| Background: Hepatocellular carcinoma(HCC)is a common highly malignant cancer which seriously threatens people's life and health.Metastasis is one of the major causes of the death in HCC patients,and regarded as a major challenge in HCC treatment.However,the mechanisms of HCC metastasis have not elucidated yet.Progress in this area is required for the development of novel anti-HCC strategies.Epithelial-to-mesenchymal transition(EMT)is an important pathological change involved the polarization of epithelial cells,which enables a switch from an epithelial phenotype to an invasive mesenchymal state.The mechanism of EMT involved in tumor metastasis is complicated and has not been elucidated.It is vital to further investigate the specific molecular mechanism of EMT in HCC and is crucial for the prevention and treatment of liver cancer metastasis.Abnormal gene regulation leads to the occurrence and development of various cancer including liver cancer.At present,high-throughput biotechnology combined with bioinformatics analysis such as gene chip has been widely used to explore the mechanism of tumor formation at the molecular level.This study found that FEN1 may be a key gene in the development of liver cancer with bioinformatics screening and clinical sample validation.FEN1 is a structure-specific nuclease involved in DNA replication,synthesis,and damage repair,and is essential for maintaining genome stability.Previous studies have shown that FEN1 is abnormally highly expressed in various cancers and is involved in the regulation of various biological processes.However,the functional significance of FEN1 in HCC has not clarified at the molecular level.It is significant to explore the underlying mechanism of FEN1 on the biological process of liver cancer for the prevention and treatment of HCC.Part ? FEN1 is up-regulated in HCC and is associated with HCC metastasis based on bioinformatics screening and clinical sample validationObjective: 1.To identify hepatocellular carcinoma-related core genes by screening the liver cancer-related gene chip datasets in the GEO database.2.To verify the expression of FEN1 in hepatocellular carcinoma and to analyze its correlation with clinicopathological parameters.Methods: 1.The liver cancer-related gene chip datasets in GEO database were downloaded.The differentially expressed genes(DEGs) were analyzed using GEO 2R and intersected using Funrich software.2.Database for Annotation,Visualization and Integrated Discovery(DAVID) database was applied to investigate Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis.3.The protein-protein interaction network(PPI) of DEGs was obtained using String database,and visualized using Cytoscape software.Cyto Hubba plug-in analyzed hub gene and MCODE plug-in constructed key clustering modules(cluster).The GO and KEGG analysis were performed on the genes in the modules.Cyto Hubba plug-in analyzed the core genes in the most critical cluster.4.The clustering and expression of core genes in TCGA liver cancer tissues were analyzed using UCSC Cancer Genomics Browser.The GEPIA database validated the expression of core genes and analyzed the effects on the survival and prognosis of liver cancer.5.The PPI of the core genes was analyzed using cbioportal database,and the expression of core genes with interaction in UCSC TCGA liver cancer database was further analyzed.6.The expression of FEN1 in tumor tissues including liver cancer was investigated in Oncomine database and Firebrowse database.7.The expression of FEN1 in HCC was validated by immunohistochemistry(IHC) and RT-qPCR analysis,and its correlation with clinical pathological parameters was analyzed.Results: 1.The GEO 2R online analysis tool was utilized to investigate the DEGs in four datasets(GSE14520,GES29721,GSE60502,GSE45267).The overlapped DEGs among the four datasets were identified and demonstrated in Venn software,including 142 upregulated and 267 downregulated genes.2.GO analysis showed that the biological process(BP)of up-regulated DEGs was mainly related to cell division,cell cycle,DNA replication,etc;cellular component(CC) analysis showed that it was mainly involved in nucleoplasm,nucleus,cytoplasmic,etc;molecular function(MF) analysis showed that it mainly had the function of protein binding,ATP binding,DNA helicase activity and chromatin binding,etc.The BP analysis of the down-regulated DEGs showed that it was mainly involved in the redox process,the cytochrome 450 pathway and the negative regulation of growth;CC analysis showed that it was involved in the composition of extracellular bodies and organelle membranes;MF results showed oxidoreductase activity,monooxygenase activity,etc.KEGG signaling pathway analysis showed that the up-regulated DEGs were mainly enriched in cell cycle,DNA replication,P53 signaling pathway,tumor pathway,etc.,while the down-regulated DEGs were mainly enriched in metabolic pathway,fatty acid degradation,chemical carcinogenesis,PPAR signaling pathway,etc.3.The PPI of DEGs was constructed and visualized using String database combined with Cytoscape software.185 Hub genes were identified with cyto Hubba plug-in.MCODE plug-in filtered out three clustering modules(cluster1-3).4.GO and KEGG analysis were performed on three modules.BP showed that cluster 1 mainly involved in cell differentiation and DNA replication;cluster 2 mainly involved in plasminogen activator and complement activation regulation;cluster 3 mainly involved in steroid metabolism and exogenous Processes such as drug catabolism.CC results showed that cluster 1 mainly involved in nucleoplasm and nucleus;cluster 2 mainly involved in exosomes and extracellular regions;cluster 3 mainly involved in organelle membrane and endoplasmic reticulum membrane.MF showed that cluster 1 functioned as protein binding and DNA binding;cluster 2 mainly functioned as endopeptidase activity and transcription factor binding;cluster 3 mainly functioned as aerobic binding and heme binding.KEGG analysis showed that cluster 1 was mainly enriched in cell cycle,DNA replication,P53 signaling pathway,etc.;cluster 2 was mainly enriched in the complement system;cluster 3 was mainly enriched in chemical carcinogenesis and metabolic pathways.5.The cyto Hubba analysis was performed on tumor related cluster 1.The first forty genes obtained by twelve different algorithms were intersected and finally screened nine core genes including BIRC5,DLGAP5,DTL,FEN1,KIAA0101,KIF4 A,MCM2,MKI67 and RFC4.The UCSC Cancer Genomics Browser database showed that nine core genes were highly expressed in HCC tissues,and the results were consistent with that in GEPIA database.Meanwhile,the high expression of nine core genes indicated poor prognosis of HCC patients.The PPI of the core gene analyzed by cbioportal database showed an interaction between FEN1,MCM2,RFC4 and BIRC5,and there was a significant positive correlation between FEN1 and MCM2 or RFC4 or BIRC5 expression in TCGA liver cancer tissues.6.Oncomine and Firebrowse databases showed that FEN1 was highly expressed in a variety of tumor tissues,including liver cancer.Otherwise,FEN1 was highly expressed in three different liver cancer related datasets in Oncomine database.7.The highly expression of FEN1 in HCC was verified by immunohistochemistry and RT-q PCR analysis.Furthermore,the expression of FEN1 in metastatic liver cancer tissues was significantly higher than that in non-metastatic liver cancer tissues.Correlation analysis showed that highly expressed FEN1 was significantly correlated with tumor size and metastasis status.Conclusions: Nine liver cancer-related core genes were screened,of which FEN1 was highly expressed in liver cancer and correlated with liver cancer metastasis.Part ? Effects of FEN1 on invasion,migration and EMT of hepatoma cellsObjective: 1.To explore the expression of FEN1 in six different hepatoma cells and normal liver cells HL7702.2.To investigate the effects of FEN1 on invasion,migration and EMT of hepatoma cells.Methods: 1.Firstly,we detected the expression of FEN1 in six hepatocellular carcinoma cell lines(SK-HEP-1,MHCC-97 H,Hep G2,SMMC-7721,HCCLM3,Huh7) and normal hepatocyte HL7702 by RT-q PCR and Western blot analysis.2.SMMC-7721 cells were transfected with lentiviral FEN1 knockdown and overexpression vector,and stable cell lines were screened by puromycin.The hepatoma cell lines stably overexpressing and knocking down FEN1 were verified by RT-q PCR and Western blot analysis.3.We performed the scratch healing and transwell chamber assays to explore the effects of FEN1 on the invasion and migration of hepatoma cells after FEN1 overexpression or knockdown.The expression of EMT markers(E-cadherin,N-cadherin,Vimentin),MMP2 and MMP9 were detected by Western blot analysis.4.The effects of FEN1 on the growth of transplanted tumors and lung metastasis of liver cancer were explored in nude mice.The effects of FEN1 on EMT markers was also detected by IHC analysis in tumor tissues.Results: 1.The expression of FEN1 in six liver cancer cells was significantly higher than that in normal liver cells HL7702.2.The hepatoma cell lines stably overexpressing and knocking down FEN1 were successfully constructed.3.Using genetic interference,we demonstrated that FEN1 silencing inhibited HCC cells EMT,invasion and migration in vitro.Conversely,overexpression of FEN1 had an opposite effect.4.Meanwhile,we demonstrated that FEN1 silencing significantly suppressed tumor growth and metastasis in vivo.Results from IHC analysis suggested that EMT process was blocked after FEN1 knockdown.Conversely,overexpression of FEN1 in HCC cells had an opposite effect.Conclusions: We demonstrated that FEN1 promoted HCC EMT in vitro and in vivo.Part ? mi R-140-5p targets FEN1 to inhibit HCC EMTObjective: 1.To search the upstream micro RNAs that regulate FEN1 expression.2.To verify whether mi R-140-5p can regulate FEN1 expression in hepatoma cells.3.To discuss whether mi R-140-5p involved in the FEN1 induced EMT.Methods: 1.We predicted micro RNAs that regulated FEN1 by Target Scan database.2.Dual luciferase reporter gene was used to verify whether mi R-140-5p binds to the 3'-UTR region of FEN1.3.Firstly,SMMC-7721 and MHCC-97 H stably overexpressing mi R-140-5p were constructed by lentiviral transfection,then mi R-140-5p inhibitor was transfected.The expression of mi R-140-5p was detected by RT-q PCR,and the expression of FEN1 was detected by RT-q PCR and Western blot.4.The expression of mi R-140-5p in HCC tissues was detected,and the correlation between FEN1 and mi R-140-5p in HCC tissues was analyzed.5.The viral vectors containing mi R-140-5p and FEN1 were co-transfected into liver cancer cells.Transwell chamber assay was used to investigate the effect of mi R-140-5p on the invasion and migration of hepatoma cells stably overexpressing FEN1.Western blot was used to detect the effect of mi R-140-5p on the FEN1 induced EMT.6.The SMMC-7721 and MHCC-97 H cells overexpressing FEN1 were transfected with mi R-140-5p,then cells were injected into the axilla of nude mouse to construct a subcutaneous tumor formation model.The effects of mi R-140-5p on FEN1 regulation of tumor growth and EMT was verified in vivo.Results: 1.Target Scan database showed that mi R-140-5p may be involved in the transcriptional regulation of FEN1.2.Dual luciferase reporter gene showed that mi R-140-5p binded to the 3'UTR region of wild-type FEN1,but had no effect on the 3'UTR region of mutant FEN1.3.The hepatoma cell lines overexpressing mi R-140-5p were successfully constructed,and mi R-140-5p inhibitor significantly inhibited the expression of mi R-140-5p.RT-q PCR and Western blot suggested that mi R-140-5p inhibited FEN1 expression,while mi R-140-5p inhibitor restored FEN1 expression.4.We demonstrated that mi R-140-5p was down-regulated in HCC tissues,and the expression of mi R-140-5p in metastatic liver cancer tissues was significantly lower than that in non-metastatic liver cancer tissues.Correlation analysis indicated that FEN1 in liver cancer tissues was negative correlation with mi R-140-5p expression(r=-0.465,P=0.034).Meanwhile,the low expression of mi R-140-5p indicated poor prognosis of HCC patients.5.Transwell chamber assay showed that mi R-140-5p attenuated the invasion,migration and EMT induced by FEN1 in vitro.6.The results showed that mi R-140-5p attenuated the growth of subcutaneous tumors and inhibited EMT induced by FEN1 in vivo.Conclusions: mi R-140-5p targetes FEN1 to inhibit HCC EMT.Part ? TGF?1 acts through mi R-140-5p to up-regulated FEN1 in regulating HCC EMTObjective: 1.To verify the regulatory effect of TGF?1 on HCC EMT.2.To investigate the regulatory effects of TGF?1 on mi R-140-5p and FEN1 in hepatoma cells.3.To determine whether TGF?1 regulates FEN1 via mi R-140-5p in the EMT process.Methods: 1.To determine the effect of TGF?1 on HCC EMT,we treated HCC cells with 10 ng/ml TGF?1 for 48 h.Then the morphology change was observed and the expression of EMT markers and FEN1 was detected by Western blot.RT-q PCR was used to detect the expression of mi R-140-5p and FEN1.2.The FEN1 knockdown hepatoma cells were treated with 10 ng/ml TGF?1 for 48 h,then the expression of EMT markers and FEN1 was detected by Western blot.3.The mi R-140-5p overexpressing hepatoma cells were treated with 10 ng/ml TGF?1 for 48 h,then the expression of EMT markers and FEN1 was detected by Western blot.Results: 1.The morphology of hepatoma cells became fibrillar after 48 h of TGF?1 treatment.Simultaneously,the expression of E-cadherin decreased while the expression of N-cadherin and Vimentin increased.2.RT-q PCR showed that TGF?1 down-regulated the expression of mi R-140-5p while up-regulated the expression of FEN1 in hepatoma cells.Western blot analysis showed that TGF?1 promoted the expression of FEN1.3.FEN1 knockdown attenuated the TGF?1 induced EMT.4.mi R-140-5p attenuated the TGF?1 induced EMT and inhibited the positive regulation of TGF?1 on FEN1.Conclusions: TGF?1 acts through mi R-140-5p to up-regulated FEN1 in promoting HCC EMT. |
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