| PurposeWith its high disease burden and high mortality rate,gastric cancer remains a huge global health challenge.Tumour metastasis is a major cause of increased mortality from gastric cancer.Therefore,clarifying the molecular mechanisms of gastric cancer invasion and metastasis is an urgent need to guide individualised treatment.Preliminary study: human tissue microarrays screened for differential genes in gastric and paraneoplastic tissues.Tropomyosin 4(TPM4)is an actin-binding protein that provides structural stability of actin filaments and regulates the cytoskeleton.the literature reports that TPM4 is aberrantly expressed in multiple tumours and exhibits different biological behaviours.t PM4 acts as an oncogene in gastric cancer and can Based on the above background,the scientific question: Explore the role of TPM4 as a potential molecular target in pan-cancer,including gastric cancer? Explore the mechanism of how TPM4 regulates gastric cancer invasion and metastasis through targeting proteins?In the first part of this thesis,the role of TPM4 in gastric cancer is explored in a comprehensive pan-cancer analysis.In the second part,the role of TPM4 in gastric cancer invasion and metastasis was verified by in vivo and ex vivo experiments;the correlation between the expression of TPM4 and the clinicopathological characteristics of gastric cancer was analysed;the target proteins(related complexes)and regulatory mechanisms of TPM4 affecting gastric cancer cell invasion and metastasis were clarified.This paper enriches the scientific connotation of gastric cancer invasion and metastasis,and provides a theoretical basis for expanding the diagnosis and treatment of metastatic gastric cancer.Methods1.Comprehensive pan-cancer analysis of TPM4 in gastric cancer.The expression,diagnosis,prognostic value,epigenetic alterations,and immune cell infiltration of TPM4 in pan-cancer were analyzed by bioinformatics methods on databases such as The Cancer Genome Atlas(TCGA),Genotype Tissue Expression Project(GTEX),and Immunoassay Tool(EPIC)in a comprehensive manner.The regulatory network of lnc RNAs-mi RNAs-TPM4 in gastric cancer was constructed by databases such as RNA22,mi RWalk,mi RDB and Cytoscape software.The genomic data analysis platform(GSCALite)and the connectivity map(c Map)database were used to analyze the sensitivity of TPM4 expression to antitumor drugs.2.Mechanisms by which TPM4 regulates invasion and metastasis of gastric cancer cells2.1 Effect of TPM4 on invasion and metastasis of gastric cancer cells.RT-PCR and Western-Blot were used to detect the m RNA levels and protein levels of TPM4 in several gastric cancer cells.Lentiviral technology was used to construct gastric cancer cell lines with stable knockdown of TPM4,and the migration and invasion ability of TPM4 on gastric cancer cells was detected by cell scratch assay,Transwell and Migration assay.A model of intraperitoneal metastasis of human gastric cancer cells in nude mice was constructed to observe the metastatic ability of TPM4 gene on nude mice.The correlation between TPM4 expression and the clinical and pathological features of gastric cancer was analysed in clinical specimens and databases.2.2 Proteomic identification and validation of TPM4 target proteins affecting gastric cancer invasion and metastasis.Tandem Mass Tag(TMT)was used to identify differential proteins in gastric cancer cell lines with/without knockdown of the TPM4 gene that promote gastric cancer invasion and metastasis.Parallel Reaction Monitoring(PRM)(Parallel Reaction Monitoring(PRM)technology was used to verify whether the differential protein identified by TMT was the target protein of TPM4 for gastric cancer invasion and metastasis.2.3 TPM4 promotes invasion and metastasis of gastric cancer cells via Arp2/3complex/F-actin.The correlation of TPM4 with Arp2/3 complex and actin nucleating factor was detected by Western-Blot.Immunofluorescence assay was performed to detect the effect of TPM4 on actin(F-actin).Results1.TPM4 has high diagnostic and prognostic value in digestive system tumours.Correlations with DNA methylation,m RNA modification,immune infiltration,tumour mutational load,microsatellite instability,neoantigens,and immune checkpoint genes existed in TPM4 expression in pan-cancer.The Lnc RNA-mi RNA-TPM4 regulatory network exists in gastric cancer,and TPM4 is a potential therapeutic target for a variety of tumors,including gastric cancer,and is involved in chemotherapy and small molecule targeted drugs.2.1 Cell function assay shows that TPM4 promotes invasive migration of gastric cancer cells.The positive expression rate of TPM4 in gastric cancer(stage II-III)was31.50%(63/200).univariate analysis showed that TPM4 expression correlated with pathological staging,pathological stage,T stage,N stage,Her-2 and MMR expression of gastric cancer(P<0.05).multivariate regression analysis showed that the effect of TPM4 positive expression was Her-2(OR=4.609),MMR(OR=0.924),N stage(OR=0.581),pathological stage(OR=0.422),T stage(OR=0.298),and pathological staging(OR=0.291),in that order.The expression of TPM4 was elevated in lymphatic metastases and progressive gastric cancer compared to early gastric cancer(P < 0.05).2.2 Of the differential proteins screened by TMT in gastric cancer cells with/without knockdown of TPM4,232 were up-regulated and 129 were down-regulated.The differential proteins were subjected to GO and KEGG functional enrichment analysis suggesting that TPM4 promotes invasive metastasis of gastric cancer,which may be related to Arp2/3 complex-mediated actin polymerisation function.PRM targeting technique verified that TPM4 targets ARPC3 protein to regulate invasive metastasis of gastric cancer2.3 In gastric cancer cells,TPM4 was positively correlated with and polymerization factors(Arp2/3 complex,WASL,CDC42,PFN1).TPM4 may form a pseudopod through Arp2/3-mediated actin polymerization and thus promote invasion and metastasis in gastric cancer cellsConclusionBioinformatics identified TPM4 as a potential biomarker with diagnostic,prognostic and therapeutic value(immunotherapy,chemotherapy and small molecule-targeted drugs)in pan-cancer including gastric cancer.lnc RNA-mi RNA-TPM4 regulatory network is involved in the development of gastric cancer.In vitro and in vivo experiments showed that TPM4 promotes invasive metastasis of gastric cancer.The positive expression rate of TPM4 in stage II-III gastric cancer was 31.50%.The positive expression of TPM4 was associated with the pathological staging,pathological stage,T stage,N stage,Her-2 and MMR of gastric cancer,and the expression of TPM4 was elevated in metastatic lymph nodes and invasive gastric cancer.TPM4 may be involved in gastric cancer through Arp2/3 complex-mediated actin polymerization to form pseudopods.ARPC3 is its target protein. |
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