Effect Of TPPP3 On Apoptosis And Cell Cycle Of Nasopharyngeal Carcinoma And Its Mechanism

Objective:In our previous study,we found that TPPP3 mRNA and protein expression level in nasopharyngeal carcinoma(NPC)were significantly lower than that in normal nasopharyngeal epithelial tissue,and overexpression of TPPP3 in NPC cell line could significantly inhibit the proliferation and invasion of NPC cells.Based on the previous study,we further studied the effect of TPPP3 on apoptosis and cell cycle of NPC cells,and applied it Bioinformatics analysis and high-throughput transcriptome sequencing were used to explore the possible mechanism of its in NPC.Methods:1.The apoptosis of TPPP3 Overexpressed NPC 5-8F cells NPC 5-8F cells(5-8F-TPPP3),control empty cells(5-8F-NC)and 5-8F cells(which did not transfected by lentivirus)were detected by Annexin V-APC/7-AAD.2.The cell cycle of Each experimental NPC cell group were detected by propidiumiodide(PI)staining.3.Based on GSE12452,a data set of the Gene Expression Omnibus(GEO),the expression profile data of 31 NPC tissue samples were grouped by the mean value of TPPP3 expression in the samples,and the Gene set enrichment analysis(GSEA)was carried out to analyze to study the biological processes involved in the expression of TPPP3 gene in NPC and the possible related pathways with the molecular functions and biological processes of Gene Ontology(GO)and Kyoto Encyclopedia of genes and genes(KEGG).4.Immunocell AI was used to predict the infiltration of immune cells of NPC tissues in the four data sets of GSE68799,GSE64634,GSE102349 and GSE13597 of GEO database,the expression of TPPP3 in each sample was extracted,and the correlation between the expression of TPPP3 and the infiltration of each immune cell were analyzed.5.The total RNA of5-8F-TPPP3 and 5-8f-NC cells was extracted and detected by high-throughput transcriptome sequencing(RNA-seq).The mRNA expression differences of5-8F-TPPP3 cells compared with 5-8F-NC cells were analyzed.The genes and transcripts of the differences were analyzed.The function and pathway enrichment were analyzed by online gene enrichment tool G:profiler,difference between 5-8F-TPPP3 and 5-8F-NC cell alternative splicing(AS)were also analyzed.Results:1.The apoptosis rate of 5-8F-TPPP3 group was 15.597±1.107,5-8F-NC group was 6.903±1.0130,5-8F group was 6.553±1.166,5-8F-TPPP3group was significantly higher than 5-8F-NC group and 5-8F group(P<0.05).2.The cell cycle was detected by flow cytometry.The results showed that the cell ratios of(G2+M)phase of the three groups of 5-8F,5-8F-NC,and 5-8F-TPPP3were(12.610±1.992)%,(13.353±1.472)%,(18.923±3.158)%,the5-8F-TPPP3 group was significantly higher than the 5-8F-NC group,the difference was statistically significant(p<0.05),and the(G0+G1)phase of the three groups of cells There was no significant difference in the proportion of cells in S phase(p>0.05).3.According to results of GSEA,compared with TPPP3 low expression group,TPPP3 high expression NPC sample group enriched to 19 up-regulated KEGG pathways and 11 down regulated KEGG pathways.up-regulated:Antigen processing and presentation,Complement and coagulation cascade,Calcium signaling pathway,Autoimmune thyroid disease,B cell receptor signaling pathway,Leishmania infection,Viral myocarditis,Hematopoietic cell line,Graft-versus-host disease,Cell adhesion molecule,Leukocyte migration across endothelial cells,Intestinal immune network,Asthma,Systemic lupus erythematosus,Type I diabetes,Allograft rejection,Primary bile acid biosynthesis,Chemokine signaling pathway,Dilated cardiomyopathy.Down regulated:Glycosylphosphatidylinositol biosynthesis,Mismatch repair,Homologous recombination,Cell cycle,Splicing,Selenite Metabolism,Galactose metabolism,DNA replication,Glyoxylate and dicarboxylic acid metabolism,Pentose phosphate pathway,Amino sugar and nucleotide sugar metabolism.There was no enrichment in GO molecular function and GO biological process.4.Analysis of the correlation between TPPP3 expression and immune cell infiltration showed that the expression of TPPP3 in GSE68799 was significantly positively correlated with CD4~+T cells,Tr1 cells,B cells,natural killer(NK)cells,naive CD4~+T cells,and macrophages,Monocytes and neutrophils were significantly negatively correlated.In GSE64634,TPPP3 was significantly positively correlated with CD4~+T cell,and was significantly negatively correlated with neutrophils,Th2cells,and natural regulatory T cells(n Treg).In GSE102349,TPPP3 is significantly positively correlated with naive CD4~+T cells,central memory T cells,CD4~+T cell,Tr1,B cells,follicular helper T cells(Tfh),Th17 cells,NK cells,and adaptive regulatory T cells(i Treg),γδT cells,depleted T cells,and macrophages were significantly negatively correlated.In GSE13597,the expression level of TPPP3 was significantly positively correlated with Tfh cells,CD8~+T cell cells,NK cells,Tr1 cells,naive CD4~+T cells,CD4+T cell,B cells,but was associated with macrophages,monocytes,neutrophils,Natural killer T(NKT)cells were significantly negatively correlated.5.29 differentially expressed genes were screened,including 7 up-regulated genes and 22down-regulated genes.420 transcripts with significant differences were obtained,including 204 up-regulated transcripts and 216 down-regulated transcripts.The analysis of pathways and functional enrichment of 29 genes were mainly enriched in three biological processes of GO:Cytokine mediated signaling pathways,Lack of There are the negative regulation of signal transduction in ligand and the negative regulation of apoptotic signal pathway in absence of ligand,but there is no enrichment in go molecular function and KEGG pathway.6.The analysis of AS on the sequencing data showed that there was no significant difference in the number of overall AS between the 5-8F-TPPP3group and the 5-8F-NC cells.Significant differences were found in Alternative exon ends(Approximate)of RAP1B,Skipped exon and Skipped exon(Approximate)of IL24,Skipped exon of IL24,Transcription start site and Transcription terminal site of LCP1,Alternative exon ends of CDK3(p<0.05).Analysis of the differences in the transcripts of the four genes RAP1B,IL24,LCP1,CDK3 showed that RAP1B-207,IL24-206,LCP1-202 were significantly down-regulated and LCP1-201 was significantly up-regulated(p<0.05),but there was no significant difference in the expression levels of CDK3transcripts(p>0.05).Conclusions:1.TPPP3 overexpression in NPC cell line 5-8F may promote cell apoptosis by down regulating the expression of UNC5B and IL1A.2.TPPP3overexpression can inhibit cell proliferation by blocking the cell in G2/M phase,which may be caused by TPPP3 over polymerization of microtubules,or by up regulating ITM2A.3.Based on the expression profile data of NPC in GEO database and the bioinformatics analysis of our team’s RNA-seq,TPPP3 may be involved in the regulation of immune response in NPC;4.After TPPP3overexpression,AS of RAP1B,IL24,LCP1 and CDK3 changed,and the transcriptional expression of RAP1B,IL24 and LCP1 changed significantly.TPPP3 may regulate the expression of these three genes in NPC by regulating AS of RAP1B,IL24 and LCP1.