| Objective: Gastric cancer is a malignant tumor with a high incidence and highly aggressive nature in China.In this study,we combined The Cancer Genome Atlas(TCGA)database and Gene Expression Omnibus(GEO)database to use transcriptomic data from a large sample of gastric cancer patients to screen for long-stranded non-coding RNA(lncRNA),microRNA(miRNA)and messenger RNA(mRNA)that are closely related to the degree of immune infiltration and Lauren typing.Long noncoding RNA(lncRNA),microRNA(miRNA)and messenger RNA(mRNA),and constructed a competing endogenous RNA(ceRNA)regulatory network associated with the genetic characteristics of the adjuvant therapy beneficiary population.The mRNA non-coding region/circular RNA(circRNA)/lncRNA-miRNA-mRNA regulatory network was constructed.Preliminary screening of key molecules and validation of their effects on gastric cancer cell function will lay the molecular biological foundation for finding novel diagnostic and prognostic targets for gastric cancer.Methods: In this study,the transcriptome information of gastric cancer patients in the combined TCGA-STAD and GSE62254 datasets was used to analyze the degree of immune cell infiltration in each patient using the CIBERSORT algorithm,and patients were classified for survival analysis by means of cluster analysis;the impact of different Lauren typing on patients’ sensitivity to adjuvant therapy was analyzed.Combined with Lauren typing,this study screened the differentially expressed lncRNA,miRNA and mRNA between different typing,and constructed ceRNA regulatory network accordingly.The functions and signaling pathways enriched by mRNAs in the ceRNA network were analyzed by GO database and KEGG,and key regulatory genes were extracted by PPI network analysis.The expression differences of key genes in cancer and paraneoplastic tissues were analyzed,and the mRNAs of downstream genes playing key roles in the ceRNA network were sequentially screened by combining the correlation between patient prognosis and the expression levels of immune checkpoint PD1/PD-L1.This study verified the expression of key genes by immunohistochemistry experiments,and detected the expression of core regulatory genes in gastric cancer cell lines using Western blot and q RT-PCR.The expression of core regulatory genes in gastric cancer cell lines was examined by Western blot and q RT-PCR.Combined with patient clinical data and prognostic relevance analysis,this study targeted the most relevant ESPL1 for in-depth study,explored its effect on the biological behavior of gastric cancer cells using CCK8,scratch and Transwell experiments,and predicted the relevant functional pathways by GSEA and validated them.Results: 1.Based on published data sets TCGA-STAD(n=375)and GSE62254(n=300),patients were classified into three categories according to the degree of immune cell infiltration,and there was a significant difference in prognosis among the three categories;patients were classified into two categories based on Lauren’s staging,in which patients with diffuse type had a significantly worse prognosis than those with intestinal type,and there was a significant difference in median survival between the two categories(36 months vs.74 months,P=0.015),and patients with diffuse gastric cancer had a significant improvement in survival with adjuvant therapy(P=0.004),while patients with intestinal type had no survival benefit(P=0.877).2.28 lncRNAs,23 microRNAs and 107 mRNAs with target binding relationship and closely related to the degree of immune infiltration and Lauren’s typing were analyzed.microRNA and 107 mRNAs,and based on this,the ceRNA regulatory network was constructed.Using GO analysis and KEGG analysis,we found that the mRNAs in this network were mainly enriched in PI3K-AKT,MAPK and HIF-1 signaling pathways;PPI protein interaction analysis obtained CCNA2,AURKA,TTK,ESPL1,CXCR4,COL1A2,CCR7,DEPDC1,IGF1 and VEGFA,among which CCNA2,AURKA,TTK,ESPL1,CXCR4,COL1A2,CCR7 and DEPDC1 were highly expressed in gastric cancer tissues and significantly correlated with the degree of immune cell infiltration.A literature search screened the ESPL1,TTK and DEPDC1 genes as the focus of this study,and correlation analysis revealed that the expression levels of the three were significantly and positively correlated with the expression level of PD-L1,while the expression level of ESPL1 was also positively correlated with PD-1.3.The homologous proteins encoded by the ESPL1,TTK and DEPDC1 genes were highly expressed in gastric cancer tissues with poor prognosis.The expression level of ESPL1 can be used as an independent risk factor to assess the prognosis of gastric cancer patients,and accordingly,ESPL1 gene was screened as a target gene,and interference with endogenous ESPL1 expression significantly downregulated the proliferation,migration and invasion ability of gastric cancer cells.4.The results of GSEA enrichment analysis showed that ESPL1 can be involved in regulating epithelial-mesenchymal transition(EMT)in gastric cancer.The expression of endogenous ESPL1 gene was significantly upregulated by E-cadherin,Zonula Occludens-1(ZO1),β-catenin,and Recombinant Claudin 1(CLDN1)in SGC-7901 and MGC-803 cells,down-regulated N-cadherin and Snail-related zinc-finger transcription factor(Slug)Conclusion:In the present study,we successfully constructed a ceRNA regulatory network associated with the degree of immune infiltration and Lauren typing in gastric cancer patients.Ten key genes,including CCNA2,AURKA,TTK,ESP1,CXCR4,COL1A2,CCR7,DEPDC1,IGF1 and VEGFA,were screened out from them,which had important effects on the degree of immune infiltration and Lauren typing in gastric cancer.Among them,high expression of TTK,ESPL1 and DEPDC1 genes was associated with poor patient prognosis,while the expression level of ESPL1 could be used as an independent risk factor to assess patient prognosis.Further experiments showed that the ESPL1 gene may affect the proliferation,migration and invasive ability of gastric cancer cells by regulating the EMT signaling pathway.These findings are expected to provide a new theoretical basis and therapeutic targets for the treatment and prognosis assessment of gastric cancer. |
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