| Lung cancer(LC)has the highest morbidity and mortality in the world.It is a disease that seriously endangers human health.Lung cancer is divided into small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC)according to the pathological type,accounting for 20% and 80% of the total number of lung cancers,respectively.Patients with non-small cell lung cancer can undergo surgical treatment at an early stage,and surgery,chemotherapy,and radiation therapy are often used in the middle and advanced stage.Not only are the patients suffering,prone to drug resistance,but the overall cure rate is still less than 20%.Therefore,it is urgent to explore and find new treatment methods.Trichinella spiralis(T.s),also known as Trichinella,can cause trichinellosis.Recent studies have found that Trichinella can inhibit tumor growth.In vivo and in vitro experiments have confirmed that the active protein of Trichinella spiralis can inhibit tumor cells.proliferation.The research of the research group showed that Excretory-secretory proteins(ESPs)from Trichinella spiralis muscle larvae can effectively inhibit tumor cell proliferation.Transcriptome is a collection of all transcripts produced by a species or a specific cell type.It can study gene function and structure at an overall level,and reveal molecular mechanisms in specific biological and disease processes.It has been widely used in clinical diagnosis,basic research,and drug development.There are no reports about the gene expression levels and signal pathways of Trichinella spiralis muscle larva ESPs in A549 cells.RNA-seq to investigate the effects of Trichinella spiralis muscle larva ESPs on gene expression and signal pathways of human non-small cell lung cancer A549 cells,and provide experimental evidence for the development of anti-tumor drugs.In addition,the research team used mass spectrometry to screen out six antitumor components in Excretory-secretory proteins(ESPs)of Trichinella spiralis muscle larvae.This experiment screened one of them,namely the splicing polyadenylation specific factor subunit 2(Cleavage and Polyadenylation Specificity Factor subunit 2,CPSF2).The CPSF2 recombinant protein and plasmid were constructed.It was observed whether the CPSF2 recombinant protein and eukaryotic expression plasmid could induce the apoptosis of A549 cells and the possible mechanism.Part ? Transcriptomics study on the effects of ESPs on A549 cell of Trichinella spiralis muscle larva.Purpose:Transcriptome sequencing technology was used to investigate the changes of gene expression and signaling pathway in A549 cells treated by Trichinella spiralis larva ESPs..Methods:1 Construction of Library: according to the instructions of NEB next poly(A)m RNA magnetic isolation module kit,enrich the m RNA of each group of samples.The treated product RNA was constructed by KAPA strutted RNA SEQ library prep Kit(Illumina).2 Sequencing: sequence with Illumina novaseq 6000 sequencer.3 Quantitative analysis of transcriptional expression,screening of differentially expressed genes,enrichment analysis of go function significance,and enrichment analysis of pathway significance were performed on the sequence data of RNA-Seq transcriptome from 6 samples of experimental group and control group.Results:1 Three samples in the control group and three samples in the experimental group were 31475828,28699647,33661971,33000876,25627123 and 27549020 read pairs.2 After differential analysis,2860 differentially expressed genes were obtained,including 1634 up-regulated genes and 1226 down-regulated genes.3 Among the up-regulated genes,the three most abundant genes are the development process,protein binding and cell component classification.Among the down regulated genes,the three most abundant genes are metabolic process in biological process classification,amide binding in molecular function classification and intracellular in cell component classification.4 2860 differentially expressed genes participated in 74 signaling pathways,71 of which were significantly enriched(P<0.05).The most up-regulated gene enrichment difference pathway is cancer pathway.Metabolic pathway is the most important pathway for down regulated gene enrichment.Conclusion:1 Trichinella spiralis ESPs affected gene expression and signaling pathway in A549 cells.After differential analysis,2860 differentially expressed genes were obtained,including 1634 up-regulated genes and 1226 down-regulated genes.2 Differentially expressed genes cover three aspects: cell components,molecular functions and biological processes.It was mainly concentrated in 71 signaling pathways.Part ? Construction of CPSF2 eukaryotic expression plasmid and its effect on apoptosis and cycle of A549 cellPurpose:By constructing CPSF2 prokaryotic and eukaryotic expression vectors,to study the effects of recombinant proteins and eukaryotic expression plasmid on the proliferation and apoptosis of non-small cell lung cancer A549 cells,and to explore the possible mechanism of CPSF2 recombinant proteins and eukaryotic expression plasmid inducing apoptosis of non-small cell lung cancer A549 cells.Methods:1 Annexin V-FITC/PI double staining detection rate of A549 cells apopt osis: the experimental group 2?g,4?g plasmid transfection respectively after A549 cells develop 24 h,the empty plasmid group to join 100?L serum free me dium and 10?l transfection reagent,transfection cultivate 24 h after A549 cells,negative control group without any treatment,after collecting cells with PI dy eing and dye FITC,cells through the detection of apoptosis of A549 cells.2 PI staining method to detect A549 cell cycle arrest: experimental group2?g,4?g plasmid transfection respectively after A549 cells develop 24 h,the empty plasmid group to join 100?L serum free medium and 10?L transfection reagent,transfection cultivate 24 h after A549 cells,negative control group without any treatment,with PI dyeing liquid after collecting cells,cells through the detection of A549 cell cycle arrest.3 Real-time PCR method to detect apoptosis of A549 cells related factor mRNA transcription level: the experimental group 2?g,4?g plasmid transfection respectively after A549 cells develop 24 h,the empty plasmid group to join100 ?L serum free medium and 10?L transfection reagent,transfection 24 h after A549 cells in culture,nucleic acid detection Bip,Atf4,Chop,Perk,Elf2?,Bax,Caspase-3,Caspase-9 gene transcription level.4Statistical analysis: SPSS19.0 statistical software was used to analyze the data.One-way anova was used to process the data.P<0.05 means the difference is statistically significant.Results:1 FITC Annexin V/PI double staining detection A549 cell apoptosis rate:the control group,the empty plasmid group,low concentration of plasmid transfection group,the high concentration of plasmid transfection group total apoptosis rate were 35.89%,34.15%,38.76%,49.8%,and the empty plasmid group total apoptosis rate,compared with low concentration of plasmid transfection group,the high concentration of plasmid transfection group total apoptosis rate is higher than the empty plasmid group and the gradually increasing trend,and give priority to with the late apoptosis,statistically significant difference(P<0.05).Control group,the empty plasmid group,low concentration of plasmid transfection group,the high concentration of plasmid transfection group of late apoptosis rate were 29.46%,29.46%,34.15% and 43.81% respectively,compared with the empty plasmid group late apoptosis rate,low concentration of plasmid transfection group,the high concentration of plasmid transfection group of late apoptosis rate is higher than the empty plasmid group and the gradually increasing trend,statistically significant difference(P<0.05)A549 cell cycle arrest was detected by PI staining: the proportion of G0/G1 cells in the control group was(49.6±0.87)%.The empty plasmid group was(48.64±0.13)%.The transfection group was(42.0±0.35).The transfection group was(53.34±1.02)%.Compared with the empty plasmid group,the proportion of G0/G1 phase cells in the transfection group with high concentration of plasmid increased(P<0.05),indicating that the cells in the transfection group with high concent ration of plasmid were blocked in G0/G1 phase.The proportion of cells in Sphase was(35.2±6.5)% in the control group.In the empty plasmid group(41.88±0.82)%,and in the low-concentration plasmid transfection group(48.70±2.79)%.The transfection group was(36.11±6.48)%.Compared with the empty plasmid group,the proportion of S stage in the low concentration plasmid transfection group was higher(P<0.05),indicating that the cells in the low concentration plasmid transfection group were blocked in S stage.Conclusion:1 CPSF2 eukaryotic expression plasmid transfected into human non-small cell lung cancer(NSCLC)A549 cell line can induce apoptosis of A549 cells,mainly at late stage.It may work by upregulating Bip,Perk,and Elf2?.2 CPSF2 eukaryotic expression plasmid was transfected into human non-small cell lung cancer cell line A549.The low concentration plasmid transfection group and the high concentration plasmid transfection group blocked A549 cells in phase S and phase G0/G1,respectively. |
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