The Expression Level,Functional,and Molecular Mechanism Research Of A Liver-specific Long Non-coding RNA FAM99B In Hepatocellular Carcinoma

ObjectiveAn increasing amount of research have shown that liver-specific long non-coding RNAs(lncRNAs)play an important role in the occurrence and development of hepatocellular carcinoma(HCC).Our study aims to investigate the expression level,clinical significance,and its biological functions of a liver-specific lncRNA FAM99B in HCC.At the same time,bioinformatics analyses were applied to predict the underlying molecular mechanism of FAM99B in HCC.Methods1.The Cancer Genome Atlas(TCGA)database:(1)The RNA sequencing(RNA-seq)data of 371 HCC tissues and 50 adjacent tissues were downloaded from the TCGA database.The edge R package of R language was used to screen out the differentially expressed lncRNAs between carcinoma tissues and paracancerous tissues.(2)Online survival analysis was performed using the GEPIA website to identify those liver-specific lncRNAs were associated with the prognosis of HCC patients.(3)The receiver operating characteristic(ROC)curve was performed to assess the efficiency of FAM99B in the diagnosis of HCC tissues.The Chi-square(c~2)test was conducted to assess the relationship between the expression level of FAM99B and clinical characteristics in HCC patients,and the Kaplan-Meier plot was utilized for survival analysis.2.Gene Expression Omnibus(GEO)database:The microarray data sets of lncRNAs in HCC tissues and paracancerous tissues were retrieved from the GEO database,and then the expression level of FAM99B was extracted from the included GEO datasets.To mediate the small sample effect of individual data sets,a meta-analysis was performed to reach a comprehensive and reliable conclusion using the STATA 11.0 software.A forest plot was conducted to obtain the standardized mean difference(SMD)with its 95%confidence interval(CI).Thec~2 test and I~2 tests were implemented to evaluate the heterogeneity among the included GEO datasets.Furthermore,a funnel plot and Begg’s test were utilized to assess the publication bias.3.Validation of the FAM99B expression level in tissue samples:Biologicalsamples of HCC tissues and their corresponding peritumoral normal tissues were collected from 80 HCC patients who underwent hepatectomy in the Affiliated Cancer Hospital of Guangxi Medical University.The relative expression levels of FAM99B in the tissues were detected using the real-time quantitative polymerase chain reaction(qRT-PCR)method.Thec~2 test was used to explore the association between FAM99B expression level and clinicopathological characteristics in HCC patients.4.Lentivirus transfection and cell functional experiments:The lentiviral vectors containing the full-length sequence of human FAM99B(Lv-FAM99B)and the empty control(Lv-NC)were constructed and transfected into HCCLM3cells.After successful transfection,complete culture medium containing puromycin was added to screen out the stable transfection cell lines.The proliferation capacity of the HCCLM3 cells was evaluated using the clone formation assay and CCK-8 assay,while the Transwell chambers were used to measure the migration and invasion abilities of the HCCLM3 cells.5.Pathway enrichment analyses and protein-protein interaction(PPI)network construction:Three online websites,c Bio Portal,MEM,and TANRIC,were used to predicting the co-expressed genes of FAM99B.Afterwards,the KOBAS 3.0database was implemented to perform Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses.A PPI network was accomplished by utilizing the STRING database to reveal the inter-association among the co-expressed genes of FAM99B.Results1.TCGA database:(1)A total of 697 differentially expressed lncRNAs were identified by the differential analysis using the edge R package,of which 541lncRNAs were up-regulated and 155 lncRNAs were down-regulated.Among the down-regulated lncRNAs,six liver-specific lncRNAs were found.Online survival analysis by the GEPIA website found that only LINC01093,FAM99B and LINC02499 were significantly associated with the prognosis of HCC patients.(2)The expression level of FAM99B in HCC tissues was lower than that in para-carcinoma tissues(P<0.001),with an area of 0.828(P<0.0001)under the ROC curve(AUC).In addition,the expression level of FAM99B was also associated with vascular invasion(P=0.001),histological grade(P=0.001),and T stage(P=0.045).Kaplan-Meier survival analysis showed that lower FAM99B expression was significantly correlated with poor overall survival(OS)(P=0.034)and development-free survival(DFS)(P=0.031)in HCC patients.2.GEO database:A total of 18 eligible GEO datasets were enrolled in our study.As there was significant heterogeneity among these eighteen trials(I~2=58.0%;P=0.001),so the random-effect model was chosen for the meta-analysis.The combined result indicated that FAM99B displays a lower expression level in HCC tissues compared with normal tissues(SMD=-1.394;P<0.001).Meanwhile,no obvious publication bias was observed in the meta-analysis(P=0.449).3.Validation of our HCC tissue samples:The results of qRT-PCR demonstrated that compared with adjacent normal tissues,the FAM99B expression level was decreased in HCC tissues(P<0.001).The AUC of ROC curve was 0.707(P<0.0001).Similarly,the expression of FAM99B was remarkably related to microvascular invasion(P=0.014)in patients with HCC.4.Effects of cell biological behavior:The results of qRT-PCR showed that FAM99B displayed a lower expression in cells with metastatic potential(HCCLM3,MHCC97L,MHCC97H)than those without or with little metastasis potential(Huh-7,Hep G2,Hep3B).The lentivirus transfection results exhibited a significantly higher FAM99B expression in the Lv-FAM99B group than that in the Lv-NC group(P<0.001).In vitro cell experiments showed that overexpression of FAM99B could notably reduce the number of clones of HCCLM3 cells(P=0.006)and inhibit the proliferation viability(P<0.001),migration ability(P<0.001),as well as invasion capacity(P<0.001)of HCCLM3 cells.5.Pathway enrichment analyses and the recognition of core genes:Co-expressed genes of FAM99B were mainly enriched in the pathways of“Metabolic pathways”,“Fatty acid elongation”,“Complement and coagulation cascades”and so on.At the same time,PPI network identified six core genes(F2,SRC,HNF4A,F9,MPP3,and SPP1)that might interact with FAM99B in HCC.ConclusionFAM99B is down-regulated in HCC tissues and demonstrated a high performance in the diagnoses of HCC tissues.Besides,its decreased expression level is significantly correlated with the malignant phenotype and poor prognosis of HCC patients.In addition,FAM99B could suppress the proliferation,migration,and invasion ability of HCC cells in vitro.The results of bioinformatics analyses suggest that FAM99B may play an important role in inhibiting tumor formation and development of HCC by regulating the metabolism and coagulation cascades pathways.These results may contribute to our understanding of the molecular mechanisms underlying the development of HCC and may contribute to the early diagnosis and prognosis prediction of HCC patients.