| Background:Hepatocellular carcinoma(HCC)is a kind of primary malignant liver tumor that displays a high incidence rate and poor therapeutic effect.With the emergence of targeted therapeutic drugs(like sorafenib and lenvastinib)and immune checkpoint inhibitors,the prognosis of HCC has been improved,but the 5-year survival rate remains low.Competitive endogenous RNA(ceRNA)has revealed a new mechanism of RNA interaction,that is,non-coding RNAs(ncRNAs),such as long non-coding RNA(lncRNA),circular RNAs(circRNA),ect.can regulate mRNA by competitively binding microRNA(miRNA).This regulatory mechanism plays an important role in cancer pathogenesis.However,the competitive mechanism of ceRNA in HCC remains unclear so far,and more efforts are needed to explore the new regulatory axis of ceRNA.LncRNA CYTOR,a non-coding gene,has been proved to play a role in promoting cell proliferation and migration in various tumors;in addition,it can also be used as an endogenous sponge for a variety of miRNAs.However,its role as a ceRNA in the occurrence and development of HCC,together with the underlying regulatory network,is still unclear.Therefore,this study focused on lncRNA CYTOR to explore the application of ceRNA regulatory mechanism in HCC.Materials and methods:The Cancer Genome Atlas(TCGA)database was searched to obtain the expression data and clinical information of lncRNAs,mRNAs and miRNAs in human HCC.At the same time,the HCC-related GSE62232 and GSE84402 RNA datasets were downloaded from the Gene Expression Omnibus(GEO)database for subsequent verification.Afterwards,we analyzed the differential expression of IncRNA CYTOR between HCC tissues and adjacent normal tissues obtained from TCGA database,and its expression in various tumor types as well as other HCC-related datasets based on the online database.In addition,the relationships between IncRNA CYTOR expression and the overall survival(OS)of HCC patients from TCGA database were analyzed.Additionally,the correlations of IncRNA CYTOR with the common clinicopathological parameters,such as gender,age,tumor grade,clinical stage and TNM stage,were also explored.Furthermore,these parameters were incorporated into univariate and multivariate Cox regression analyses to explore whether lncRNA CYTOR was able to independently predict patient prognosis.After that,the differentially expressed miRNAs in HCC were identified based on TCGA database,and the target miRNAs of lncRNA CYTOR were predicted by the ENCORI software(starbase 3.0).Later,the down-regulated miRNAs in differential analysis were selected to intersect with the predicted miRNAs,so as to obtain the candidate miRNAs.In the meantime,differentially expressed mRNAs in HCC were also analyzed by TCGA database.Six databases(including PITA,miRNAmap,DIANA-microT,miRanda,PicTar and TargetScan)were utilized to predict the candidate miRNAs-targeting mRNAs.The up-regulated mRNAs in differential analysis were selected to intersect with the predicted mRNAs.Moreover,gene set enrichment analysis(GSEA)was conducted to identify the gene sets and pathways associated with the highly expressed mRNAs selected,and to explore their potential functions.Results:The expression of lncRNA CYTOR was up-regulated in a variety of cancer tissues(including colon adenocarcinoma,kidney renal papillary cell carcinoma,bladder urothelial carcinoma)and several HCC datasets(such as GSE54236,GSE64041,GSE36376).In TCGA database,the expression of lncRNA CYTOR in HCC tissues was significantly higher than that in adjacent normal tissues(P<0.001),and the OS of patients with high lncRNA CYTOR expression was significantly shorter than that in low expression patients(P<0.001).According to correlation analysis of clinicopathological parameters,the expression of lncRNA CYTOR was significantly correlated with grade,and there were significant differences between G2 and G1(P<0.01),G3 and G2(P<0.05),G3 and G1(P<0.001),as well as G4 and G1(P<0.05).Besides,the expression of lncRNA CYTOR at clinical stage ? was significantly up-regulated compared with that at clinical stage I(P<0.001),and that at T2 and T4 was significantly higher than that at T1(P<0.001;P<0.05).Independent prognostic analysis indicated that lncRNA CYTOR was able to predict the prognosis of HCC patients independent of the above clinical parameters(P<0.05).ENCORI software analysis revealed that miR-125b-5p might serve as the target miRNA of lncRNA CYTOR.Further,the integrated analysis of the following six databases showed that KIAA1522 and PODXL might be used as the candidate target mRNAs of miR-125b-5p.After differential,survival and correlation analyses with miR-125b-5p,KIAA1522 was identified as the target mRNA.Compared with the adjacent tissues,KIAA1522 expression significantly increased in HCC tissues of GSE62232 and GSE84402 datasets(P<0.001).Additionally,GSEA suggested that gene sets related to the cell cycle signaling pathway were significantly enriched in the phenotype with high KIAA1522 expression.Conclusion:It is an effective way to explore the expression and biological function of lncRNA and construct a novel lncRNA/miRNA/mRNA regulatory axis,so as to investigate the pathogenesis of HCC.The expression of lncRNA CYTOR in HCC is significantly upregulated,which is related to a variety of clinicopathological parameters,and it can be used as a potential prognostic biomarker for HCC patients.In addition,lncRNA CYTOR can regulate KIAA1522 by targeting miR-125b-5p,thus participating in the occurrence and development of HCC.Background:Hepatocellular carcinoma(HCC),an inflammation-related cancer induced by a variety of etiological factors,is still one of the most prevalent and lethal cancers in human beings.Therefore,it is urgently needed to find the novel and clinically effective biomarkers for the diagnosis and prognosis of HCC.This study aimed to explore the role of lncRNA CYTOR/miR-125b-5p/KIAA1522 in regulating the development of HCC.Methods:After transfection into normal hepatocytes(HHL-5)and several HCC cell lines(SK-Hep-1,Hep3B and Huh-7),the expression of lncRNA CYTOR,miR-125b-5p and KIAA1522 in HCC cells was detected by real time quantitative polymerase chain reaction(RT-qPCR).Afterwards,the expression levels of KIAA1522 in HCC and adjacent normal tissues were detected by immunohistochemistry(IHC).Dual-luciferase reporter assay was conducted to confirm the binding relationship among lncRNA CYTOR,miR-125b-5p and KIAA1522.Thereafter,cell counting kit-8(CCK-8)was adopted to detect the proliferation of HCC cells under different transfection conditions.Changes in the cell cycle of HCC cells after transfection were analyzed by flow cytometry.In addition,terminal deoxynucleotidyl transferase mediated nick end labeling(TUNEL)assay was performed to observe the apoptosis of HCC cells after transfection.Meanwhile,the expression levels of several proteins related to cell proliferation,cell cycle and apoptosis were detected by western blotting(WB).Results:Compared with HHL-5 cells,the above genes were most significantly expressed in Hep3B cells,so Hep3B cells were selected for subsequent experiments.The results of dual-luciferase reporter gene assay showed that the luciferase activity of Hep3B cells significantly decreased in miR-125b-5p mimic+CYTOR wt co-transfection group and miR-125b-5p mimic+KIAA1522 wt co-transfection group.Moreover,lncRNA CYTOR directly acted on miR-125b-5p,while miR-125b-5p directly targeted KIAA1522.At the same time,results from CCK-8 and WB assays indicated that knockdown of lncRNA CYTOR inhibited the proliferation of HCC cells and down-regulated the protein expression of Ki-67 and PCNA;while inhibiting the over-expression of miR-125b-5p or KIAA1522 had opposite effects.Flow cytometric analysis combined with WB assay revealed that knockdown of lncRNA CYTOR decreased the proportion of cells at S phase and G2/M phase,down-regulated the expression of cyclin D1 and cyclin E,and up-regulated that of P21;while inhibiting the over-expression of miR-125b-5p or KIAA1522 had opposite effects.As revealed by TUNEL and WB analysis results,knockdown of lncRNA CYTOR promoted the apoptosis of Hep3B cells,down-regulated the expression of Bcl-2,and up-regulated that of Bax,caspase 3,caspase 9,cleaved caspase 3 and cleaved caspase 9;while inhibiting the expression of miR-125b-5p or overexpression of KIAA1522 had opposite effects.Conclusion:In conclusion,lncRNA CYTOR up-regulates the expression of KIAA1522 by targeting miR-125b-5p,promotes the proliferation of HCC cells,affects cell cycle and inhibits apoptosis.This study sheds new lights on the pathogenesis of HCC. |
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