| Background and ObjectiveHepatocellular carcinoma(HCC)is a primary malignant tumor originating from parenchymal cells of the liver.Hepatitis B virus and hepatitis C virus are the major causes of HCC.HCC is the fifth most common tumor in the world,and the number of deaths each year ranks second among all malignant tumors.China has a high incidence of HCC,accounting for more than half of the world’s HCC cases and deaths every year.In recent years,with the continuous development of imaging and surgery technology,great progress has been made in the diagnosis and treatment of HCC,but there are still some problems,such as lack of effective diagnostic markers,high postoperative recurrence rate,low postoperative survival rate and so on.Therefore,screening the key molecular markers that play a role in the occurrence and development of HCC is of great significance to improve the level of diagnosis and treatment of HCC.Methods(1)Download the microarray dataset GSE55092 from the Gene Expression Omnibus(GEO).Bioinformatics software was used to analyze the data to find differentially expressed genes(DEGs).The differential genes were analyzed by the gene ontology(GO)enrichment analysis,kyoto encyclopedia of genes and genomes(KEGG)pathway analysis,ingenuity pathway analysis(IPA)and protein-protein interaction(PPI)network analysis.The key differential genes were identified by Centiscape2.2 in Cytoscape software(Cytoscape＿v3.7.2)and molecular complex detection(MCODE).And use gene expression profiling interactive analysis(GEPIA)database to verify the expression and clinical significance of the screened differential genes.(2)Our research group selected 5 samples of tumor tissues,paracancerous tissues and distant normal tissues of patients with primary liver cancer,and analyzed 719 differentially expressed long non-coding RNAs(lncRNAs)using Affymetrix m RNA-lncRNA gene chip.Six lncRNAs were selected according to the selection criteria of the intergenic region type lncRNA and the calibration p-value sorting,including ENST00000514608.1,NONHSAT138156,NONHSAT024276,NONHSAT124357.2,NONHSAT053785 and cancer susceptibility candidate 7(CASC7).The serum samples of 50 patients with confirmed HCC,50 patients with chronic hepatitis B(CHB)and 50 normal controls were collected.Droplet Digital PCR(dd PCR)was used to verify the expression of these six lncRNA.By comparing the clinical parameters of these lncRNAs in the diagnosis of HCC,to identify the target molecules for further study.The sample size of the three groups was expanded to continue the study of the clinical value of the target molecule in HCC.Results(1)Based on the microarray data set GSE55092 of the GEO database,2264 m RNAs were differentially expressed,of which 764 were up-regulated and 1500 were down-regulated.GO analysis showed that these DEGs were related to small molecule metabolic process,xenobiotic metabolic process and cellular nitrogen compound metabolic process.KEGG pathway analysis showed that DEGs was related to metabolic pathways,complement and coagulation cascades.Disease and biological function analysis show that DEGs is mainly related to cancer,organismal injury and abnormalities,gastrointestinal disease,and hepatic system disease.The top 10 upstream regulators were predicted to be activated or inhibited by Z-score.The top 10 genes were identified as key genes.Cox regression analysis showed that the 10 genes(CDC20,BUB1 B,KIF11,TTK,EZH2,ZWINT,NDC80,TPX2,MELK,KIF20A)were associated with the overall survival rate of HCC patients.(2)Six lncRNAs screened by expression microarray based on fresh liver cancer tissues were verified,and four lncRNAs,CASC7,NONHSAT053785,NONHSAT024276 and NONHSAT138156,were differentially expressed,among which CASC7 had the highest accuracy in the diagnosis of HCC.Therefore,CASC7 is regarded as a target molecule for further research.dd PCR has been successfully established and optimized to detect the expression of CASC7 in serum of patients with HCC,CHB and normal controls.The expression of CASC7 in serum of patients with HCC was significantly higher than that of patients with CHB(median: 8.8 versus 2.2 copies/μl,p < 0.001)and healthy controls(median:8.8 versus 3.8 copies/μl,p < 0.001).The high expression of serum CASC7 was significantly correlated with tumor number(p = 0.005),intrahepatic metastasis(IM)(p < 0.001),tumor size(p = 0.007)and tumor-node-metastasis(TNM)stage(p = 0.008).The area under the receiver operating characteristics(ROC)curve of CASC7 to distinguish IM from non-IM was0.811(95% CI: 0.714-0.909).However,AFP could not distinguish between these clinicopathological parameters.Correlation analysis of CASC7 with other clinical indicators showed positive associated with carcino-embryonic antigen(CEA)(r = 0.322,p = 0.004),alkaline phosphatase(ALP)(r = 0.467,p < 0.001),gamma-glutamine transferase(γ-GT)(r =0.29,p = 0.009),lactate dehydrogenase(LDH)(r = 0.275,p = 0.014),White blood cell(WBC)(r = 0.296,p = 0.008),platelet(PLT)(r = 0.279,p = 0.012)and Monocyte(r = 0.295,p =0.008),and negative associated with cholinesterase(CHE)(r =-0.231,p = 0.039).The AUC of CASC7 to distinguish HCC patients from CHB patients and healthy controls was 0.808(95% CI: 0.742-0.874)at the cut-off value of 7.24 copies/μl with 63.8% sensitivity and95.2% specificity.Conclusion:Key candidate genes associated with HCC progression were identified by bioinformatics analysis,which is helpful in the search for biomarkers and therapeutic targets for HCC.At the same time,CASC7 was screened based on the expression microarray,and it was verified that CASC7 was significantly up-regulated in the serum of HCC patients,and was closely correlated with tumor number,IM,tumor size and TNM stage,which may be a promising diagnostic biomarker. |
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