| Objective:The diagnostic value,prognostic ability and biological function of hypoxia-induced LncRNA CTD-2510F5.4 in hepatocellular carcinoma(HCC)would be discussed in this study,which could provide a theoretical basis for targeted therapy of HCC,in the meanwhile,guide the individualized medication of HCC patients to achieve precise treatment.Methods:1.Bioinformatics methods:(1)The expression data of LncRNAs were mined from the Gene Expression Omnibus(GEO)dataset(GSE155505)and the Cancer Genome Atlas(TCGA)database,and the R "limma" package was used to analyze the differentially expressed LncRNAs in GSE155505 and TCGA datasets to identify the hypoxia-induced differentially expressed LncRNAs(HIDELs);(2)Identification of prognostic related HIDELs and establishment of a nomogram model for HCC patients:Univariate and multivariate COX regression analyses were performed to identify prognostic related HIDELs based on TCGA HCC survival information.Then,the clinicopathological characteristics such as gender,age,grade,TNM stage and HIDELs were analyzed by univariate and multivariate Cox regression analysis.Finally,a HCC nomogram model was established and used to visualize independent prognostic factors,and the calibration curve was used to evaluate the accuracy of the nomogram;(3)Prediction of biological function of LncRNA CTD-2510F5.4:protein-coding genes(PCGs)intersecting with CTD-2510F5.4 were identified by calculating Pearson correlation coefficient between each gene,and gene correlation was displayed by heat map,biological process(BP)and Kyoto Encyclopedia of Genes and Genomes(KEGG)Enrichment analysis was performed using the "cluster Profiler" package on R,PCGs were ranked based on their Pearson correlation with CTD-2510F5.4 expression level and Gene set enrichment analysis(GSEA)was done using the “cluster Profiler” based on h.all.v7.2.symbols.gmt [Hallmarks] gene sets on molecular signatures database(MSig DB);(4)Methods for analyzing the mutation characteristics,immune characteristics and treatment response prediction between different CTD-2510F5.4 groups :The R package "Maftools" was used to analyze the mutation quality between high and low-risk CTD-2510F5.4 groups.The level of immune cell infiltration in each HCC sample was estimated using a cell sorting tool(CIBERSORT).Immune-related functions were analyzed by "GSVA" and "GSEABase" software packages.The Tumor Immune Dysfunction and Exclusion(TIDE)score was calculated on the web platform(http:// tide.dfci.harvard.edu/)to predict immunotherapy response.The chemotherapy response of each sample was predicted using GDSC.The half-maximal inhibitory concentration(IC)was used to evaluate the response effect.2.Tests in vitro:(1)The overexpressed lentivirus vector pLVX-CTD-2510F5.4-001-IRES-GFP-puro was constructed,and the DNA-transfection reagent complex,helper plasmid mixture(PSPAX2 and PMD2G)and lentivirus expression plasmid were prepared and thoroughly mixed,then the cell lines were infected with mixture and amplified;(2)The effect of LncRNA CTD-2510F5.4 on HCC proliferation was investigated by CCK-8 cell proliferation assay using LV-CTD-2510F5.4,LV-control cells,respectively.The migration ability of HCC cells was detected by scratch assay,and invasion ability of HCC cells was determned by transwell chamber test.Results:(1)Hypoxia-induced differentially expressed LncRNAs were identified by bioinformatics methods,and further Cox univariate and multivariate analysis showed that CTD-2510F5.4 was an independent prognostic factor for OS in HCC patients;(2)The results of biological function prediction analysis showed that CTD-2510F5.4 was correlated with tumor signaling pathway,such as HALLMARK E2 F TARGETS,HALLMARK G2 M CHECKPOINT,HALLMARK MITOTIC SPINDLE,HALLMARK MYC TARGETS V1,HALLMARK PROTEIN SECRETION,suggesting that CTD-2510F5.4 may be involved in cell proliferation and tumor-related signaling pathways;(3)Gene mutation characteristics and immune function analysis suggested that CTD-2510F5.4 was closely related to the immunosuppressive state of tumor,and might predict the therapeutic response of commonly used drugs in HCC;(4)It showed that overexpression of CTD-2510F5.4could promote the proliferation,invasion and metastasis of HCC cells in vitro.Conclutions:1.Hypoxia-induced LncRNA CTD-2510F5.4 is abnormally expressed in HCC,and identified as an independent prognostic factor for OS in HCC patients and has a good predictive ability for HCC prognosis.2.LncRNA CTD-2510F5.4 is involved in cell proliferation and tumor-related signaling pathways.3.CTD-2510F5.4 is closely related to the immunosuppressive state of the tumor,and can predict therapeutic response of commonly used drugs in HCC,which can be used to guide the individualized medication of HCC patients.4.Overexpression of CTD-2510F5.4 can promote the proliferation,invasion,and metastasis of HCC cells. |
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