanism of TM eukaryotic expression plasmid on SCLC H446 cells was discussed.Methods:1.The plasmid of TM overexpression group was constructed into pc DNA3.1-TM group,no-loaded plasmid pc DNA3.1 group(overexpression negative control group,NC group)and blank control group.The transfection was performed according to the instructions of the transfection reagent,the quantity of transfection plasmid was 2 μl,and the transfection time was 48 h.2.The m RNA transcription level of TM in SCLC H446 cells after TM overexpression was detected by q-RT-PCR;The expression level of His-tag protein in H446 cells after TM overexpression was detected by WB.3.To detect the effect of TM overexpression on the malignant phenotype of H446 cells(1)The proliferation ability of H446 cells after TM overexpression was detected by CCK8 assay.(2)Colony formation ability of H446 cells after TM overexpression was detected by plate cloning assay.(3)The migration ability of H446 cells after TM overexpression was detected by cell scratch assay.4.The effect of TM overexpression on H446 cell apoptosis was detected(1)Hoechst 33258 staining was used to detect the apoptosis of H446 cells after TM overexpression.(2)Apoptosis of H446 cells after TM overexpression was detected by flow cytometry with Annexin V/PI double staining(3)m RNA transcription levels of mitochondrial apoptosis-related factors Apaf1,Bax,Cyt-c,Caspase3,Caspase9 and Bcl-2 in H446 cells after TM overexpression were detected by q-RT-PCR.m RNA transcription levels of endoplasmic reticulum stress-induced apoptosis pathway related factors EIF2α,Bip/GRP78,Chop,ATF4 and p-erk.(4)The protein expression levels of mitochondrial apoptosis-related factors Bax,Bcl-2,Cyt-c,Caspase3 and Caspase9 in H446 cells after TM overexpression were detected by WB assay.Protein expression levels of endoplasmic reticulum stress-induced apoptosis pathway related factors p-erk,EIF2α,Bip/GRP78,Chop and ATF4;Protein expression levels of JAK/STAT pathway related factors JAK1 and STAT3.(4)The endoplasmic reticulum stress-induced apoptosis pathway of H446 cells was detected by immunofluorescence assay with EIF2α,Chop and ATF4 after TM overexpression.Expression levels of mitochondrial apoptosis pathway related factor Bax and JAK/STAT pathway related factor STAT3.5.The effect of TM overexpression on H446 cell cycle was detected(1)The cell cycle distribution of H446 after TM overexpression was detected by flow cytometry with PI staining.(2)The protein expression levels of cyclin A1 and cyclin E1 were detected by WB6.Statistical analysis: Experimental data were statistically analyzed using Graph Pad Prism 7.0,and comparison between groups was conducted using One-way ANOVA.Experimental results were expressed as mean ± standard deviation((?)±s),and P < 0.05 was considered statistically significant.Results:1.After overexpression of TM,the m RNA expression level and His-tag protein expression level of pc DNA3.1-TM group were higher than those of NC group and control group(P < 0.05).2.Effect of TM overexpression on malignant phenotype of H446cellsCompared with NC group and control group,proliferation,migration and clonogenesis of pc DNA3.1-TM group were decreased(P < 0.05).3.Effect of TM overexpression on apoptosis of H446 cells(1)Compared with NC group and control group,Hoechst 33258 staining showed that pc DNA3.1-TM group showed more apoptotic cells with strong blue fluorescence.(2)Compared with NC group and control group,flow cytometry showed that the apoptosis rate of pc DNA3.1-TM group was significantly increased(P < 0.05).(3)Compared with NC group and control group,transcription levels of mitochondrial apoptosis pathway related factors Apaf1,Bax,Cyt-c,Caspase3 and Caspase9 m RNA were increased(P < 0.05),while transcription levels of Bcl-2 m RNA were decreased(P < 0.05)compared with control group.Compared with NC group,the m RNA transcription level of Bcl-2 was not statistically significant.Compared with NC group and control group,m RNA transcription levels of ER stress-induced apoptosis pathway related factors EIF2α,Bip/GRP78,Chop and ATF4 were increased(P < 0.05),but there was no statistical significance in P-ERK m RNA transcription levels.(4)Compared with NC group and control group,the expression levels of mitochondrial apoptosis pathway related proteins Bax,Cyt-c,Caspase3 and Caspase9 of H446 cells in pc DNA3.1-TM group of experimental group were increased(P < 0.05).The expression level of Bcl-2 protein was decreased(P < 0.05).The protein expression levels of related factors P-ERk,EIF2α,Bip/GRP78,Chop and ATF4 were increased(P < 0.05)in endoplasmic reticulum stress-induced apoptosis pathway.The protein expression levels of JAK/STAT pathway related factors JAK1 and STAT3 were decreased(P < 0.05).(5)Compared with NC group and control group,immunofluorescence results showed that the expression levels of endoplasmic reticulum stress-induced apoptosis pathway related factors EIF2α,Chop,ATF4 and mitochondrial apoptosis pathway related factor Bax in pc DNA3.1-TM group were increased(P < 0.05).The expression level of JAK/STAT pathway related factor STAT3 was decreased(P <0.05).4.The effect of TM overexpression on H446 cell cycle was detected(1)Flow cytometry showed that the cell cycle of H446 in pc DNA3.1-TM group was blocked in S phase.(2)WB showed that the expression level of cyclin A1 protein was decreased(P < 0.05);cyclin E1 protein expression level was increased(P< 0.05).Conclusion:1.TM eukaryotic expression plasmid transfected human SCLC H446 cells could inhibit their proliferation,clonogenesis and migration ability and promote their apoptosis.TM could block H446 cells in S phase.2.TM may promote apoptosis of human SCLC H446 cells by activating mitochondrial apoptosis pathway,endoplasmic reticulum stress-induced apoptosis pathway and JAK/STAT pathway.Part Ⅱ:Proteomic study of Trichinella spiralis recombinant protein TM on human small cell lung cancer H446 cellsPurpose:Objective to investigate the effect of Trichinella spiralis recombinant protein TM on the protein components of human small cell lung cancer H446 cellsMethods:1.Cell sample collection and grouping: The cells were divided into pc DNA3.1 null carrier negative control group(n=3)and pc DNA3.1-TM experimental group(n=3).The cells were treated according to the transfection method described in Part I 2.4.2,and the transfection time was 24 h and 48 h.The integrity of the two histones was detected by SDS-PAGE,and the bands of the cells changed significantly at 48 h.Cells transfected for 48 h were selected,frozen in liquid nitrogen for 1 h,placed in dry ice,and sent to Beijing Huada Protein Gene Co.,Ltd.for proteomic sequencing.2.The sequencing data was qualitatively analyzed by using Mascot software,and the differential proteins were screened by Uniport database.GO functional enrichment was used to analyze the biological process,cell components and molecular functions of different proteins.KEGG pathway was used to analyze the signaling pathways involved in differential proteins.Cytoscape software was used to map PPI networks to screen central node proteins.3.The protein expression level of N-myc downstream regulatory gene 1(NDRG1)in H446 cells after TM overexpression was detected by WB.Results:1.Changes of protein components in human SCLC H446 cells treated by TMThe results of SDS-PAGE showed that the protein had good integrity with clear bands and no degradation.According to ANOVA analysis of variance,Proteins with P < 0.05 and differences greater than1.2 times were selected as Differentially Expressed Proteins(DEPs).A total of 70 differentially expressed proteins(DEPs)were detected,including 34 upregulated and 36 downregulated.2.Biological function analysis of DEPs obtained after TM action on human SCLC H446 cells(1)GO analysis showed that the cellular components involved in differential proteins were mainly prolactose bodies,small subunit precursors,transcription extension factor complex,cyclin K-CDK12 complex,etc.The biological processes involved mainly included lipid localization,regulation of transcription extension by RNA polymerase II promoter,and lymphocyte mediated immune regulation,etc.The main molecular functions involved are sugar derivative binding,ribonucleotide binding,purine nucleotide binding and so on.(2)KEGG database pathway enrichment analysis showed that differentially expressed proteins were involved in 26 signaling pathways,and the first two signaling pathways with significant enrichment(P<0.05)were taurine and taurine metabolism and Staphylococcus aureus infection.(3)The interaction data of proteins were obtained using STRING database,and Cytoscape software was used to further analyze the interaction between proteins and screen out 10 central node proteins.They are GAK,HIST2H3PS2,MAP2K2,SYMPK,AURKB,PKN2,CDK12,MRPL4,NDRG1 and AURKA.The AURKB was revised up,and the GAK,HIST2H3PS2,MAP2K2,SYMPK,PKN2,CDK12,MRPL4,NDRG1 and AURKA were revised down.3.WB detected the protein expression level of N-myc downstream regulatory gene(NDRG1)in H446 cells after TM overexpressionAfter overexpression of TM,the protein expression level of NDRG1 in pc DNA3.1-TM group was lower than that in NC and control groups(P<0.05).Conclusion:1.The protein components of H446 cells changed significantly after TM overexpression.TM could change the protein expression profile of human SCLC H446 cells by upregulating AURKB and downregulating GAK,HIST2H3PS2,MAP2K2,SYMPK,PKN2,CDK12,AURKA,MRPL4 and NDRG1.2.Overexpression of TM may affect the growth of H446 cells by down-regulating the expression level of NDRG1 protein in human SCLC H446 cells.