| BackgroundPrimary liver cancer is one of the most common malignant tumors worldwide,and its incidence and mortality rate are increasing year by year.Hepatocellular carcinoma(HCC)accounts for approximately 85%-90%of all cases of primary liver cancer.Insidious onset,high degree of malignancy and easy metastasis and recurrence contribute to gloomy outcome of HCC.Therefore,it is important to explore the molecular biological mechanisms for the occurrence and development of HCC,and identify new therapeutic targets and treatment strategies.Centromere protein U(CENPU)plays a key role in promoting mitotic progression and regulating kineto-microtubule attachment.Recently,CENPU has been found to play a pro-cancer role in a variety of cancers.However,the role of CENPU in HCC remains unclear.MicroRNA(miRNA)are a class of non-coding RNA molecules that regulate target gene expression at the post-transcriptional level via binding to the 3’-untranslated region(3’UTR)of target mRNA resulting in translational inhibition or mRNA degradation.Multiple studies have confirmed that miRNAs are involved in regulating various biological processes such as cell differentiation,proliferation and survival.Numerous studies have demonstrated that abnormal expression of miRNA is associated with the development and progression of various types of human cancer.In this study,we first analyzed the expression and prognostic value of CENPU in HCC by analyzing public databases.Subsequently,we examined the expression of CENPU in HCC tissues and cell lines using data from our center.Further,we systematically explored the effects of CENPU expression changes on the proliferation,migration,and invasion ability of HCC cells in vitro cell tests and in vivo animal experiments.Finally,we applied the miRNA online databases to predict that CENPU was a potential target of miR-127-5p.We validated that CENPU was the target gene of miR-127-5p by performing the dual luciferase reporter assay and other experimental approaches.In addition,the gene expression of miR-127-5p was examined in HCC and the effects of miR-127-5p were investigated on the biological properties of HCC,as well as how miR-127-5p regulates the expression and function of CENPU in HCC.Part Ⅰ Expressional and prognostic value of CENPU in hepatocellular carcinoma via bioinformatics analysisObjectiveTo investigate the expression and prognostic value of CENPU in HCC by bioinformatics analysis.Methods1.CENPU expression level and its relationship with the prognosis of digestive tract cancers were determined using the TCGA and GTEx databases.2.The expression of CENPU in HCC was analyzed using HCC expression data from TCGA and GEO databases.3.GEPIA and Kaplan-Meier plotter online databases were used to analyze the relationship between CENPU expression and survival prognosis of HCC patients.4.The association between CENPU expression and clinicopathological characteristics of HCC patients was analyzed by TCGA database.5.The top 100 genes related to CENPU expression were obtained using the Ualcan online database and GO and KEGG enrichment analysis were performed by DAVID website.Protein-protein interaction(PPI)network maps for the above 100 genes were generated through the STRING database.Results1.Analyses of TCGA and GTEx data demonstrated that LIHC,CHOL,ESC A,STAD,COAD,READ,and PAAD tissues had significantly higher levels of CENPU expression than normal tissues(P<0.001).Univariate Cox regression analysis indicated that CENPU was significantly correlated with prognosis of LIHC and PAAD patients(P<0.05),while CHOL,ESCA,STAD,COAD and READ patients were not associated with poor prognosis(P>0.05).2.TCGA data showed that CENPU expression was significantly higher in HCC tissues(n=374)than in normal tissues(n=50)(P<0.001).Three HCC datasets of GSE46408,GSE46408,and GSE84402 derived from GEO database revealed that the expression of CENPU in HCC tissues was significantly higher than that in normal tissues(P<0.01,P<0.001,P<0.001).3.GEPIA database analysis showed that the overall survival and disease-free survival of HCC patients in the high CENPU expression group were significantly shorter than those in the low expression group(P<0.05,P<0.001).Kaplan-Meier plotter database analysis revealed that HCC patients with high expression of CENPU had significantly decreased overall survival,disease-free survival,progression-free survival and recurrence-free survival compared to those with low expression(P<0.05,P<0.001,P<0.001,P<0.001).Based on the data from TCGA database,high expression of CENPU was associated with histological grade(P<0.01),TMN stage(P<0.01),and depth of tumor infiltration(P<0.001)in HCC patients,but not with age,gender,M stage,or N stage(P>0.05).Univariate Cox regression analysis suggested that TMN stage(P<0.001),depth of tumor infiltration(P<0.001),distant metastasis(P<0.05)and CENPU expression level(P<0.01)were influential factors for poor overall in HCC patients.Multifactorial Cox regression analysis showed that CENPU expression level(P<0.05)was an independent prognostic factor for overall survival in patients with HCC.4.The UALCAN database was used to select the top 100 co-expressed genes of CENPU.DAVID website was used to analyze the GO and KEGG enrichment analysis of these 100 genes.The results of the GO analysis revealed that these 100 genes were mainly enriched in the nucleus,cytoplasm,and condensed chromosomal mitoses at the cellular component class.Within the biological process category,the enriched genes were mainly associated with the cell differentiation,mitotic nuclear division,DNA repair and sister chromatid adhesion.In the molecular function,protein binding,ATP binding,NA binding,and microtubule binding were significantly enriched.KEGG enrichment analysis showed that seven pathways included cell cycle,DNA repair,oocyte meiosis,Fanconi anemia pathway,mismatch repair,luteinizing hormone-mediated oocyte maturation,and miRNA in cancer.The 100 genes were used to construct a PPI network based on analysis using the STRING online tool,in which the top 10 genes were ranked as TOP2A,BUB1,CCNB2,TTK,NCAPG,NDC80,CDCA8,EXO1,ASPM,and TPX2.Conclusions1.Bioinformatics analysis indicates that CENPU was highly expressed in HCC tissues and its high expression was associated with the prognosis and clinicopathological characteristics of patients with HCC.2.With GO,KEGG and PPI analyses,we can determine the main function distribution and interaction of genes and we constructed a regulatory network of CENPU-related genes in HCC and screened 10 core genes.Part Ⅱ Expression and functional role of CENPU in hepatocellular carcinomaObjective1.To explore CENPU expression level in HCC tissues samples and cell lines.2.To investigated the effects of CENPU on proliferation,migration,and invasion of HCC cellsMethods1.RT-qPCR,Western blot,Immunohistochemistry were used to detect the expression of CENPU mRNA and protein in 10 paired HCC tissues and corresponding paracancerous tissues.The mRNA and protein expression of CENPU was measured in five HCC cell lines(Huh7,HepG2,Bel7402,HCCLM3,and SMCC7721)and human normal hepatocyte line LO2 via RT-qPCR and western blot assays.2.CENPU overexpression plasmid was constructed using the GV658 vector and for RNAi construction,the vector GV493 was used.Transfection efficiency was checked by Western blot analysis and Immunohistochemistry.3.The CCK-8 assay and plate colony formation assays were utilized to detect the effect of CENPU on cell proliferation.The effects of CENPU on migration and invasive ability of HCC cells were determined using transwell and scratch wound assays.4.To establish stable CENPU-knockdown cell lines,SMCC7721 cells were transduced with lentiviral RNAi vector GV493.Subcutaneous xenograft tumor model was adopted to evaluate the effects of CENPU-knockdown on tumor growth.Subcutaneous xenograft tumor model was adopted to evaluate the effects of CENPU-knockdown on tumor growth.Results1.RT-qPCR results showed that CENPU mRNA expression was significantly upregulated in HCC tissues compared with that in paracancerous tissues(P<0.001).Western blot and immunohistochemistry results indicated that CENPU was markedly higher in HCC tissues than that in paracancerous tissues.RT-qPCR and western blot analyzed that CENPU expression was elevated in five HCC cell lines including Huh7,HepG2,Bel7402,HCCLM3,and SMCC7721 compared with that in the human normal hepatocyte line LO2.2.Western blot results showed that CENPU overexpression could obviously attenuate expressions CENPU expression in Huh7 cells.In contrast,the expression of CENPU was significantly decreased after CENPU knockdown in Bel7402 and SMCC7721 cells.3.CCK-8 and plate clone formation assays indicated that CENPU overexpression promoted Huh7 cell proliferation,while CENPU knockdown inhibited Bel7402 and SMCC7721 cell proliferation.Transwell and scratch wound assays revealed that the migrated and invasive abilities of CENPU overexpressed Huh7 cells were obviously increased,while the migrated and invasive capacity of Bel7402 and SMCC7721 cells with knockdown of CENPU were significantly reduced.4.Tumor xenograft in nude mice revealed that tumor growth rate in the CENPU knockdown group was substantially repressed compared with the control group and the volume and weights of the transplanted tumors significantly decreased compared with control mice.Conclusions1.CENPU is relatively highly expressed in HCC tissues and cell lines.2.CENPU promotes the proliferation,migration,and invasion capacities of HCC cells.Part Ⅲ miR-127-5p regulate biological functions of hepatocellular carcinoma cells by targeting CENPUObjective1.To predict and validate the targeting relationship between miR-127-5p and CENPU.2.To investigate the expression of miR-127-5p in HCC tissues and paraneoplastic tissues.3.To explore the biological function of miR-127-5p in HCC as well as the regulation of miR-127-5p on CENPU expression and function.Methods1.TargetScan,miRWalk,mirDIP,and miRDB databases were used to predict upstream miRNAs of CENPU.miRanda algorithm was applied to verify the binding ability of the predicted 5 miRNAs to CENPU.RT-qPCR was performed to detect the expression of the predicted 5 miRNAs in 10 HCC tissues and corresponding paraneoplastic tissues.2.Dual-luciferase gene reporter assay was conducted to verify the targeting relationship between miR-127-5p and CENPU.3.RT-qPCR was used to detect the expression of miR-127-5p in five HCC cell lines(Huh7,HepG2,Bel7402,HCCLM3,and SMCC7721)and human normal hepatocyte line LO2.RT-qPCR and western blot were performed to monitor the changes of miR-127-5p,CENPU mRNA,and CENPU protein expression after miR-127-5p overexpression.4.CCK-8,plate clone formation assay,and subcutaneous xenograft tumor model of nude mice were carried to detect the effect of miR-127-5p overexpression on proliferation ability of HCC cells.Transwell and cell scratch assay were performed to assess the influence of miR-127-5p overexpression on migration and invasion ability of HCC cells.5.Rescue experiments were used to demonstrate miR-127-5p regulate biological functions of HCC cells by targeting CENPU.miR-127-5p mimics and CENPU overexpression in Bel7402 and SMCC7721 cells were co-transfected with lentiviral particles,then we determine the changes in cell proliferation,migration and invasion ability.We also observe whether CENPU overexpression could reverse the effect of miR-127-5p mimics on biological properties of HCC cells.Results1.Five potential miRNAs including miR-3158-5p,miR-1322,miR-127-5p,miR-1256,and miR-1200 were identified after prediction and taking intersections by TargetScan,miRWalk,mirDIP,and miRDB databases.miRanda algorithm results suggested that the binding score between miR-127-5p and CENPU was the highest.RT-qPCR results showed that the expression of miR-127-5p was markedly lower in HCC tissues than in paracancerous tissues,while miR-3158-5p,miR-1322,miR-1256,and miR-1200 expression have no obvious difference in HCC tissues and paracancerous tissues.2.The dual luciferase gene reporter assay revealed that luciferase activity was significantly reduced following co-transfection with miR-127-5p mimic and wild-type CENPU 3’UTR compared with the control group.However,the luciferase activity was not significantly changed following co-transfection with miR-127-5p mimics and the mutant CENPU 3’UTR.These results suggested that miR-127-5p binds with 3’UTR of CENPU.3.RT-qPCR results suggested that the expression of miR-127-5p was significantly higher in human normal hepatocyte line LO2 than five HCC cell lines(Huh7,HepG2,Bel7402,HCCLM3,and SMCC7721).The results found that miR-127-5p expression was significantly increased,while CENPU mRNA and protein expression levels were decreased after miR-127-5p mimics transfection in Bel7402 and SMCC772 cells.4.CCK-8,plate clone formation assay,and subcutaneous xenograft tumor model of nude mice indicated that the cell proliferation ability was obviously repressed after miR-127-5p mimics transfection in Bel7402 and SMCC772 cells.Transwell and cell scratch assays showed that the migration and invasion ability of Bel7402 and SMCC772 cells were significantly reduced after miR-127-5p mimics transfection in Bel7402 and SMCC772 cells.5.Rescue assay results showed that CENPU overexpression could significantly reversed the inhibitory effect of miR-127-5p on the proliferation,migration and invasion of Bel7402 and SMCC772 cells.Conclusions1.CENPU is a direct target of miR-127-5p.2.miR-127-5p inhibits the proliferation,migration,and invasion ability of HCC cells.3.miR-127-5p negatively regulates the effect of CENPU on the proliferation,migration,and invasion ability of HCC cells. |
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